293E cells for transient transfection to recover 1g/L of protein [[link|http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2528171/]]
3T3 is a fibroblast cell line. The '3T3' designation refers to the fact that this cell line was originally grown at a rate of 3 x 105 cells per 20-cm² dish (the first '3'), with a transfer interval (the 'T') of 3 days (the second '3').
Actin binding proteins. Includes [[Arp2/3]], [[Profilin]], [[alpha-actinin]], [[N-WASP]], [[Spire]], [[Formins]]. See wikipedia [[Actin-binding proteins|http://en.wikipedia.org/wiki/Actin-binding_protein]].
The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. The APC gene product binds through its armadillo repeat domain [[ARM]] to a Rac GEF [[Asef]] [[Akiyama 2000|http://www.ncbi.nlm.nih.gov/pubmed/10947987?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Armadillo repeats are named after the β-catenin-like Armadillo protein.
c-Abl activates WAVE2 via tyrosine phosphorylation to promote actin remodeling in vivo and that [[Abi1]] forms the crucial link between these two factors. [[Yuan 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16899465&query_hl=28&itool=pubmed_docsum]]\nAbl KO neuroepithelial cells display gross alterations in their actin cytoskeleton. [[
See [[Abi1]].
Abelson kinase interacting protein. [[Abi1]] (also known as E3b1) recruits PI3K, via [[p85]], into a multimolecular signaling complex that includes [[Eps8]] and [[SOS]]. The recruitment of [[p85]] to the ~Eps8-Abi1-Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate ([[PIP3]]), the catalytic product of PI3K, concur to unmask its ~RacGEF activity in vitro [[Scita 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12515821&query_hl=25&itool=pubmed_docsum]]. Abi1 and Abi2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia [[Stradal 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11516653&query_hl=31&itool=pubmed_docsum]].
See [[Abelson kinase]].
Monomeric ~ATP-binding protein, which assembles into filaments at the critical concentration. See wikipedia article [[Actin|http://en.wikipedia.org/wiki/Actin]]. Great review on actin and actin binding proteins [[Pollard 2003 review|http://www.ncbi.nlm.nih.gov/pubmed/12600310?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Actin filaments may also anneal end to end in-vitro [[Pollard 1988|http://www.ncbi.nlm.nih.gov/pubmed/3384850]]
Materials:\n1. Intracellular buffer: prepare 10X stock with 1.4 M KCl, 10 mM MgCl2, 20 mM EGTA, 200 mM Hepes pH 7.5. Prepare 2X working solution by dilution of two parts with eight parts water; store at 4°C. Add 0.4% low endotoxin albumin from human serum (A-5843, Sigma) fresh and let sit at room temperature for 30 min before mixing (see Note 5).\n2. 2X Fixation buffer: 2X intracellular buffer plus 640 mM sucrose and 7.4% formaldehyde (F-1268, Sigma); store at 4°C (see Note 6).\n3. Stain buffer: intracellular buffer plus 0.2% triton and 2 µl/ml rhodamine phalloidin (R415, Invitrogen) (see Note 7).\n\n\nMethod:\n1. Stimulate adherent cells with micropipette or uniform chemoattractant. Alternatively, you can stimulate cells in solution.\n2. Add 2X fixation buffer to cells. (Thus, if you had 125 µl cells in RMPI media, you would add 125 µl fixation buffer to give a final concentration of 320 mM sucrose and 3.7% formaldehyde.) Fix for 20 min at 4°C (see Note 19).\n3. Aspirate supernatant (adherent cells) or spin down cells at 400xg for 1 min and then aspirate supernatant (suspended cells).\n4. Permeabilize cells with 125 µl stain buffer and protect from light for 20 minutes (Fig. 6).\n5. Remove supernatant, replace with intracellular buffer, and image (see Note 20).\n\n[img[stain|http://img508.imageshack.us/img508/5748/figure6rgbpi4.gif]]\n\n''Fig. 6. Staining the actin cytoskeleton.'' Add 2x fixation buffer to plated cells and fix for 20 min at 4°C. Remove fixation buffer and replace with stain buffer for 20 min; protect from light. Shown is an example of an HL-60 cell stained with rhodamine phalloidin visualized with structured illumination microscopy.
Actin fused to YFP.
Loss of receptor phosphorylation (and subsequent internalization) in Dicty does NOT prevent chemotaxis [[Devretoes 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9341180&query_hl=19&itool=pubmed_docsum]].
Adducin is a membrane-skeletal protein that promotes the binding of [[Spectrin]] to actin filaments and is concentrated at the cell-cell contact sites in epithelial cells. [[Myosin phosphatase]] binds adducin. Rho-kinase ([[ROCK]]) phosphorylates alpha-adducin in vitro and in vivo and the phosphorylation of alpha-adducin by [[ROCK]] enhances the interaction of alpha-adducin with actin filaments in vitro [[Kaibuchi 1998|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=9488679&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus]].
The cells include [[Lymphocyte]], [[Monocyte]], and [[Macrophage]]
Actin-interacting protein1 - related to cofilin. Whereas control cells mostly formed a single lamellipodium in response to chemokine stimulation, Aip1 knockdown cells abnormally exhibited multiple protrusions around the cells before and after cell stimulation. This indicates that Aip1 plays an important role in directional cell migration by restricting the stimulus-induced membrane protrusion to one direction via promoting cofilin activity [[Mizuno 2008|http://www.ncbi.nlm.nih.gov/pubmed/18494608]].\n\nThe cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro. Additionally, in aip1Delta mutant cells, lat A-insensitive actin assembly is significantly enhanced, which implies that there is some latA-insensitive polmerization of actin oligomers [[Drubin 2010|http://www.ncbi.nlm.nih.gov/pubmed/20231387]].
Showed that [[Ras]] diffusion was suppressed after activation by visualizing single molecules [[Kusumi 2004|http://www.ncbi.nlm.nih.gov/pubmed/15123831]].\n\nVisualized the colocalization of two single molecules in living cells [[Kusumi 2004|http://www.ncbi.nlm.nih.gov/pubmed/15596511]].\n\nShowed that GPCRs undergo rapid hop diffusion between cytosketetal-bound membrane compartments [[Kusumi 2005|http://www.ncbi.nlm.nih.gov/pubmed/15681644]].\n
Akt has an N-terminal pleckstrin homology ([[PH]]) domain that binds to the lipid products of phosphoinositide 3-kinase (PI3K), phosphatidylinositol-3,4-bisphosphate [[PI(3,4)P2]] and phosphatidylinositol-3,4,5-trisphosphate [[PI(3,4,5)P3]]. This binding locates it at the plasma membrane.
Showed that microinjection of CA Rho caused cells to adopt a distinct and novel phenotype with a contracted
config.options.chkHttpReadOnly = false;\n
Aluminum fluoride increases lamellipodia formation in B16F1 cells [[Small 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11257497&query_hl=6&itool=pubmed_DocSum]]. Aluminum fluoride binds reversibly to proteins sites normally occupied by phosphate [[Chabre 1990|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=2499311&query_hl=9&itool=pubmed_DocSum]]. Previously shown to indirectly activate the Arf1 GTPase, aluminum fluoride (AIF) treatment of Arf6-transfected cells resulted in a redistribution of both Arf6 and actin to discrete sites on the membrane. Aluminum fluoride causes rapid activation of Rac within 5 minutes combining 30 mM NaF with 100 µM AlCl3 [[Coffer 1999|http://www.ncbi.nlm.nih.gov/pubmed/10419906?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Showed that Fluoride can activate heterotrimer GPCRs [[Gilman 1981|http://www.ncbi.nlm.nih.gov/pubmed/6271754]] in an aluminum dependent way [[Gilman 1982|http://www.ncbi.nlm.nih.gov/pubmed/6289322]].
Andrew Houk\n12/3/07\n\n5E6 cells per reaction\n\nMake the media: 20% FBS, 1x antibiotic/antimycotic, 1x glutamine in monocyte media.\n\nSpin the cells (each reaction has its own tube), 10 min @ 90g\n\nDuring spin:\nPut 2mL media in a 12-well plate into incubator for 5 minutes to get CO2, temperature levels right.\n\nMix tfxn soln. with supplement (mix entire amounts of each with each other)\n\n100uL of soln/supplement + 2ug DNA for each reaction, flick to mix\n\nGet plastic pipette @ RT\n\nSet “Y001” on the Amaxa machine\n\nTransfer 500uL media from the 12-well plate into an eppie for each reaction.\nGet electroporation chambers ready.\n\nAfter spin, suck off all the media with aspirator\n\nUse P1000 to put 100uL transfection solution onto cells, resuspend gently and put cells in electroporation chamber.\n\nPut chamber into machine and press Start\n\nUse plastic pipette to suck up 500uL recovery media from eppie, flush the chamber, suck up cells and put back into the eppie.\n\nIncubate at 37C for 30 minutes.\n\nFor each reaction put 1.25mL media into each of two wells on a 12 well plate. Put plate in incubator.\n\nAfter 30 minutes transfer 500 uL/well to each 1.25-1.5 mL media in the 12-well plate.\n\n[img[Amaxa|http://img508.imageshack.us/img508/6123/figure2rgbhu1.gif]]\n\n''Fig. 2. Transient transfection of HL-60 cells with amaxa nucleofection.'' Spin ~5 million cells at 100xg. Aspirate supernatant and resuspend pellet in 100 µl transfection solution per reaction and nucleofect with amaxa program “Y-001”. Flush with prewarmed recovery media and incubate in an eppendorf tube for 30 min. Transfer to a 6-well dish with 1.5 ml of recovery media; expression occurs after 2 hours. Shown is an example of HL-60 cells 5 hours after transfection with GFP visualized with DIC and fluorescence microscopy.\n\nNote: I tend to see expression and good cell viability between 7-13 hours. AM 5/10/09
Is a douchebag
Showed that CXCL1 induces [[Cdc42]] and [[Pak1]] activation in CXCR2-expressing HEK293 cells [[Richmond 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12033944&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that RhoB siRNA impaired CXCR2-mediated chemotaxis [[Richmond 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17405813&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that VASP binds CXCR2 in a stimulus dependent fashion, which triggers phosphorylation of VASP by [[PKA]] and PKCdelta and that knockdown of VASP in HL-60 cells results in severely impaired CXCR2-mediated chemotaxis and polarization [[Richmond 2009|http://www.ncbi.nlm.nih.gov/pubmed/19435808]].\n\nShowed that a novel protein LASP-1 (for LIM and SH3 domain containing) binds CXCR2 under both basal and stimulated conditions. Knockdown of this interaction impairs cell migration [[Richmond 2010|http://www.ncbi.nlm.nih.gov/pubmed/20419088]].
Showed that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both beta1 and beta3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment [[Huttenlocher 1997|http://www.ncbi.nlm.nih.gov/pubmed/9407041]].\n\nShowed that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient in HL60 cells and neutrophils [[Huttenlocher 2007|http://www.ncbi.nlm.nih.gov/pubmed/17192410]].\n\nShowed that mAbp1 (mammalian actin binding protein) directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. The interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1 [[Huttenlocher 2009|http://www.ncbi.nlm.nih.gov/pubmed/19910490]].\n\nShowed that a genetically encoded photoactivatable Rac is sufficient to direct migration with precise temporal and spatial control in zebrafish [[Huttenlocher 2009|http://www.ncbi.nlm.nih.gov/pubmed/20159593]]
Is a ~FirstYear //year// ''graduate'' student [[her paper|http://www.ncbi.nlm.nih.gov/pubmed/19303437?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]
Showed that CA Rho rapidly stimulated stress fiber and
Arap3 is a dual [[GAP]] for RhoA and [[Arf6]] that is regulated by phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], a product of the phosphoinositide 3-kinase (PI3K) signalling pathway. ~ARAP3-deficient cells were not capable of producing lamellipodia upon
Arf proteins compose a family of related small guanosine triphosphatases (GTPases) in the [[Ras]] superfamily that were originally isolated on the basis of their ability to stimulate adenosine diphosphate (ADP) ribosylation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by cholera toxin. These proteins play a role in vesicle budding from internal organelles. Arf family members also play a role in activation of phospholipase D. Arf6, an unusual member of the family, is located at the
Arf1 mediates [[Paxillin]] recruitment to focal adhesions. Leakage of endogenous Arf from permeabilized cells was coincident with loss of ~GTPgammaS- induced redistribution of [[Paxillin]] to focal adhesions, and the response was recovered by addition of Arf1 [[Cockcroft 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9864369&query_hl=15&itool=pubmed_docsum]].
The ~ADP-ribosylation factor 6 (Arf6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. Arf6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by [[Rac]]. Additionally, Arf6 at the PM induces the formation of actin-containing protrusions. Two residues in the amino-terminal half of Arf6, Q37 and S38, when substituted into the (amino-terminal [[Arf1]] and a carboxy terminal Arf6) 1-6 chimera allowed protrusion formation [[Donaldson 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10913182&query_hl=2&itool=pubmed_DocSum]]. Signaling by insulin, endothelin 1, or G(alpha)11(~Q209L) mobilizes cortical F-actin in cultured adipocytes. Interestingly, expression of a dominant inhibitory form of the actin-regulatory GTPase Arf6 [Arf6(~T27N)] in cultured adipocytes selectively inhibits F-actin formation in response to endothelin 1 but not insulin [[Czech 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11438680&query_hl=2&itool=pubmed_DocSum]]. Endogenous [[Rac1]] colocalized with Arf6 at the plasma membrane and on the Arf6 recycling endosome in untransfected HeLa and primary human fibroblast cells. Co-expression of the dominant negative, GTP binding-defective Arf6 ~T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing [[Rac1]], and the constitutive ruffling observed in cells expressing the activated Rac1Q61L mutant [[Donaldson 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10036235&query_hl=2&itool=pubmed_DocSum]]. Arf6 binds to and activates ~PIP5KI, leading to the recruitment of clathrin coats in synaptic-vesicle preparations [[De Camilli 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=12847086&dopt=Abstract]]. Arf6-mediated knockdown using siRNAs revealed that Arf6 regulates the internalization of most G-protein-coupled receptors, irrespective of its route of entry [[Claing 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15590645&query_hl=21&itool=pubmed_docsum]].\n\nActivation of Arf6 by [[Arno]] stimulates
Depletion of endogenous the Arf6 GEF Brag2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of beta1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of beta1 integrin and reduced adhesion and spreading [[Casanova 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16461286&query_hl=19&itool=pubmed_docsum]]. Another exchange factor for Arf6 (Efa6) contains Sec7 and pleckstrin homology domains. Expression of EFA6 induces actin-based membrane ruffles [[Chavrier 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10075920&query_hl=22&itool=pubmed_docsum]].
Three related Arf-like GTPases of unknown function, Arl4a, Arl4c, and Arl4d, are able to recruit ARNO and other cytohesins to the
Expression of the ArfGEF [[Arno]] in MDCK cells induces robust activation of [[Rac]], the formation of large lamellipodia, and the onset of motility. Arno-dependent activation of [[Rac]] is mediated by a bipartite Rac GEF, the [[Dock180]] [[Elmo]] complex. Both [[Dock180]] and [[Elmo]] colocalize extensively with ARNO in migrating [[MDCK]] cells [[Santy 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16213822&query_hl=10&itool=pubmed_docsum]]. [[Arno]] (Arf nucleotide-binding site opener) is a member of a growing family of ArfGEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on [[Arf1]] and [[Arf3]]. [[Arno]] can stimulate nucleotide exchange on both [[Arf1]] and [[Arf6]] and [[Arno]] is localized to the plasma membrane in mammalian cells rather than the Golgi [[Casanova 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9417041&query_hl=2&itool=pubmed_DocSum]].
Seven member complex, which nucleates actin assembly; binds to the sides of actin filaments to cause branching [[Pollard 2003 review|http://www.ncbi.nlm.nih.gov/pubmed/12600310?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
The Arp3 component of the Arp2/3 complex fused to GFP.
Arthur Millius = arthur.millius@ucsf.edu\n\nWorks on: [[Permeabilized Cells]] and [[Bead assay]] and [[Microscopy]] and [[Biochemistry]]\nEducation:\n2005 – present University of California – San Francisco\n2001 – 2005 Rice University B.A. in Biochemistry, B.S. in Biophysics\n\nProfessional Background:\n2005 – present Graduate Research Advisor Orion Weiner, UCSF\n Cell polarity in neutrophil chemotaxis\n2002 - 2005 Undergraduate Research Advisor HHMI Professor Bonnie Bartel, Rice University\n Arabidopsis auxin homeostasis in the peroxisome\n\nHonors/Awards:\n2010 NSF East Asia and Pacific Summer Institutes Research Fellowship\n2007 National Defense Science and Engineering Graduate Research Fellowship\n2006 UCSF Genetics T.A. teaching award\n2005 Jenessa Shapiro Award for Outstanding Undergraduate Research\n2005 Frederick B. Rudolph Award for Outstanding Biochemistry and Cell Biology Senior \n2005 National Science Foundation Graduate Research Fellowship Program Honorable Mention \n2005 Rice University Cum Laude Graduate\n2004 American Society of Plant Biologists Summer Undergraduate Research Fellowship \n \nPublications:\n1. Millius, A., Dandekar, S., Houk, A.R., and Weiner, O.D. “Neutrophils establish rapid and robust WAVE complex polarity in an actin-dependent fashion.” Curr Biol. 2009 Feb 10;19(3):253-9.\n2. Millius, A. and Weiner, O.D., “Chemotaxis in neutrophil-like HL-60 cells.” Methods Mol Biol. 2009;571:167-77.\n3. Houk, A.R., Millius, A., and Weiner, O.D. “Compete globally, bud locally.” Preview, Cell 2009 Nov 13; Vol. 139, Issue 4\n4. Zolman, B.K, Martinez, N., Millius, A., Adham, A.R., and Bartel, B. “Identification and characterization of Arabidopsis indole-3-butyric acid response mutants defective in novel peroxisomal enzymes.” Genetics. 2008 Sep;180(1):237-51.\n5. Adham, A.R., Zolman, B.K, Millius, A., and Bartel, B. “Mutations in Arabidopsis thaliana acyl-CoA oxidase genes reveal overlapping and distinct roles in b-oxidation.” Plant J. 2005 Mar;41(6):859-74.\n\nTalks:\n2008 Millius, A., Houk, A., Dandekar, S., and Weiner, O. “How cells estabish polarity and guide movement in response to external cues.” FASEB Summer Research Conference, New Haven, CT\n\nPoster Presentations:\n2009 Millius, A., Dandekar, S., Houk, A., and Weiner, O. “Neutrophils establish rapid and robust WAVE complex polarity in an actin-dependent fashion.” Gordon Conference – Gradient Sensing and Directed Cell Migration, Galveston, TX\n2008 Millius, A., Houk, A., Dandekar, S., and Weiner, O. “Analysis of Scar/WAVE membrane dynamics in chemotactic gradients and in response to external cues.” Keystone Symposia – Cell Migration in Invasion and Inflammation, Taos, NM\n\nOutreach and Extracurricular Activities:\n2008-2009 Studying Japanese Language and Culture at CCSF\n2009 UCSF Science and Health Education Partnership, Biochem Teach\n2008 UCSF Science and Health Education Partnership, City Science Program\n2007 UCSF Science and Health Education Partnership, Quattro Program\n2006 UCSF Science and Health Education Partnership, Stat Program\n
[[APC]] promotes Asef GEF activity to Rac in vitro and the formation of ruffles in MDCK cells [[Akiyama 2000|http://www.ncbi.nlm.nih.gov/pubmed/10947987?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
A Rac GEF that when knocked down in HT1080 cells reduces cell migration [[Webb 2009|http://www.ncbi.nlm.nih.gov/pubmed/19934221?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1]].
B cells are lymphocytes that play a large role in the humoral immune response as opposed to the cell-mediated immune response that is governed by T cells. The abbreviation "B" comes from bursa of Fabricius that is an organ in birds in which avian B cells mature. The principal function of B cells is to make antibodies against soluble antigens. B cells are an essential component of the adaptive immune system.
Non-migratory mouse melanoma cell line.
Migratory mouse melanoma cell line.
BAR (Bin/amphiphysin/Rvs) domains contain two pairs of basic residues, which mediate their interaction with negatively charged phospholipid heads in membranes. BAR domains dimerize, generating a crescent-shaped structure in which one of the basic residue pairs in each monomer is located on the concave surface and the other at the edge of the crescent [[McMahon 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14645856&query_hl=126&itool=pubmed_docsum]]. BAR proteins bind strongly to PIP2 and PIP3 [[Takenawa 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16418535&query_hl=128&itool=pubmed_docsum]].
[[Antonny 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12471028&query_hl=28&itool=pubmed_docsum]]\n[[Nakamoto 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10716190&query_hl=32&itool=pubmed_DocSum]]\n[[Swartz 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14764880&query_hl=37&itool=pubmed_docsum]]\n\nShowed that anionic lipids are required for Rac dissociation from GDI. Showed that RacGTPGDI is more susceptible to dissociation from anionic lipids than RacGDPGDI [[Pick 2006|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16702219&ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]
Bem1 directly interacts with [[Cdc42]] and [[Cdc24]] [[Bender 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=7962098&dopt=Abstract]] and is required for landmark independent (without Bud1p/Rsr1) [[Cdc42]] polarization. [[Lew 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14625559&query_hl=37&itool=pubmed_docsum]]. Bem-1 scaffolds Cdc24 and Pak to activate Cdc42.
Several papers discuss the [[WAVE complex]]. One recent one showed that activation of the WAVE2 complex requires simultaneous interactions with prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation [[Kirschner 2009|http://www.ncbi.nlm.nih.gov/pubmed/19917258]].
Inhibits myosin II. Causes mutiple pseudopod formation in fMLP-treated cells, but PhAkt is not as bright at the leading edge [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887922&query_hl=11&itool=pubmed_docsum]]. Inhibition of myosin II alone decreases retrograde flow by 51% in neuronal growth cones [[Forscher 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed]].
Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the RhoGTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates [[Gerhard 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11680784&dopt=Abstract]], [[Aktories 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7777059&query_hl=2&itool=pubmed_DocSum]], [[Aktories 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9548761&query_hl=4&itool=pubmed_docsum]].
C. elegans polarity – summary\n\nEverything I am talking about occurs within a single cell embryo after fertilization.\n\nThe new players: Par1-6, aPKC, ECT-2 (Rho GEF) - The Par proteins, together with aPKC, are thought to dictate which regions of the embryo adopt "anterior" and "posterior" fates.\n\nFamiliar players: Cdc42 and Rho\n\n“Anterior complex”\n\nPar3, Par6 and aPKC all localize to the anterior of the embryo in a mutually dependent manner – They may form a complex (review evidence for this)\n\nPosterior \n\nPar1 (kinase) localizes to posterior, it doesn’t appear to play an active role in localization of the other Par proteins, but it is important that Par1 is localized to the posterior because it helps set cell-fate decisions for subsequent posterior-derived cells. \nPar2 localizes to posterior. Par2 plays an important role in the localization of the other Par proteins.\n\nUniform localization\n\nPar4 and Par5 are uniform. Par5 is a 14-3-3 protein, in its absence there is considerable overlap between Par2 and Par3/6/PKC. I don’t know much about Par4\n\nNow that we have a little background, here is my understanding of how the Par proteins become properly localized\n\nChronology of events:\n\nIt appears as though "cortical localization", i.e. localization at or near the membrane, is associated with activity of the various proteins. Presumably the cortical localization of particular Par proteins at a given region of a cell confers either "anterior" or "posterior" identity on that region.\n\nAt fertilization Par1,2,3,6 all colocalize over the entire cortex. \n\nAt meiosis II Par2 leaves the cortex uniformly – I believe this removal requires Par3/6/PKC. The removal of Par2 definitely requires Cdc42.\n\nApproximately 30 minutes after fertilization, actomyosin and Par3/6/PKC flow towards the anterior – probably driven by actomyosin contraction. Par3/6/PKC enhances the flow rate!\n\nOnce Par3/6/PKC are restricted to the anterior, Par2 now returns to the posterior cortex. If Par3/6/PKC is not removed from posterior cortex by actomyosin then Par2 will never return to posterior cortex!\n\nThe antagonism between Par2 and Par3/6/PKC is mutual (i.e. in par2 mutants, Par3/6/PKC flow to the anterior – however they then creep back towards the posterior and eventually become uniform). \n\nMutual antagonism between Par2 and Par3/6/PKC maintains their asymmetric distributions. Par2 also contributes to the flow by preventing myosin clump assembly in the posterior – without Par2 the flow rates don’t change but there is still myosin formation at posterior which prevents maximal segregation of Par3/6/PKC. \n\nPar5 is required for Par2 and Par3/6/PKC to antagonize one another, because when Par5 is eliminated, the distributions of Par2 and Par3/6/PKC overlap spatially. Par5 is also required for maximal initial asymmetric localization of Par3/6/PKC. It could be required either for Par3/6/PKC to be segregated to anterior by actomyosin flow or it may be required for the flow itself as well. However, the effects on initial Par3/6/PKC could be via the effects of Par5 on Par2 crosstalk as well. Thus it is hard to define the exact role of Par5 from the existing data.\n\n_____________________________________________________________________\nWhat initiates the asymmetric cytoskeletal flow? It is certainly directed by the sperm centrosome at the posterior. The centrosome is required to initiate polarity; however, it is not required to maintain polarity. Upon fertilization, there is uniform myosin activation which depends on Rho (Rho is activated by the GEF Ect-2). The sperm provides a cue which relaxes cortical contraction at the posterior. This cue appears to be the Rho-GAP Cyk-4. Paternal Cyk-4 at the posterior locally inhibits Rho causing local relaxation of the cortex and net cortical flow towards the anterior.\n\nThere is some debate over whether microtubules mediate the effect of the sperm centrosome. \n\nThe evidence against MTs comes primarily from (Hyman, Nature, 2004) where RNAi of gamma-tubulin or alpha-tubulin or beta-tubulin. Also, nocodazole doesn’t prevent polarity.\n\nThe evidence for MT involvement is presented nicely in (Wallenfang, Nature, 2000) and (Ahringer, JCB, 2007). First, in MeiosisI arrested mutants, there is very strong spatial correlation bt. Par2 and meiotic spindle posn., when spindle is off-center so is the Par2 – also sometimes patches of Par2 colocalize with MTs in random cellular loci. Secondly, Spd2/5 mutants “centrosome maturation defective” have delayed MT nucleation and thus don’t establish posterior domain of Par2, rather they establish an anterior domain of Par2 (which is where the meiotic spindle is located) this polarity is abolished upon combination of tubulin mutation and tubulin RNAi – thus MTs can be sufficient to induce polarity. Finally, if you knockdown both a/b tubulin you get a delay in aster formation and a correlated delay in Par2 polarity.\n\nBasically the question is whether the tubulin perturbations ever completely eliminate MTs. Lots of strong correlations between MTs and sites of Par2 accumulation, there is also the convincing spindle/Par2 colocalization which is MT dependent. \n----------------------------------------------------------------------------------------------------------\n\nWhat does Cdc42 do? When Cdc42 is eliminated, the actomyosin flow still occurs (although flow is slower) and Par6-GFP becomes localized to the anterior (although it is less pronounced). Cdc42 is required to keep Par6 at the cortex – when Cdc42 is gone, the anterior cortical Par6-GFP eventually gets removed by Par2. Par3 and PKC are probably effected exactly like Par6.\n\nWhat does Rho do? Rho is required for the cortical flow. Ect-2 (a Rho GEF) is also required for the flow. Ect-2 is locally excluded from the posterior pole in a centrosome-dependent manner. As flow proceeds, the domain from which Ect-2 is excluded increases in size. Proteins like Par3, Cdc42 and Rho itself are required for the expansion of the domain from which Ect-2 is excluded.\n\nSee [[Hall 2003 review|http://www.ncbi.nlm.nih.gov/pubmed/12517706?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n
A chemoattract and intermediate product in the [[Complement Cascade]].
C5a receptor fused to GFP.
Chinese Hamster Ovary
[[Casein kinase2]] is a serine/threonine-selective protein kinase, which phosphorylates WAVE2. When these phosphorylation sites are mutated to non-phosphorylatable alanine residues, it inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells [[Cory 2009|http://www.ncbi.nlm.nih.gov/pubmed/19012317]].
[[CK2]] interacting protein, contains a [[PH]] domain, and may play a role in inactivating [[Akt]] [[Takenawa 2007|http://www.ncbi.nlm.nih.gov/pubmed/17942896?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Dicty protein named for Cytosolic regulator of adenylate cyclase. No obvious homologs in humans, flies, worms, and yeast. Recruited by [[cAR1]] through a [[PH]] domain to the front of the cell and is required to activate adenylate cyclase. Intriguingly, adenlyate cyclase is localized at the back and in small vesicles. CRAC recruitment can undergo adaptation in Dicty is prestimulated with cAMP [[Devreotes 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7790361&query_hl=17&itool=pubmed_docsum]].
[[Cdc42]] [[Rac]] induced binding domain.
See wikipedia [[CXCR2|http://en.wikipedia.org/wiki/CXCR2]]. IL8 receptor.
Calcium is one of the most important cellular signals. Numerous cytoskeletal regulators are, in turn, regulated by calcium. However, the role of calcium in neutrophil polarity and chemotaxis appears to be limited. I will go into all of these things in more detail below. First, I will give a brief summary of what I know about “canonical” calcium signaling. Then I will go into a bit more detail about a few cases where calcium affects important regulators of the cytoskeleton. Then I will discuss the role of calcium in cell motility.\n\nGeneral stuff about what calcium regulates.\n\nCalcium is present in the cytosol of resting cells at ~50nM. The extracellular calcium concentration is much higher than this (I don’t have a specific number). There is also an enormous amount of calcium stored in the endoplasmic reticulum (ER). The calcium concentration within the ER is 400uM. Calcium signaling can occur by opening channels at the plasma membrane or at the ER. Plasma membrane calcium channels are probably gated by numerous different mechanisms and it is widely thought that membrane or cytoskeletal tension is one of these gates. From conversations with Julius lab members, no calcium channels are proven to be tension-gated in vivo. ER calcium channels are also probably by numerous mechanisms. The main ligand for ER calcium channels that I know of is inositol-(1,4,5)-triphosphate (IP3), which is produced from phospholipase C- mediated cleavage of PIP2. If anyone knows more about how calcium channels are gated then elaborate on this paragraph.\n\nHow do elevated cytosolic calcium levels get transduced? As far as I know, the primary transducer of cytosolic calcium levels is a protein called calmodulin (CaM). CaM binds calcium directly. Calcium binding induces conformational changes within CaM that cause it to bind numerous effectors. It is pointless for me to try to list all of the CaM effectors but some of the important ones are: calmodulin-dependent kinases (CaMKII or CaMKIV, for example), calcineurin (a phosphatase), and myosin light chain kinase (MLCK). Thus, calcium can activate myosin (via CaM and MLCK).\n\nIn addition to the “canonical” calmodulin-dependent signaling pathways there are other ways that calcium concentration talks to the cytoskeleton. Gelsolin is an actin-severing protein (the severing of actin filaments can increase the number of free barbed ends and it helps recycle actin monomers to maintain a large pool of monomeric actin). Gelsolin generally caps the severed filaments, unless phosphoinositides are around to prevent capping. I don’t know the physiological consequences of gelsolin depletion/hyperactivation.\n\nI know a bit more about calpain, which is a calcium-dependent protease. There are two types of Calpain (Calpain 1 = mu-calpain, Calpain 2 = m-calpain). Different cell types contain different relative amounts of each calpain. Calpain cleaves numerous proteins that regulate the cytoskeleton, such as talin, ezrin, myosin X and WASP. I will go into more detail about calpain later on.\n\n\nThe role of calcium in motility\n\nIn fibroblasts and keratocytes, calcium appears to play a significant role in motility. First, calpain cleaves talin in a calcium-dependent manner, which is essential for focal adhesion turnover in fibroblasts. Calcium is also required for tail detachment/retraction in keratocytes (possibly calpain-mediated). Generally, calcium concentration is highest at the rear of cells; however, many calcium sensitive proteins are localized to the lamella/lammelipod. A recent study by Wei et al (Nature, 2008) found that there are also transient calcium flickers in the lamella/lamellipod of fibroblasts. The functional role of the flickers was not explored in the study, but the frequency, magnitude and distribution of flickers were shown to be sensitive to chemoattractant. Interestingly, calcium flickers and more global calcium influxes can be triggered mechanically in both fibroblasts and keratocytes – by stretching the substrate or by applying fluid flow. \n\nThere are known to be calcium spikes during neutrophil migration; however, the importance of these spikes to neutrophil migration was hotly debated for some time. There have been various contradictory reports about the requirement of calcium for neutrophil migration. A careful study by Maxfield in JCB (1991) found that the requirement for calcium is largely dependent on the adhesivity of the surface. On adhesive surfaces like fibronectin or vitronectin, calcium is strictly required for movement. In contrast, on albumin coated glass or serum coated glass, calcium is not required for cell movement. Intriguingly, the combination of EGTA with Quin-2 treatment abolishes movement on albumin, even though neither treatment does anything on its own. This could indicate that neutrophil migration requires basal calcium levels. I consider Maxfield, JCB, 1991 to be the authoritative work on the significance of calcium.\n\nWhy is calcium required for movement on adhesive surfaces?\n\nOne interesting story by the Maxfield group (in JCS, 2000) found that calcium depletion impairs myosin activation in neutrophils. In this study, they also show that myosin inhibition phenocopies calcium depletion by causing tail retraction problems and cell elongation. According to them, plating on less adhesive substrates rescues this phenotype, even when myosin inhibitors are used! Myosin inhibition causes a redistrubition of a talin from the front to the tail of polarized neutrophils (presumably calcium does this too). Both the Maxfield and Huttenlocher groups find that calpain is not required for neutrophil movement on either fibronectin or fibrinogen. Huttenlocher says in the discussion section that adhesion turnover is very different in neutrophils than it is in fibroblasts and integrin endocytosis at the rear plays a larger role. Thus, it appears that calcium influences tail retraction in neutrophils by activating myosin (as opposed to calpain mediated degradation of adhesions at the rear).\n\nWhat does calpain do in neutrophils? Is it important at all? 2 Huttenlocher papers and Dewitt/Hallett\n\nDewitt and Hallett think that calpains regulate the reservoir of plasma membrane that is trapped in wrinkles. They have a paper showing that calcium influx is important for phagocytosis and that calpains are a major player downstream of calcium. Specifically, they show that calpain activity liberates integrins, so that more integrins can bind the bead and accelerate phagocytosis. There is no mention of wrinkles in this paper; it seems to be an idea that they proposed in a much later review. \nHuttenlocher has studied the role of calpains in neutrophil migration. Specifically she finds that calpain inhibition causes quiescent neutrophils to start moving. She also finds that calpain activity is high in resting neutrophils. The inhibition of mu-calpain but not m-calpain causes quiescent cells to become active. This isn’t just an artifact of adhesion regulation either, as calpain inhibition of cells in suspension activates Rac and Cdc42. She also finds that calpain inhibition reduces directional persistence (but not speed, questionable) in a micropipette delivered chemoattractant gradient. In a later paper, she examines m-calpain and finds that it localizes to the pseudopod (mu-calpain localizes in a diffuse, cytosolic manner that may be enriched in the back). In this study, she finds that m-calpain may cleave talin (no genetic knockout though?) in neutrophils – the significance of which is unknown. Interestingly, overexpression of WT m-calpain causes the pseudopod to expand; meanwhile, overexpression of protease-dead m-calpain induces multiple pseudopodia. M-calpain may also distribute to lipid rafts in these cells. The above papers, suggest that calpains participate in polarity/chemotaxis; but are probably redundant with other signals – since inhibition of calpain does not prevent polarity and chemokinesis.\n\nThere are some people who believe that calcium signaling is very important for macrophage polarity. One example is Evans and Falke (2007, PNAS), who perform their studies in a macrophage-like line called RAW. These cells polarize on glass and stick so tightly that they don’t translocate at all. Somehow, despite their inability to translocate, their fronts are stable for >15 minutes. They show some of the same properties of neutrophils, namely the corequirement of PIP3 and F-actin. Inhibiting the calcium influx disrupts both PIP3 and actin accumulation at the leading edge. Unfortunately, they don’t do any convincing experiments to show that calcium increases cause PIP3/F-actin increases – they attempt this by adding ATP to the cells, which increases calcium, but we already know that ATP itself induces PIP3/F-actin. Calcium itself is enriched at the leading edge of these cells (in contrast to many other cell types) – I’m not sure about the specificity of their biosensor. Finally they show that PI3K activity is required for calcium enrichment at the leading edge (although latrunculin has much less of an effect than wortmannin, which doesn’t fit well with their hypothesis of a PIP3/Ca/Actin feedback loop.). Overall they show one convincing piece of data: the requirement of calcium for PIP3 and F-actin accumulation in these cells. I am not sure whether basal levels of calcium are playing a permissive role in PIP3/F-actin accumulation or a calcium spike is required for PIP3/F-actin. They never directly measure cytosolic calcium levels so you can’t tell whether EGTA, La, SKF are completely eliminating cytosolic calcium, or just reducing it to non-elevated levels.\n\n
Caps actin barbed ends. Capping protein increases branching by promoting nucleation through Arp2/3 complex [[Mullins 2008|http://www.ncbi.nlm.nih.gov/pubmed/18510928?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. [[VASP]] may antagonize capping protein.
Cappuccino and [[Spire]] were originally identified in a Drosophila screen. Both mutants had defects in cytoplasmic streaming [[Therkauf 1994|http://www.ncbi.nlm.nih.gov/sites/entrez?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8091233&dopt=AbstractPlus]]. Cappuccino contains a formin-homology2 domain ([[FH2]]), which binds to a domain on [[Spire]], which promotes actin nucleation [[Mullins 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17923532&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. Both Cappuccino and [[Spire]] have microtubule crosslinking activity. SpireD inhibits Cappucino's microtubule crosslinking activity, but activated Rho1 suppresses this [[Parkhurst 2006|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16518391&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
See [[CK2]].
WAVE2, Hem1, and Abi1 all have predicted casein kinase 2 substrate sites according to scansite. This recognition site occurs at the c-terminus of WAVE2 and is conserved in both WAVE1 and WAVE3. Furthermore, CaseinKinase2 phosphorylates WASP on its VCA, increasing its affinity for Arp2/3 [[Ridley 2003|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=12769847&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. CaseinKinase2 inhibitors prevent retinal endothelial cell migration and proliferation [[Castellon 2004|ttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15557471&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
GEF for [[Cdc42]].
Cdc42 is a small GTPase involved in the regulation of the cytoskeleton and cell polarity, but not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells [[Brakebusch 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16014609&query_hl=38&itool=pubmed_docsum]]. Activated Cdc42 pulls down [[IQGAP]] [[Cerione 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9305904&query_hl=39&itool=pubmed_DocSum]]. Cdc42 binds to and controls the cellular distribution of the [[IRSp53]]-[[Eps8]] complex [[Scita 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17115031&query_hl=7&itool=pubmed_docsum]] and induces filopodia by promoting the formation of an IRSp53:[[Mena]] complex [[Hall 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=11696321]].
CA Cdc42, which is deficient at GTP hydrolysis.
[[293]] \n[[3T3]]\n[[B16F1]]\n[[B16F10]]\n[[B cell]]\n[[CHO]]\n[[Dictyostelium]]\n[[Fibroblast]]\n[[GpG]]\n[[Granulocyte]]\n[[HeLa]]\n[[HL60]]\n[[HT-1080]]\n[[Keratocyte]]\n[[Leukocyte]]\n[[Lymphocyte]]\n[[Macrophage]]\n[[MDCK]]\n[[Monocyte]]\n[[Natural killer cells]]\n[[Neutrophil]]\n[[PAE]]\n[[Spanish PAE]]\n[[Swiss3T3]]\n[[T cell]]
Chaotropic agents are those that disrupt molecular structure, especially nonbinding forces such as Van der Waals force and hydrophobic effects.
Regulates the bacterial flagellar motor and establishes a precedence for a strict input-output relationship between a protein and behavior. [[Leibler 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10698740&dopt=Abstract]]
Chemokinesis involves changes in speed, alterations of amplitude or frequency of motile character, but has a random direction of migration.
Movement toward certain chemicals in the environment.
Chronophin is a novel ~HAD-type serine protein phosphatase, which regulates cofilin-dependent actin dynamics [[Bokoch 2005|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15580268&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and localizes with [[LIMK]] and [[Cofilin]] at membrane protrusions [[Bokoch 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17500066&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Developed correlation FSM to show the relationship between focal adhesion proteins and f-actin retrograde flow [[Waterman-Storer 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17204653&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that TIRF speckle microscopy is superior to wide-field speckle microscopy [[Waterman-Storer 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15516225&query_hl=4&itool=pubmed_DocSum]].
Papers to read:\n\n[[Coupling |http://www.nature.com/msb/journal/v6/n1/full/msb201092.html]]
Severs actin filaments [[see Condeelis 2009 review|http://www.ncbi.nlm.nih.gov/pubmed/19862699]]. Phosphorylated cofilin/ADF no longer effectively binds to F-actin. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts [[Mitchison 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9087446&query_hl=14&itool=pubmed_docsum]].\n\nCofilin is dephosphorylated by two phosphatases, [[Slingshot]] and [[Chronophin]] [[Bokoch 2006 review|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16337782&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and phosphorylated by [[LIMK]].\n\nCofilin is localized just behind the lamellipodia in the leading edge of a keratocyte [[Svitkina 1999|http://www.ncbi.nlm.nih.gov/pubmed/10352018?ordinalpos=20&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and is required for chemotaxis in cancer cells [[Condeelis 2006|http://www.ncbi.nlm.nih.gov/pubmed/17113383]].\n\nCofilin may act by generating free barbed end of Arp2/3 dependent nucleation or by recycling monomers. If cofilin's primary role was to generate free barbed ends, then injection of labeled actin monomers in cofilin knock-down cells should not lead to their incorporation - but actin monomers do get incorporated [[Mizuno 2006|http://jcb.rupress.org/content/177/3/465.full]].
Colchicine inhibits microtubule polymerization by binding to tubulin. It does not inhibit neutrophil polarity, but instead induces chemokinesis. This depolymerization causes membrane recruitment and activation of ROCK [[Niggli 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12571279]].
Competent cells\n\n• Plate out your favorite strain on an LB plate (or take a few uL from frozen stock)\n• Grow O/N in 5 mL TYM (37 degrees, 300 rpm)\n• Inoculate 250 mL TYM\n• Shake until OD550 = 0.5 (don’t go over 0.55!!)\n• Divide into (6) 50 mL Falcons, spin 3000 rpm, 12 min, 4 degrees\n• Keep everything cold from here on\n• Resuspend cells with 15 mL TFB I per tube\n• Incubations: 20 min for BL21, 15 min for Top10, 10 min for DH5alpha\n• Spin 3000 rpm, 8 min, 4 degrees\n• Resuspend cells with 2 mL TFB II per tube\n• Aliquot cells; flash freeze in liquid nitrogen (store –80 degrees)\n\n\n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n\nRecipes\n\nTFB I\n30 mM potassium acetate\n50 mM manganese chloride\n100 mM potassium chloride\n10 mM calcium chloride\n15% glycerol\n\nTFB II\n10 mM Na-MOPS, pH 7.0\n75 mM calcium chloride\n10 mM potassium chloride\n15% glycerol\n\nTYM Broth (250 mL)\n5 g Bacto Tryptone\n1.25 g Bacto Yeast Extract\n2.5 mL Magnesium chloride or magnesium sulfate (1M stock)\n1 g sodium chloride\n
Replenishing your stock of competent cells\n\n• Plate out your favorite strain on an LB plate (or take a few uL from frozen stock)\n• Grow O/N in 5 mL TYM (37 degrees, 300 rpm)\n• Inoculate 250 mL TYM\n• Shake until OD550 = 0.5 (don’t go over 0.55!!)\n• Divide into (6) 50 mL Falcons, spin 3000 rpm, 12 min, 4 degrees\n• Keep everything cold from here on\n• Resuspend cells with 15 mL TFB I per tube\n• Incubations: 20 min for BL21, 15 min for Top10, and 10 min for DH5alpha\n• Spin 3000 rpm, 8 min, 4 degrees\n• Resuspend cells with 2 mL TFB II per tube\n• Aliquot cells; flash freeze in liquid nitrogen (store –80 degrees)\n~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n\nRecipes\n\nTFB I\n30 mM potassium acetate\n50 mM manganese chloride\n100 mM potassium chloride\n10 mM calcium chloride\n15% glycerol\n\nTFB II\n10 mM Na-MOPS, pH 7.0\n75 mM calcium chloride\n10 mM potassium chloride\n15% glycerol\n\nTYM Broth (250 mL)\n5 g Bacto Tryptone\n1.25 g Bacto Yeast Extract\n2.5 mL Magnesium chloride or magnesium sulfate (1M stock)\n1 g sodium chloride\n
The complement system is a biochemical cascade of the immune system that helps clear pathogens from an organism. It is derived from many small plasma proteins that work together to form the primary end result of cytolysis by disrupting the target cell's plasma membrane. C3 cleaves C5 into [[C5a]] and C5b.
1. Shigella IpgB1 mimics the role of RhoG to hijack ~Rac1-Elmo1-Dock180 to promote ruffles and enhance invasiveness [[Sasakawa 2006|http://www.ncbi.nlm.nih.gov/pubmed/17173036?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n\n2. Here is the [[Chaos paper by Jim Ferrell|http://www.ncbi.nlm.nih.gov/pubmed/12779456?ordinalpos=34itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] that Sheel referred to during his group meeting on bistability in cell signaling. Another paper Jim likes to use as a reference is his TiBS review from 1996 on [[how to convert graded inputs into switch-like outputs|http://www.ncbi.nlm.nih.gov/pubmed/9009826?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]], which happens to have a cool title.\n\n3. Here are a couple of Q&As published in the Journal of Biology by Jim Ferrell, one about [[cooperativity|http://www.ncbi.nlm.nih.gov/pubmed/19589184?ordinalpos=1itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and the other on [[systems biology|http://www.ncbi.nlm.nih.gov/pubmed/19222866?ordinalpos=3itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. For cooperativity, he goes over the different models of cooperativity, and how cooperativity is useful in biological systems. A nice combination of easy to read text and differential equations. The systems biology one much more general.
Coronin genes that can be separated into three types: type I (coronin 1A, 1B and 1C), type II (coronin 2A and 2B), and type III (coronin 7, also known as POD) [[Bear 2006|http://www.ncbi.nlm.nih.gov/pubmed/16806932?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]\n\nCoronin is an actin-binding protein associated at the leading edge. Coronin 1B interacts simultaneously with [[Arp2/3]] complex and [[Slingshot]] (~SSH1L) phosphatase, two regulators of actin filament formation and turnover, respectively. Coronin 1B also directs ~SSH1L to lamellipodia where ~SSH1L likely regulates [[Cofilin]] activity via dephosphorylation. Coronin 1B inhibits filament nucleation by [[Arp2/3]] complex and this inhibition is attenuated by phosphorylation of Coronin 1B at Serine 2, a site targeted by ~SSH1L [[Bear 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17350576&query_hl=11&itool=pubmed_DocSum]]. Coronin-1 accumulates at the leading edge of migrating neutrophils and at the nascent phagosome [[Grinstein 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17442961&ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nUnlike type I coronins, coronin 2A localizes to stress fibers and some focal adhesions, and is excluded from the leading edge. Depletion of coronin 2A in MTLn3 cells decreases cell motility and turnover of focal adhesions. Surprisingly, none of the pathways known to regulate focal-adhesion turnover are affected by depletion of coronin 2A. Depletion of coronin 2A does, however, increase phospho-cofilin, suggesting that misregulation of cofilin might affect adhesion dynamics [[Bear 2009|http://www.ncbi.nlm.nih.gov/pubmed/19654210?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Cortactin interacts with the Arp3 subunit of the Arp2/3 complex and with f-actin, which suggests that cortactin may be an NPF [[Daly 2004 review|http://www.ncbi.nlm.nih.gov/pubmed/15186216?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments [[Condeelis 2009|http://www.ncbi.nlm.nih.gov/pubmed/19704022?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Cortactin and active VCA fragment of N-WASp synergistically enhance Arp2/3-induced actin filament branching [[Cooper 2001|http://www.ncbi.nlm.nih.gov/pubmed/11267876]].
An adaptor protein with SH2 and SH3 domains that binds receptor tyrosine kinases. [[SOS]], [[Abl]], and [[Eps15]] also bind the SH3 domain of Crk.
[[Cyfip1/2]] (Cytoplasmic FMRP Interacting Proteins) [[Mandel 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11438699&query_hl=56&itool=pubmed_docsum]]. There are two known CYFIP proteins in humans, CYFIP1 (also p140Sra) and CYFIP2 (also Pir121). See [[Sra1]] or [[Pir121]].
Cytochalasin B shortens actin filaments by blocking monomer addition at the fast-growing end of polymers.
Cytochalasin D-induces an increase in the initial rate of polymerization, but a decrease in it's final extent. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate [[Frieden 1986|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3023337&dopt=Abstract]].
Cell material without a nucleus. These can still chemotax [[Malawista 1986|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=3944258&query_hl=5&itool=pubmed_docsum]].\n\nDelivery ideas:\n\nRadiofrequency ablation of carbon nanotubes [[Smalley 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17960610&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] or of other metals (iron oxides) [[Kaiser 2000|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10719826&ordinalpos=19&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and [[Kaiser 2005|http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TJJ-4FMK6F3-H&_user=4430&_coverDate=05%2F31%2F2005&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059594&_version=1&_urlVersion=0&_userid=4430&md5=8efe74126be824257bc7d0f64d66d90e]].
Catalytic dbl homology ([[DH]]) domain.
Related to Zizimin-1. DOCK11 is GEF specific for Cdc42 [[Cerione 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16968698&query_hl=39&itool=pubmed_DocSum]].
Diaphanous-related formins
Drosophila disabled. Dab2 interacts with [[Grb2]], which can interrupt it's binding with [[SOS]] [[Hsieh 2001|http://www.ncbi.nlm.nih.gov/pubmed/11371563]]. The C-terminal of Dab2, which contains the [[Grb2]] binding site, can bind [[MyosinVI]] with 1:1 stochiometry [[Ikebe 2002|http://www.ncbi.nlm.nih.gov/pubmed/11371563]]. The central region of Dab2 binds to the clathrin adaptor protein AP-2.
Constructed a photoactivatable actin [[Fletcher 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15826098&query_hl=43&itool=pubmed_docsum]].\n\nA review on cell motility for physicists [[Fletcher 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16204816&query_hl=43&itool=pubmed_docsum]].\n\nShowed that the growth velocity of a branched actin network against increasing forces is load-independent, but when force was decreased on a growing network, the velocity increased to a value greater than the previous velocity [[Fletcher 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16299496&query_hl=43&itool=pubmed_docsum]].\n\nShowed that membrane-bound actin networks alone can control when and where membrane domains form [[Fletcher 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16963509&query_hl=43&itool=pubmed_docsum]].\n\nShowed that using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), stress stiffening occurs followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network [[Fletcher 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17230186&query_hl=43&itool=pubmed_docsum]].\n\nShowed that keratocyte shape correlated with Ena/Vasp levels [[Fletcher 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17760506&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Lew showed that the scaffold protein Bem1 was required for symmetry breaking in yeast bud formation and that polarization was dependent on GTP hydrolysis by Cdc42, suggesting that assembly of a polarization site involves cycling of Cdc42 between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch [[Lew 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14625559&query_hl=71&itool=pubmed_docsum]].
Showed that phosphorylation of the [[MLC]] plays a role in motility and polarity during Dicty chemotaxis [[Soll 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11950890&query_hl=26&itool=pubmed_DocSum]].\n\nShowed that acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP [[Soll 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15821137&query_hl=26&itool=pubmed_DocSum]].
Showed that photo-activatable Rac could control collective cell migration in Drosophila border cell migration [[Montell 2010|http://www.ncbi.nlm.nih.gov/pubmed/20473296]].
Denoising protocol\n12/15/08 ARH \n\nBeginning with a multichannel timelapse movie. Isolate your channel of interest (in this example, YFP channel for Hem-1). \n\nPull your channel out onto the background and it makes its own single channel movie. \n\nExport your single channel movie as a series of tifs in a unique folder. \n\nFor example, you have a 888 frame movie: myfilename_yfpt001dc1.tif, myfilename_yfpt002dc1.tif, 003dc1… 888dc1.tif etc. within folder /myfolder/\n\nPut this folder (myfolder) onto saki – Try from "Connect to server"


 at the prompt, type in user name and password as usual; for
 "WORKGROUP" name, use MSG.\n\nUsername = weinerlab\nPwd = ucsf1234\n\nssh weinerlab@saki ucsf1234\n\nonce you are on saki, change directory into the directory with all of your tiffs. Make a “denoised” directory within myfolder/ on saki.\n\nUseful linux commands:\n\nType "ls" and then press enter - this lists the folders in the weinerlab saki folder.\nType "cd ", then the first letter of the directory, and then tab to display the folder name. Press enter to change directory to that folder.\nType "cd ../" and then press enter to move up a directory.\nUp and down arrows will move through a history of your commands. When denoising more than one file, these can help navigate back to the ndsafir command line, so you do not have to retype everything.\n\nTo run the program:\n\nndsafir –i myfilename_yfpt%03dc1.tif –o myfolder/myfilename_denoised_yfpt%03dc1.tif –first 1 –last 888 -2dt true –p 3\n\n-i specifies the input files\n-o specifies where the output will go. The output will be another series of tiffs\n-first specifies the number of the first frame\n-last specifies the number of the last frame\n-2dt true specifies that you have a series of 2d frames and they are a series of different time points (I think, not 100%)\n-p 3 specifies patch size, a parameter used by the denoising algorithm\n\nThe %03d part of the filename refers to the number of digits in your filename suffix. In this case their were 888 frames and thus three digits in the suffix (t001dc1, t002dc1 etc.). If you have 99 frames or less you may need to have:\n\n-i myfilename_yfpt%02dc1.tif and the same for the output file\n\n\n
Dicty Summary 4/5/09\n\nDictyostelium discoidum – aka Dicty – are also known by the following street names: “Slime”, “Icky Dicty” and “D-mold”. As I understand things, Dicty spend most of their lives as unicellular, haploid organisms in the soil. They eat bacteria or smaller eukaryotes. When food supplies run low, all of the Dicty in a given region send out a chemical signal (cAMP) to one another. As the Dicty chemotax towards cAMP, they also release cAMP which relays the signal to the Dicty behind them. As a result, they often march towards the source in single-file columns, known as streams. Eventually, all of the Dicty in the region to converge and form a multicellular mushroom like structure known as a fruiting body. Some of the Dicty within the fruiting body become spores. These spores then get dispersed, some of them end up in a region where there is more food and they can then proliferate as unicellular organisms again.\n\nI will roughly divide the rest of this up into a few parts:\n\nPhenomenological studies of how they respond to the gradient\n\nEarly on people studied Dicty chemotaxis (and neutrophils for that manner) in steep gradients. These early studies showed that a quiescent cell responds to a strong gradient by making its first pseudopod up the gradient. In shallow gradients, however, the cells make pseudopodia relatively independently of the gradient and retain the ones that are pointed up-gradient. In this case, there may be directional bias of new pseudopod generation to the up-gradient side of the cell, but this bias is small. It is also clear that new pseudopodia generally form as bifurcations of existing pseudopodia as a result of cellular polarity (only certain regions of the polarized cell are capable of forming pseudopodia). These shallow gradient experiments were quite recent and it is starting to appear that cellular polarity functions to increase the persistent migration of cells up the gradient once the cells get aligned. It is thought that in the absence of polarity, random receptor fluctuations could initiate a lot of misaligned pseudopodia in shallow gradients.\n\nThe manner in which the polarity circuit responds to the gradient has been under debate. The primary issue under contention is whether the cell polarizes along the gradient or whether the cell polarizes in a random direction and then steers this polarity in the direction of the gradient later on as it migrates. In reality, the steepness of the gradient determines the cellular behavior. In steep gradients, the polarization is directed by the gradient. In shallow gradients, the polarization and pseudopod generation process is relatively independent of the gradient; and the polarized cell is ‘steered’ in the right direction by retention of the correct pseudopodia. There is disagreement on the degree to which pseudopod generation is biased by the gradient. Peter van Haastert thinks that pseudopod generation is biased by the gradient while Insall thinks that pseudopod generation is not biased by the gradient. Recent genetic and pharmacological experiments are showing which signals regulate pseudopod generation, gradient sensing and polarity/directional persistence. I’ll get more into that later on. . .\n\n\n\n\nOverview of the signaling cascade\n\nChemoattractant (cAMP) is sensed by the GPCRs cAR1 and cAR2 (I’m not sure if there other receptors, besides cAR1 and 2 but I’m pretty sure that there are. The relevant heterotrimer in Dicty is composed of G-alpha-2 and G-gamma-?, as well as G-beta (there is only one type of beta subunit in Dictyostelium). Somehow, heterotrimer activation causes RasC and RasG to become active specifically at the leading edge. The GEF Aimless is thought to be involved specifically in RasC activation. RasC then activates TorC2 while RasG activates PI3K. PIP3 is confined to the front in Dicty by the phosphatase PTEN, which localizes to the sides/rear in Dicty. Somehow, PIP3 promotes actin assembly at the front, but it is not clear how. PIP3 activates Protein Kinase B (PKBA) in Dicty but deletion of this kinase has only a minor effect on chemotaxis, so there are likely to be other PIP3 effectors besides PKBA.\n\nIt is now known that several parallel pathways can mediate chemotaxis in Dicty. If PIP3 is eliminated, Dicty can still chemotax but their chemotaxis now requires phospholipase A2 (PLA2) – if PI3K signaling is intact, then chemotaxis does not require PLA2. If Dicty are allowed to differentiate for long periods of time, both PI3K and PLA2 become dispensible and chemotaxis can occur through soluble guanylyl cyclase (sGC). Surprisingly, the protein sGC and its catalytic product cGMP, appear to have distinct jobs in the front and the back respectively. Perhaps sGC has some type of scaffolding function in the front while cGMP activates myosin in the back. \n\nThe TorC2 complex has recently been shown to be important for chemotaxis in Dicty, deletion of Pianissimo (aka Rictor, a member of the complex) causes a significant speed and directionality defect. TorC2 appears to be active at the leading edge of chemotaxing Dicty. In mammals, PIP3 and Tor converge to activate PKB/Akt. In Dicty, there are two PKB isoforms (PKBA and PKBR1). PKBA is co-dependent on PIP3 and TorC2, while PKBR1 is PIP3-independent and TorC2-dependent. These PKBs then phosphorylate substrates, which promote actin assembly at the front. Phosphorylation of the currently identified substrates appears to be mediated by PKBR1. Substrates include talin, a couple of Ras GEFs, a Rho GEF and PI4P-5-kinase. These substrates are phosphorylated in response to chemoattractant. This phosphorylation requires PKB and TorC2 but does not require PIP3 (indicating that PKBR1 can phosphorylate all of the known substrates by itself). However, PKBA deletion produces a minor chemotaxis defect so there must be other relevant substrates of PKB whose identities are unknown. \n\nPeter van Haastert’s lab has studied the relative role of each of these signals in pseudopod generation and orientation of pseudopodia with respect to the gradient. They found that PLA2 is required for the generation of pseudopodia by splitting and suppression of lateral pseudopod generation. Also, PLA2 was unimportant for the gradient-biased pseudopod generation. They found cGMP to be important for suppression of lateral pseudopodia (but not for pseudopod splitting or gradient-biased pseudopod generation. Meanwhile, sGC itself is important for pseudopod formation to be biased towards the gradient (but not for splitting or suppression of lateral pseudopodia. In this study, the elimination of PIP3 does not alter either the rate of splitting or lateral pseudopod formation, but it does affect gradient-biased pseudopod generation. This last result clashes with Insall’s observation that LY294002 treatment affects the splitting frequency but not chemotactic index. These differences could arise in many places: longer differentiation times, steep vs. shallow gradients, off-target effects of LY (esp. Tor), different analysis techniques (Insall’s pseudopod detection technique is probably better).\n\nFuture directions for research:\n\nOne thing that is not well understood is what conveys spatial information in Dicty. By “spatial information” I mean the ability of a molecule to induce pseudopod production/uropod production/ tail retraction etc. in the region where the molecule is active. PIP3 can convey spatial information, because when PTEN is eliminated the PIP3 distribution expands throughout the cell and additional pseudopodia are produced. PIP3 is clearly not the only molecule that does this in these cells, because when it is eliminated, the cells can still chemotax. This chemotaxis is PLA2 dependent but PLA2 activity is probably not conveying spatial information because uniform arachidonic acid (the catalytic product of PLA2) can rescue PLA2-deficiency. Thus, there appear to be other molecules besides PIP3 that can convey spatial information but these molecules require PLA2 as a permissive signal. To my knowledge, no molecule besides PIP3 has been shown to be sufficient to produce pseudopodia in Dicty. It is possible that experiments have shown Ras to be capable of pseudopod induction in Dicty, but I don’t know of them. I know that no sufficiency experiments have been published for the likes of PLA2, TorC2 or sGC.\n\nThere are several other open questions in Dicty. For example, it is not known what GEFs are required for Ras activation – one of them is probably Aimless (Aim) – but that only appears to activate RasC. I don’t think any of the relevant GEFs for RasG are known. It is also not known which proteins induce actin assembly at the leading edge. The SCAR/WAVE complex is not strictly required for actin assembly, pseudopod production and chemotaxis in Dicty – although elimination of Scar does reduce actin assembly and causes multiple pseudopodia to form. The mechanism by which the cell adapts to persistent stimulation is not known and neither is the related issue of how the cell senses shallow gradients. The polarity mechanism in Dicty is not understood either. Obviously, lots of interesting questions remain to be answered.\n\n[img[http://img172.imageshack.us/img172/3185/slide1p.png]]\n\n\n
The diffusion coefficient of PIP2 in the plasma membrane is ~1 um2/s by FCS [[McLaughlin 2008|http://www.ncbi.nlm.nih.gov/pubmed/18256277?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n\nThe diffusion coefficient of HRas is 0.5 um2/s (human embryonic kidney, 22 degrees) or 1.13 um2/s (3T3, 37 degrees) by single-molecule measurements [[Schmidt 2004|http://www.ncbi.nlm.nih.gov/pubmed/14695305]]. RasN17 is 1 um2/s and RasV12 is 0.85 um2/s (3T3, 37deg, single molecule) [[Schmidt 2004|http://www.ncbi.nlm.nih.gov/pubmed/15860728]].\n\nThe diffusion coefficient of cAR is 0.17 um2/s (photobleached, widefield, single molecule Dicty) [[Schmidt 2008|http://www.ncbi.nlm.nih.gov/pubmed/18469015]] and 0.14 um2/s for Crac-GFP (TIRF, Dicty) [[Ueda 2006|http://www.ncbi.nlm.nih.gov/pubmed/16507590]].\n\nThe diffusion coefficient of dye measured by FCS ranges from 0.1 - 1 um2/s (membrane) and 4 - 20 um2/s (cytosol) depending on cell type [[Schwille 1999|http://www.ncbi.nlm.nih.gov/pubmed/10512844]].\n\nThe diffusion coefficient of bAR2 measured by FCS is 0.01 - 0.1 um2/s (confined) to 3-5 um2/s (free diffusing) [[Haberlein 2004|http://www.ncbi.nlm.nih.gov/pubmed/15147203]].\n\nThe diffusion coefficient of phytochrome in moss by FCS is 10 um2/s (cytosol) and 2 um2/s (membrane) [[Lamparter 2004|http://www.ncbi.nlm.nih.gov/pubmed/15345577]].\n\nThe diffusion coefficient of EGFP and PKCbeta1 in the cytosol by FCS is ~25 um2/s [[Kinjo 2003|http://www.ncbi.nlm.nih.gov/pubmed/12706832]].\n\nThe diffusion coefficient of DOPE (outer leaf of PM) by single molecule is 5 um2/s over a 100 us time window, 1.6 um2/s over 20ms time window, and 0.4 um2/s over a 100ms or 3 s time window [[Kusumi 2002|http://www.ncbi.nlm.nih.gov/pubmed/12058021]].\n\nThe diffusion coefficient of inactive Ras is 0.33 um2/s with and without lat treatment [[Kusumi 2004|http://www.ncbi.nlm.nih.gov/pubmed/15123831]]. From Kusumi's earlier paper, you would expect the diffusion coefficient to increase when treated with latrunculin, but they go on to say in their supplemental methods that this might not be the case for a variety of reasons. These reasons include a prolonged imaging time, concentration of latrunculin used, and actin aggregates causes by latrunculin treatment.\n\nThe diffusion coefficient of GPCR muOR is ~.2 um2/s or 4.2 um2/s at small time scales (75 us) [[Kusumi 2005|http://www.ncbi.nlm.nih.gov/pubmed/15681644]].\n\nThe diffusion coefficient of Lyn-GFP and Ras-GFP was 0.3 um2/s over a 100 ms time interval [[Kusumi 2010|http://www.ncbi.nlm.nih.gov/pubmed/20881966]].\n\nThe diffusion coefficient of Fab-gold is 0.22 um2/s by single molecule tracking [[Kusumi 2007|http://www.ncbi.nlm.nih.gov/pubmed/17517964]].\n\nThe diffusion coefficients of MHC, GPI, and DOPE were ~0.2 um2/s [[Kusumi 2008|http://www.ncbi.nlm.nih.gov/pubmed/18339737]].\n\nThe diffusion coefficient of PE by FCS-STED is 0.5 um2/s [[Hell 2009|http://www.ncbi.nlm.nih.gov/pubmed/19098897]].\n\nThe diffusion coefficient of PC by FCS is 1.7 um2/s and GPI is 0.9 um2/s [[Lenne 2007|http://www.ncbi.nlm.nih.gov/pubmed/17085499]].\n\nThe diffusion coefficients of a transmembrane protein and GPI are ~ 0.3 um2/s by FCS [[Lenne 2006|http://www.ncbi.nlm.nih.gov/pubmed/16858413]].\n\nThe diffusion coefficient of K+ channel is 0.14 um2/s and free QD is 2.2 um2/s [[Schwab 2008|http://www.ncbi.nlm.nih.gov/pubmed/18287336]].\n\nThe diffusion coefficient of CD59 is 0.3 um2/s at 20C and 0.45 um2/s at 37C by single molecule tracking [[Schutz 2008|http://www.ncbi.nlm.nih.gov/pubmed/17325009]].\n\nThe diffusion coefficients of all known lipid and transmembrane proteins before 1997 were between 1-20 um2/s [[Jacobson review 1997|http://www.ncbi.nlm.nih.gov/pubmed/9241424]].\n\nThe diffusion coefficient of PE and PC is ~1 um2/s [[Schutz 2000|http://www.ncbi.nlm.nih.gov/pubmed/10698931]].\n\nThe diffusion coefficient of calcium channel is 0.15 um2/s [[Schmidt 2001|http://www.ncbi.nlm.nih.gov/pubmed/11606277]].\n\nThe diffusion coefficients of T cell components ranged from 0.3-0.7 um2/s [[Vale 2005|http://www.ncbi.nlm.nih.gov/pubmed/15960980]].
Directional sensing refers to the ability of a cell to detect and asymmetric extracellular cue and generate an internal amplified response [[Janetopoulos 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12672811&dopt=Abstract]]. For instance, the amplification and asymmetric localization of [[PI(3,4,5)P3]] occurs even in the absence of a polarized cell structure in neutrophils [[Bourne 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10669415&query_hl=50&itool=pubmed_docsum]] and [[CRAC]] localization in Dictyostelium [[Devreotes 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9778249&query_hl=52&itool=pubmed_DocSum]].
11 human Dock180 homologues are termed Dock1 (=[[Dock180]])-Dock11 and the four subfamilies are denoted ~Dock-A, -B, -C and -D [[Vuori 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12432077&query_hl=15&itool=pubmed_docsum]]. [[Dock2]] is expressed selectively in hematopoietic cells including lymphocytes, dendritic cells and possibly others. By contrast, [[Dock180]] is absent from leukocytes. Dock3 (also known as Moca) is expressed predominantly in neurons and resides in growth cones and membrane ruffles. Expression of Dock4 also leads to activation of the small GTPase Rap1 and enhanced formation of adherens junctions [[Guda 2005 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16254241&query_hl=4&itool=pubmed_DocSum]].
Dock180 protein has an indispensable role in cell migration by functioning as an exchange factor for Rac GTPase via its Dock homology region (DHR)-2 domain. A form of Dock180 that lacks DHR-1 fails to promote cell migration, although it is capable of inducing Rac GTP-loading. The DHR-1 domain interacts with PtdIns(3,4,5)P(3) in vitro and in vivo, and mediates the DOCK180 signalling complex localization at sites of PtdIns(3,4,5)P(3) accumulation in the cell's leading edge [[Vuori 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16025104&query_hl=4&itool=pubmed_DocSum]]. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. The Docker domain of Dock180 specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro [[Ravichandran 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12134158&query_hl=8&itool=pubmed_docsum]]. Dock180 is ubiquitylated on the plasma membrane, and also that Elmo1 functions as an inhibitor of ubiquitylation of Dock180. Therefore, an ubiquitin-proteasome-dependent protein degradation mechanism might contribute to the local activation of Rac on the plasma membrane [[Tanaka 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16495483&query_hl=4&itool=pubmed_DocSum]].
Dock2 (expressed only in leukocytes) is a mammalian homologue of Caenorhabditis elegans ~CED-5 and Drosophila melanogaster Myoblast City, and regulates motility and polarity during neutrophil chemotaxis. Dock2 also associates with [[Elmo]] to induce [[Rac]] activation [[Fukui 2003|http://www.ncbi.nlm.nih.gov/pubmed/12829596?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and may also associate with CrkL [[Tanaka 2002|http://www.ncbi.nlm.nih.gov/pubmed/12393632?ordinalpos=25&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Dock2 and [[Elmo]] colocalize at the leading edge in HL-60 cells [[Richmond 2008|http://www.ncbi.nlm.nih.gov/pubmed/18662984?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Dock2-deficient neutrophils move toward a chemoattractant source, but exhibit abnormal migratory behavior with a marked reduction in translocation speed. In Dock2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 is severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge [[Fukui 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16943182&query_hl=15&itool=pubmed_docsum]].
[[ABA]]\n[[Aluminum Fluoride]]\n[[Blebbistatin]]\n[[C. Difficile Toxin]]\n[[C5a]]\n[[Colchicine]]\n[[Cytochalasin B]]\n[[Cytochalasin D]]\n[[fMLP]]\n[[Forskolin]]\n[[Gallein]]\n[[Kinase Inhibitors]]\n[[Jasplakinolide]]\n[[Latrunculin]]\n[[LY294002]]\n[[Methyl-ß-cyclodextrin]]\n[[Nocodazole]]\n[[Pertussis Toxin]]\n[[PMA]] \n[[PIK-90]]\n[[PP242]]\n[[Rapamycin]]\n[[Taxol]]\n[[Wiskostatin]]\n[[Wortmannin]]\n[[Y-27632]]
Showed that by analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a Kd of 7 µM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers [[Mullins 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9529382&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that full-length, recombinant human [[Scar]] protein, as well as N-terminally truncated [[Scar]] proteins, enhance nucleation by the [[Arp2/3]] complex [[Mullins 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10097107&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that activated Cdc42 or GTPγS stimulated barbed-end polymerization, whereas immunodepletion of Arp2 or sequestration of Arp2 using solution-binding antibodies blocked Rho-family GTPase-induced actin polymerization [[Mullins 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10226024&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that regulation of N-WASP requires N-WASP's constitutively active output domain [[VCA]] and two regulatory domains: a Cdc42-binding domain (CRIB) and a previously undescribed PIP2-binding domain [[Mullins 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11052943&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that [[Arp2/3]] complex bound to ADP or the nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments indicated that [[Arp2/3]] requires ATP cycling for nucleation [[Mullins 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11752435&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that [[WASP]]-family [[VCA]] domains activate the [[Arp2/3]] complex by driving its interaction with a single conventional actin monomer to form an Arp2-Arp3-actin nucleus [[Mullins 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15094799&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that Drosophila protein Spire represents a third class of actin nucleation factor. Showed that in vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex. Showed that Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer [[Mullins 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15674283&query_hl=102&itool=pubmed_DocSum]].\n\nShowed by NMR that the N-terminal half of the C domain binds monomeric actin and that both the V and C domains can bind actin independently and simultaneously [[Mullins 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16403731&query_hl=102&itool=pubmed_DocSum]].\n\nShowed that depletion of capping protein by RNAi results in the displacement of the Arp2/3 complex and disappearance of the lamellipod, but depletion of cofilin, slingshot, twinfilin, and tropomyosin, all factors that control the stability of actin filaments, dramatically expanded the lamellipod at the expense of the lamellum [[Mullins 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17331727&query_hl=102&itool=pubmed_DocSum]].
Dynamin is a GTPase thought to be responsible for endocytosis in the eukaryotic cell. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These "waves" promote an active reorganization of actin filaments in the anterior cytoplasm. IP with Dyn2 in PDGF stimulated fibroblasts pulls-down [[Arp2/3]], [[N-WASP]], and [[Rac1]], but not RhoA or [[Cdc42]] [[McNiven 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12631725&query_hl=5&itool=pubmed_docsum]].
Another name for [[Abi1]].
Protocol for charging GST-Rac1 (e. coli) with 32P hot GTP\n\nI used three conditions:\n\n1. BufferA + GTP* - Rac-GST\n2. BufferA + GTP* + 5uL Rac-GST ( 5ug Rac-GST)\n3. BufferA + GTP* + 1uL Rac-GST (1ug Rac-GST)\n\n5uCi of GTP* was used per reaction or 0.87uL GTP* per reaction\n\nBufferA = cavitation buffer w/1mM MgCl2, 10% glycerol, 1mM DTT, PI tablet, No PMSF\n\nStopping Buffer = cavitation buffer w/12mM MgCl2, 10% glycerol, 1mM DTT, PI tablet, no PMSF\n\nWashing Buffer = cavitation buffer w/3.5mM MgCl2, 10% glycerol, 1mM DTT, PI tablet, no PMSF + 1mg/mL albumin\n\nWash beads 3x in washing buffer – spin 2min @ 3000rpm, 40uL of 50% bead slurry per tube (5 tubes), spun down each tube to make sure that equal amts of beads present in each tube (20uL packed volume per tube)\n\nI had three master reactions, one for each condition. Conditions 1 and 2 were subjected to two different washing protocols (3 washes vs. 5 washes) so I had twice as much reaction for those two conditions.\n\nI added GTP* to BufferA first (enough for 6x50uL reactions), 5.22uL of GTP* in hood\n\nI added EDTA to 10mM final (10mM when BufferA/GTP* is added) to tubes containing Rac-GST and nothing else (2uL to conditions 1 and 2, 1uL to condition 3) – on ice.\n\nIn hood, Added 90uL buffer to conditions 1 and 2, 49uL buffer to condition 3, kept on ice until buffer had been added to each tube then placed each tube in 37C heat block (with water).\n\nIncubate 15 minutes at 37C\n\nStop reactions by adding an equal volume of stopping buffer (102uL to conditions 1 and 2, 51uL to condition 3)\n\nIncubate 5 minutes at 37C\n\nAdd 90uL of reaction to appropriate tube of beads (cold beads) in ice bucket.\nIncubate on ice 68 minutes (tapped once at 58 minutes). Washed two tubes (1x No Rac, 1x5uL Rac) five times. Washed all other tubes three times.\n\nAfter last wash remove all liquid – BE CAREFUL NOT TO SUCK UP BEADS!!!\n\nPump scint. Fluid into vials.\nAdd 0.5mL scint fluid from vial into each bead tube\nPut all of the beads/fluid into the scint. Tubes.\nResults\n+ 5uL Rac (3wash = 5wash) = 1E6 cpm\n- Rac 3wash = 3000 cpm\n- Rac 5wash = 1000 cpm\n+1uL Rac = 2E5 cpm\n\n
Chelates calcium.
Actin network in EM is an artifact of critical point drying [[Small 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12096898&query_hl=2&itool=pubmed_docsum]].
See [[VASP]].
See EZ-Taxis
See EZ-Taxis
Materials:\n1. RPMI culture media.\n2. Chemoattractant solution: 200 nM fMLP in RMPI culture media.\n \n\nMethod:\n1. Place “40 Glass” (41 Glass is used in conjunction with a #1.5 coverslip) in holder base. Fill with 3 ml of culture media, place small “o” ring beneath wafer housing, and add wafer housing into holder base. Place large “o” ring on top of wafer housing. Gently close inner lever.\n2. Place EZ-TAXIScan chip gently into wafer housing with forceps. Chip should fit snuggly on top of glass and between wafer holder (see Note 18). Place rubber gasket (to protect chip) beneath the wafer clamp and place wafer clamp on wafer housing. Gently close outer lever.\n3. Place assembled device on top of preheated EZ-TAXIS microscope (Fig. 5, A and B).\n4. Using syringe guide, add 1 µl of cells to lower chamber using microsyringe (MS-E10MIC, Exmire). Use a plastic pipette to draw cells into imaging surface.\n5. Add 1 µl of 100 nM – 1 µM chemoattractant to upper chamber. Begin image acquisition immediately (Fig. 5C).\n\n[img[EZtaxis|http://img508.imageshack.us/img508/5446/figure5rgbsm1.gif]]\n\n''Fig. 5.'' The components of the EZ-TAXIS system are shown in (a) with the individual components in their order of assembly from top to bottom shown in (b). (C) An example of HL-60 cells migrating toward chemoattractant in the EZ-TAXIS assay visualized with brightfield microscopy.\n\n\nEZ-TAXIS Analysis\n\n1. Go to Sheel's computer and open up MatLab.\n2. Find the directory on OWLabData and put it into the MatLab directory.\n3. Open up the CellTracker.m file in MatLab.\n4. Press "run" (the second button from the left next to "Stack".\n5. Answer the series of questions from the prompts to start the analyses. Choose "all files" to choose .jpg files for MatLab analysis.\n\n\nAddendum February 20, 2009 by Arthur Millius\n\nThere is no need to sonicate all the parts of the EZ Taxis. You only need to vortex the chip for 30 seconds to get the device completely clean.\n\n\n\n
Electroporation of HL60 cells\nArthur Millius 1/17/07\n\n''Overview''\n\nTo transiently transfect HL60 cells with your gene of interest.\n\n''Buffers''\n\nRPMI complete media (10% serum, antibiotics and antimycotics)\nRPMI serum and antibiotic free\n\n''Procedure''\n\nFive (or six) days prior to transfection, split and differentiate 50 ml of HL60 cells per transfection to a density of 0.2 x 106 cells/ml. At the time of transfection, you want 1.2 x 106 cells/ml – 2.0 x 106 cells/ml.\n\nCell were at a density of 2 x 106 cells/ml, so we decided to do three transfections.\n\nAliquot cells into (2) 50 ml tubes.\n\nSpin cells for 10 min at 400 x g.\n\nResuspend in 25 ml serum free RPMI and combine into (1) 50 ml tube.\n\nSpin cells for 8 min at 500 x g.\n\nResuspend in ~ 3 ml of serum free RPMI.\n\nTransfer 800 µl of cell suspension (80 – 100 x 106 cells total) in CellPorator cuvette (Life Technologies Cat. No. 11601-028.\n\nAdd 50 µg of circular plasmid DNA. //Note: You would want to linearize the DNA if you were going to make stable cell lines. You could do this by cutting with an enzyme, running a gel, and purifying the gel fragment.//\n\nIncubate with cells 10 min at room temperature.\n\nElectroporate on ice 310 V at 1180 µF (low resistance) on fast. Note: Only electroporate one HL60 line at a time to minimize time cells spend on ice. Use charge mode to bring voltage above 310 V (~330 V), switch to arm, wait for voltage to fall to 310 V, and then shock.\n\nPut back at room temperature for 10 minutes.\n\nTransfer cells into 20 ml of warmed fresh culture medium. Image 4 – 24 hours later.
Elmo is a critical regulator of [[Dock180]], and the ~Dock180-ELMO complex functions as a bipartite GEF for [[Rac]]. The [[PH]] domain of [[Elmo]], by binding the [[Dock180]]-[[Rac]] complex in trans, stabilizes [[Rac]] in the nucleotide-free transition state [[Ravichandran 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=15247908]]. Mammals have three [[Elmo]] proteins (Elmo1, Elmo2, Elmo3), of which two have so far been shown to interact with [[Dock180]] proteins. By binding to active RhoG, which resides at the cell membrane, [[Elmo]] can target [[Dock180]] to the cell membrane, leading to [[Rac]] activation. Alternatively, the [[Elmo]] [[PH]] domain can bind to and stabilize the nf-Rac-CZH2 complex [[Guda 2005 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16254241&query_hl=4&itool=pubmed_DocSum]]. Co-expression of Elmo and [[Dock180]] induces membrane ruffling and formation of lamellipodia with localization of both proteins to these sites, but not if the [[ARM]] repeats of [[Elmo]] are mutated to prevent interaction with [[RhoG]] [[Ravichandran 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15620647&query_hl=29&itool=pubmed_docsum]].
The sole member of the Ena/[[VASP]] family in Drosophila, Enabled (Ena), was identified through its genetic interactions with the [[Abl]] tyrosine kinase and later found to function in several signaling pathways essential for axon guidance in the developing nervous system. [[Gertler 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12432060&query_hl=16&itool=pubmed_docsum]]
Enzymatic assembly of DNA molecules (by delquin)\n\nOriginally described by Daniel Gibson, whose paper is published in Nature Methods [[Gibson 2009|http://www.ncbi.nlm.nih.gov/pubmed/19363495?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and detailed procedures are published in Nature Protocols [[Gibson Protocol|http://www.natureprotocols.com/2009/04/16/onestep_enzymatic_assembly_of.php]]. This tiddler summarizes the technique and provides basic knowledge to get your DNA assembly to work. Please see above citations for complete information.\n\nBackground:\nThis technique was developed by the Synthetic Biology Group at the J. Craig Venter Institute in order to assemble multiple, overlapping DNA molecules to generate products on the order of several hundred kilobases. The DNA fragments are joined together by the combined activities of a 5’ exonuclease, a DNA polymerase, and a DNA ligase. The exonuclease generates single stranded overhangs on the DNA molecules, allowing the overlapping regions to anneal by base pairing. The polymerase fills in any gaps, and the ligase repairs the nick and produces a circularized product. This reaction has been streamlined in which the exonuclease, polymerase, ligase, and all necessary cofactors for enzymatic activity are combined as a single mixture that can be aliquoted (typically in 15 uL volumes), frozen and stored in -20 degrees for easy access. Here, I will refer to this mixture as the Assembly Mix.\n\nHow is this useful for us?\nFor the purpose of the typical graduate student/postdoc in the research lab, we can harness the power of this molecular engineering tool to clone in your favorite gene into your desired destination vector in a few easy steps (see below for detailed protocol). As long as you start off with purified starting material, this method is highly robust and will almost certainly yield your desired product.\n\nAssembly mix.\n5X isothermal………………………320 uL\n10 U/uL T5 exonuclease…….…..0.64 uL (Epicentre, #T5E4111K, 1000U@10U/uL→ 100 uL) \n2 U/uL Phusion DNA poly…….......20 uL (NEB, #F-530L, 500U@2U/uL → 250 uL)\n40 U/uL Taq DNA ligase…….......160 uL (NEB, #M0208L, 10,000U@40,000U/mL→ 250 uL)\nwater………………………………..700 uL\ntotal 1200 uL\n\naliquot into 15 uL per 0.2 mL PCR tube. store in -20 degrees. \ncan undergo multiple freeze/thaw cycles.\n\n5X isothermal reaction buffer. \n1 M Tris-HCl pH 7.5……….......3 mL \n2M MgCl2……………………..150 uL \n100 mM dGTP………………....60 uL \n100 mM dATP………………....60 uL \n100 mM dTTP………………....60 uL \n100 mM dCTP………………....60 uL \n1 M DTT……………………….300 uL\nPEG-8000……………………...1.5 grams \n100 mM NAD………………....300 uL \ntotal 6 mL \n\nthis stuff does not seem to freeze/thaw very well. we now make fresh stuff every time.\n\n\nPCR the insert from template.\nYour primers will have specificity for your insert on one end, and your vector on the other. The only requirement is that you must have unique restriction sites available to linearize your vector. Design your primers with these points in mind:\n\n~ At least 25 bases with complementarity to the vector.\n~ Design your primers the way you want the end product to look; avoid any non-homologous junk between the vector and the insert (unless you want to introduce a new restriction site).\n~ If you keep your primers < 50 bases, you qualify for the smallest scale of production with Elim BioPharmaceuticals, or < 60 for IDT (and thus, cheapest and fastest).\n\n1. Amplify your insert by PCR. \n Meanwhile, linearize your destination vector, ideally without any bases on the ends that are non-complementary to the primer (because you don't want to introduce any useless junk between the insert and primer).\n * some have noticed that dephosphorylating your vector increases efficiency.\n\n2. Gel purify both fragments and elute in 30 uL of 70 degree water. Nanodrop spec.\n\n\nEnzymatic assembly\n• Use 1:1 insert to vector ratio (or 3:1 should work too)\n• Insert, vector, and water should add up to 5 uL (use Clontech’s molar ratio calc to optimize)\n• Take one aliquot (15 uL) of Assembly mix from the freezer.\n• Add the 5 uL DNA to the Assembly mix → incubate at 50 degrees for 1 h. (1 h yields maximum product)\n• Transform 1 (or 2) uL into homegrown Top10 competent cells. (you can bring vol up to 50 and transform 2.5, but that is the same as 1/20 here)\n• Should yield plenty of colonies!\n\nOptional: run one microliter of assembly mix on a gel alongside the input fragments. A successful assembly will reveal higher molecular weight products. This is always a neat thing to see.\n\n\n\n\n\n
Epithelial polarity\n\nOrganization of epithelial layer\n\nEpithelia can either be single layered (simple) or multilayered (stratified). All of the mammalian epithelial work that I describe is on 3D cultured cysts, which are hollow single-layered spheres of cells (models for simple epithelia). People study epithelial polarity in drosophila and c elegans, too. I assume that those are simple epithelia as well. Drosophila/elegans are the best studied systems and a lot of the stuff that I will describe was discovered in those systems and may not carry over into mammals.\n\nThe simple epithelium is a single layer of cells contacting an open lumen on one side and the basement membrane (ECM) on the other side. The luminal side of the cell is known as the apical membrane. The side contacting the basement membrane is the basal membrane. The lateral membranes are cell-cell contact zones where adhesions form.\n\nNeighboring cells adhere to one another with two types of adhesions: tight junctions and adherens junctions. Tight junctions are responsible for the barrier function of the epithelium (they keep things contained within the lumen) and they also restrict diffusion of molecules between the apical and lateral membranes. Adherens junctions are important for mechanical strength as they link the actin cytoskeletons of neighboring cells. I’m not sure about the contribution of tight-junctions to mechanical strength. There is a dense actin network under the apical membrane and behind the tight junctions. The network is less dense under the adherens junctions and basal membrane.\n\nOnce an epithelium has formed, it has a stereotyped apical-basal ordering of its adhesions. The apical surface of the cell has no adhesions (there is nothing to adhere to). In mammals, the apical-most region of the lateral surface has tight junctions. The adherens junctions are located basally to the tight junctions. The basal surface has integrin-mediated adhesions to the basement membrane. In flies, there are “septate junctions” instead of tight junctions and the ordering of septate junctions and adherens junctions is flipped.\n\nThe usual suspects: Par proteins, Cdc42 and phosphoinositides\n\nEpithelial polarity involves many of the same proteins that we find in c. elegans zygotes and elsewhere. Par6 and aPKC localize to the apical surface and they specify apical identity. In contrast to c. elegans zygotes, epithelial Par3 has a distinct localization and function from Par6 and aPKC. In epithelia, Par3 (aka Bazooka) localizes to the tight junction band – it binds to tight junctions and PIPs and promotes TJ formation. \n\nCdc42 is highly active at the apical membrane (slightly active at lateral membrane and absent from basal membrane). It is present in internal vesicles, but active Cdc42 is not vesicular. Active Cdc42 recruits Par6/aPKC to the apical membrane.\n\nRac is present at apical and lateral surfaces. It is probably active in those regions as well and promotes actin assembly. We think Par3 activates Rac via Tiam.\n\nPIP2 is present on all three surfaces but enriched apically while PIP3 is absent from the apical surface but is present on both basal and lateral surfaces.\n\nPTEN localizes to apical surface and is required for these lipid asymmetries.\n\nThe new kids on the block: Crumbs complex and Scribble complex\n\nCrumbs is a transmembrane protein that localizes to the apical surface. It binds Stardust, PATJ, Lin7 and possibly several other proteins (Spectrin/Dmoesin). The complex helps form tight junctions.\n\nThe Scribble “complex” consists of Scribble, Lethal giant larvae (Lgl) and Discs large (Dlg). I put complex in quotes because these proteins haven’t been proven to exist in a complex. They interdependently colocalize with one another, phenocopy each other and Lgl2 and Scribble coIP in mammalian cells. The role of these proteins in mammalian epithelia formation is unclear because the knockdowns have little effect, but this could be due to redundancy. In flies, these proteins are required to form the adherens zone.\n\n“Cortical” Localizations\n\nAll of the proteins listed above localize at or near the plasma membrane in their respective domains (apical, lateral etc.). It’s unclear whether these proteins bind the membrane but I think they do. Cdc42 inserts its lipid tail into membranes and binds Par6 (which, in turn, binds aPKC), so all 3 of those proteins are likely at the PM. Crumbs is a transmembrane protein, so all of its binding partners are probably membrane localized, too. I don’t know about membrane localization for Scribble, Lgl and Dlg. \n\nOne thing that I don’t know is where these proteins hang out when they are inactive (cytoplasm vs. vesicles). Cdc42-GFP localizes to vesicles in large amounts, while the Cdc42 activity readout is exclusively at the plasma membrane. To what degree does localized delivery/exocytosis contribute to the distributions of the signaling components? Crumbs is a transmembrane protein so I would expect trafficking plays a huge role in its localization. Is it locally enriched by preventing endocytosis?\n\nWhat are the external cues?\n\nAdhesion (cell-cell adhesion vs. cell-ECM adhesion) appears to be the major orientation cue. For example, cysts grown in liquid culture have their apical surfaces pointing out but collagen/laminin binding can flip this. How does this couple to the internal polarity circuit?\n\n\nDo they self-organize and if so, how?\n\nLet’s begin by reviewing the distributions of the polarity components. Starting at the apical surface and moving basally:\n\nPIP2, Cdc42, Par6, aPKC, PTEN and the Crumbs complex\n\nPar3\n\nPIP3, Scribble, Lgl, Dlg\n\nPolarized cysts will spontaneously develop under all sorts of culture conditions, including suspended liquid cultures. However, there are always differential adhesions on the various cell surfaces that could direct polarity formation. To what extent are the signaling networks (Pars, Crumbs etc.) amplifying pre-existing, adhesion-mediated patterns vs. breaking symmetry on their own. The system self-organizes but I’m not convinced that it breaks symmetry in the same sense that neutrophils do. The degree to which the signals mentioned above pattern one another isn’t clear. To my knowledge, nobody has artificially mislocalized any one of these signaling proteins to see whether it patterns the other signals. For example, does the ectopic recruitment of Par6 to the basal membrane cause Par3 to localize to the junction between basal and lateral membranes? Exogenous PIP2 at the basal membrane does cause tight junctions and gp135 to reposition themselves basally, but these relationships haven’t been examined for the other signals. One complicating factor is that when one component is deleted, the cyst is often severely deformed which may screw up the patterning all by itself. Another interesting question is the degree to which one cell’s polarity affects the polarity of its neighbors.\n\nI suspect that people think the signals pattern one another because of all their reciprocal interactions. For example, Cdc42 and Crumbs bind Par6/aPKC. aPKC phosphorylates essential residues on Crumbs. Par3 can also associate with Par6/aPKC. The complexes also directly bind PIP enzymes. Par3 recruits PTEN, which probably keeps the PIP2/PIP3 distributions correct. Scribble/Lgl/Dlg bind p85 of PI3K, which could be responsible for basolateral PIP3 accumulation. And so on, and so on.\n\nMost people think the domain boundaries are maintained by mutual cross-inhibition between complexes. For example aPKC phosphorylates Lgl to kick it off of the “cortex”. Lgl directly binds aPKC which knocks aPKC off the “cortex” and inhibits its kinase activity. Par3 is removed from the “cortex” by phosphorylation from aPKC and Par1 (which is recruited by Scribble/Lgl/Dlg). So the Crumbs/Par6 and Scribble complexes restrict Par3 on each side by scaffolding their associated kinases. Crumbs also directly dislodges Par3 from Par6 further removing it from the apical side.\n\nIMO, a huge unanswered question is how the various domains survive in the presence of antagonistic neighboring domains. I used to think that positive feedback did it and there is evidence for positive feedback. For example, Cdc42 activation requires Annexin2 (a lipid binding protein) and Cdc42 also binds Annexin2 in a GTP-dependent manner. Also, Par3 binds PP1alpha (phosphatase), which could remove inhibitory phosphates from Par3 to help it maintain/enrich itself. However, UTSW simulations suggest that systems with inhibitory crosstalk systems and positive feedback fail to maintain multiple separate signaling domains unless the positive feedback loop has an intrinsic autoinhibition mechanism. If autoinhibition is absent then if one species (e.g. Scribble) gets stronger than the others (due to random variation), it will take over the entire cell due to its positive feedback. However, positive feedback with autoinhibition is superficially inconsistent with studies showing that if you get rid of one signaling protein, its neighbors expand beyond their bounds. Perhaps a combination of positive feedback, autoinhibition and crosstalk is responsible for the pattern?\n\nOne other important point is that each domain of the cell is exposed to a different extracellular environment. The basal surface connects to the basement membrane via integrins. The lateral surface is adherens junctions. Just apical to that are tight junctions and above that is the lumen, where there are no adhesions. Par3 associates with tight junctions, at least in Drosophila (where they are called septate junctions) and perhaps that association keeps Par3 at the membrane despite its unfriendly neighbors. Similar stories could emerge for the other players as well. Perhaps adherens junctions keep Scribble at the lateral membrane. In this case, the localization patterns of the various proteins would be maintained by the structure of the epithelium itself. I suspect that some of the mislocalization/expansion effects seen upon deletion of polarity components are actually do to an overall failure to form a normally structured epithelial tube/cyst.\n\nInterface with trafficking\n\nAt the end of the day, all of these polarity signals regulate membrane trafficking to ensure that the right channels/transporters/adhesion proteins get delivered to the correct surfaces. The mechanism by which they couple to trafficking is unknown. Directed trafficking along microtubules and actin cables probably plays a role. Are microtubule plus-ends enriched on one region of the cell? Microtubule networks can be oriented by asymmetrically distributed plus-end stabilizing proteins (or severing proteins etc.) or “cortex” bound motors that can pull microtubules around. Vesicle targeting specificity is also achieved with pairwise interactions of SNAREs and also large docking/tethering molecules that capture arriving vesicles. The Rab GTPases direct all of this: they directly bind tethers/SNAREs etc. or do so via adaptors. There will often be a particular Rab on the vesicle and a complementary target Rab on the target membrane. Each Rab will bind tethers/SNAREs and other adaptors to induce tethering and fusion of the vesicle with the target membrane.\n\nGiven the known vesicular pool of Cdc42 and the fact that Crumbs is transmembrane, it is likely that membrane trafficking plays a direct role in the patterning of the signals, as occurs with Cdc42 polarization in budding yeast.\n
Eps8 contains a phosphotyrosine binding ([[PTB]]) domain, a SH3 domain, and a C-terminal effector region [[Scita 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12620401&query_hl=60&itool=pubmed_docsum]]. Full-length Eps8 caps barbed ends and is auto-inhibited in vitro, and interaction with the [[Abi1]] protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. [[Scita 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15558031&query_hl=17&itool=pubmed_DocSum]]. Eps8, through its SH3 domain, binds [[Abi]] and [[SOS]] to form a tri-complex in vivo that exhibits [[Rac]]-specific GEF activity in vitro [[Di Fiore 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10499589&query_hl=25&itool=pubmed_docsum]]. Eps8, through its pro-rich domain, binds IRSp53 in and is required for [[Rac]]-induced [[Rac]] activation [[Takenawa 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15289329&query_hl=7&itool=pubmed_docsum]]. Eps8 null mice are ethanol resistant and are resistant to the actin-remodeling activities of NMDA and ethanol [[Scita 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17018287&query_hl=60&itool=pubmed_docsum]].
See [[BAR]] domain.
Formin-homology 2
The GPCR for fMLP is called fMet-Leu-Phe receptor FPCR [[Malech 1990|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1690150&query_hl=7&itool=pubmed_DocSum]] and guanine nucleotide exchange and nucleotide-mediated regulation of the FPCR are inhibited by pertussis toxin [[Smiley 1985|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=2983319&query_hl=9&itool=pubmed_DocSum]].
FPLC Protocol AM 9/1/09\nPreparation\nDegas all buffers before use. Make up the buffer as normal, then vacuum filter, leaving the vacuum filter on while the buffer is slowly stirring for at least 15 minutes. You should not be able to see any bubbling. Add unstable compounds (i.e. ATP, DTT) after degassing. You will also need at least 500 ml degassed water and 500 ml degassed ethanol. The protein itself needs to be prepped in order to be loaded onto the FPLC. You can do the fastest speed on the table top for ~10 min and take the supernatant, or push the sample through a filter.\n\nFPLC set-up\nSign on to guest in Lim FPLC. Place both A and B nozzles into degassed water. Click manual -> pumpwashbasic ->execute. Set alarm to 1.8 Mpa and execute. Add 1 ml loop to injection valve at positions 2 and 6. Wash loop in “load” and “inject” mode. Change flow rate to 0.1 ml/min to start washing loops. You can increase the pressure as long as it does not go above 1.8 Mpa. For the loop, you can go as high as 2 ml/min. The column position should be in position1bypass. After ~5 ml (if your loop length is 1 ml), you are ready to load the column. When you are washing the loop, examine the connections. Make sure they do not drip (Note: when I tried to gel filter 488 actin, I think I lost some of the protein because the loop was no completely secure. I saw dripping, but only after I finished my experiment and I was cleaning the loop). \n\nColumn Loading\nWe used a GF S75, which resolve molecules with a MW of 75 Kd or below (a S200 would resolve molecules with 200 Kd or below). This is faster than a S200 because the S200 is a much longer column. Position the column on the akta with the column holders. Reduce the flow rate to 0.2 ml/min. Switch the column position to position2 and observe liquid flowing out. Connect the top of the column to position2 on column valve2, when you see sufficient water accumulate. Make sure you connect water drop to water drop. Note: At this point you have a very dangerous situtation – there is significant pressure building up in the column! Quickly, unscrew the bottom of the column and then wait for water to flow out of the bottom of the column. After you see a drop flowing out of the bottom of the column, connect the bottom of the column to position2 on column valve3. Wash the column with 1.5 column volumes. The GF S75 CV is 24 ml, so wash with ~36 ml of water. Use the highest flow rate that gives pressure below 1.8 Mpa. This may take an hour. We used a flow rate of 0.4 ml/min and then increased the flow rate halfway through to 0.5 ml/min. Switch to position1bypass and then stop the flow rate. Do this before stopping the flow. Your first priority is always to the column. Always switch to position1bypass before doing anything. Remove nozzleA from water and place in buffer A. Pumpwashbasic with bufferA (note you do not have to rewash nozzleB/pumpB in water). Wash the inject valve in “inject” position with BufferA. Switch back to “load” on the inject valve. Wash the GF S75 column with 1.5 CV bufferA by switchng to column position2. Start with a low pressure and then build up, but never go above 0.5 ml/min. You are now ready to load your sample.\n\nSample Loading\nSwitch to position1bypass and turn off the flow rate. Make sure injection valve is under “load”. Rinse the injection needle with water first, then fill your sample into the syringe. Push out any air bubbles and insert the needle into position3 on the injection valve. Inject the sample onto the injection loop and leave the needle in position3.\n\nRunning the sample\nGo to the method editor and set up the method. Save the method with your name and date in the title. I used the S75 method that was already available. For a 1 ml injection loop, you would empty the column with 1.5 ml from the injection loop and then ran 1.5 CV, collecting 0.5 ml aliquots. The whole run took ~1 hour. Make sure the fraction collector is set-up with 1.5 ml screwtop epee tubes. You do not have to number the tubes because the fraction collector is numbered. Position the nozzle of the fraction collector over the first tube. You need to depress the handle the fraction collector for it to move. Press run under system control. This brings up a dialog box, which I mostly click through. I make sure the file is saved with my name and date. Your intial conditions should resemble something like this:\n\n1) Tube = waste900; v1_inject = inject; v2_colpos = 2. This injects the sample currently inside the injection loop onto the column. I used 6 ml for a 5 ml loop (or 1.5 ml for a 1 ml loop). At this point the block volume will rise to 6 ml. At 6 ml the following happens:\n2) Tube = 1; v1_inject = load; v2_colpos = 2. Now the sample is flowing through the column. Verify to make sure the fraction collector is doing it’s job. If it’s not, you will have to pause and reposition the fraction collector.\n\nCleaning up\nAt the finish of the run, remove the fraction collector head from the tubes. This keeps the fraction collector from accidentally bumping the tubes during any subsequent pump washes. Run a gel on pertinent fractions and only save those. Remove needle from position3 and rinse with water. Switch to position1bypass, decrease flow rate and set alarms. Rinse the injection loop with water. Here, we had bufferB as water so we switched the gradient to 100% bufferB. Switch back to column position2 and rinse the column with 1.5 CV of water. Switch to position1bypass, decrease flow rate to 0, and place nozzles in 20% ethanol. Pumpwashbasic nozzleA and nozzleB. Wash injection loop with ethanol (injection valve = “inject”), then switch to column position2, and set the flow rate to 0.2 ml/min. The pressure will rise with ethanol over time. Rinse column with 1.5 CV 20% ethanol. Set to position1bypass and decrease flow rate to 0. First disconnect the top of the column, cap, and then disconnect the bottom of the column and cap. Place nozzleA and nozzleB in water for storage (storing nozzles in ethanol would cause ethanol to evaporate). Press end on the system control on the computer. This ensures that the UV light does not stay on (quitting the program will not turn off the UV bulb).\n
A feed forward loop (FFL) is a network where A regulates B, and A and B jointly regulate C. FFLs can be coherent (where the direction of A regulating B coincides with the direction of A and B regulating C) or incoherent (where the direction is opposite between A regulating B and A and B regulating C). FFLs have the potential to reduce noise and produce a rapid off to the system.
A fibroblast is a type of cell that synthesizes and maintains the extracellular matrix of many animal tissues. Fibroblasts provide a structural framework (stroma) for many tissues, and play a critical role in wound healing. They are the most common cells of connective tissue in animals.\n\nOrigin/General Information\nFibroblasts are derived from the primitive mesenchyme, and are thus mesodermal. Their morphology depends upon their location within the body as they have many specialized roles in different tissues. In contrast to epithelial cells, fibroblasts do not form flat monolayers and are not restricted by a polarizing attachment to the basal lamina. When crowded at high density, align and form clusters. During the growth of an organism, fibroblasts divide and secrete ~ECM. Tissue damage induces the proliferation of fibroblasts. Fibroblasts can give rise to other types of mesodermal cells, such as bone, fat and smooth muscle. Unlike epithelial cells, fibroblasts migrate over their substrate at ~30 microns per hour. Epithelial cells may undergo an epithelial to mesenchymal transition (~EMT) to turn into fibroblast-like cells, an event which is common in invasive/metastatic cancers. Inactive fibroblasts are called "fibrocytes".\n\nCommon Fibroblast "Types":\n~MEFs or Mouse Embryonic Fibroblasts - These are primary cells derived from ground up mouse embryos. They are the cells which are most similar to actual fibroblasts in the body of an animal.\n\nCos7 - Transformed fibroblasts from the kidney of an african green monkey, adherent growth plastic and glaass. They are easily transfected with plasmids.\n\n3T3 - Stands for (3E5 cells per 20cm dish w/ transfer interval of 3 days). This was one of the earliest immortal fibroblast cell lines to be established (back in the early 60s). I think either Swiss or NIH 3T3 cells are aneuploid, although I don't know which one.\n\n~NR6 - Murine fibroblast line\n\nRat2 - Rat fibroblast line, developed as a ~BrdU-resistant version of the Rat1 cell line. The Rat1 cell line was a rat version of the ~3T3 lines.\n\nMorphology\nFibroblasts typically have a branched cytoplasm and an elliptical, speckled nucleus w/1-2 nucleoli. Active fibroblasts usually have a large, rough ~ER. Fibroblasts are about 50 microns in diameter. The height of the outer 4-5 microns of a motile leading edge of a fibroblast fluctuates between 400-800nm, with ruffles that are at least 1200nm tall. The outer 500nm of a a stable (non-motile) edge is under 1000nm tall. Immediately inside that region, is a 5+ micron long region which is 1.5-2um thick and has a more uniform height than the active leading edges. This information was obtained with ~AFM in the Radmacher lab.\n\nMotility\nFibroblasts will spontaneously migrate on fibronectin in the presence of serum. Hormones and growth factors affect their movement. They move at ~30 microns per hour, although individual lamellipodia can move much faster (maybe 100 microns per hour, estimate from video). Their motility is highest in their first few hours after plating, after which time they are too adherent. People usually study their spreading on a surface instead of their net translocation. They will spread spontaneously in the presence of serum. To maximally activate their spreading pathway, people serum-starve them and then add growth factor.\n\n
10x mHBSS = 1.5 M NaCl, 40 mM KCl, 10 mM MgCl2, 100 mM glucose, 200 mM Hepes, pH 7.2 (1.5 L, sterile-filtered, store @ -4)\n\n''For differentiated HL-60 cells''\n//Note: RPMI +serum may be used as a simpler alternative to PBS and mHBSS for the washing, blocking, and plating steps.//\n\nFibronectin-coating coverslips\n1. Dissolve fibronectin (stored in 1 mg bottles in -20 C freezer) in 1 ml of sterile water. Let sit at room temperature in the hood for 1 hr.\n2. At end of hour, pipet up and down to mix gently.\n3. Dilute 1:5 in Ca/Mg-free PBS (keep fibronectin stock and unused diluted fibronectin at 4 C)\n4. Plate 100 µl diluted fibronectin per coverslip chamber (use 8 well chambers). Let sit for 1 hr at room temperature.\n5. Aspirate fibronectin.\n6. Wash wells 2 or more times with Ca/Mg free PBS.\n7. Block wells with mHBSS + 0.2% albumin for 3 -5 minutes (use albumin fresh).\n8. If using coverslips the same day, keep at room temperature.\n9. For plating cells, spin down 5 – 8 day differentiated cells (1 min at 400 x g)\n10. Wash once in warm mHBSS + 0.2% albumin\n11. Bring back up in between on volume and ¼ volume mHBSS + albumin (see how dense cells are and decide how much to use).\n12. Plate in incubator at 37 C in humidified chamber for 20 miin.\n13. Wash unbound cells 2-3x with mHBSS + albumin\n14. If important to have minimally prepolarized cells, can let incubate and additional 10 – 20 min at 37 C before using. Otherwise, cells are ready to go.\n15. Unused fibronectin-coated coverslips can also be stored at 4 C in humid chamber (give generous amounts of mHBSS + 0.2% albumin and check periodically to make sure they don’t dry out).\n\n''For adherent fibroblasts (NIH-3T3) for TIRF microscopy''\n1. dilute fibronectin to ~0.08 micrograms/mL in PBS (1:25 dilution of homemade 2 mg/mL stock).\n2. coat glass for 1 hr at RT (in the tissue culture hood).\n3. wash with PBS 2x. use immediately, or store in PBS at 4 degrees for no longer than 3 days.\n\n[img[Fibronectin|http://img508.imageshack.us/img508/9118/figure3rgbvu6.gif]]\n\n''Fig. 3. Preparing a coverslip for live cell microscopy.'' (A) Dissolve 1 mg of bovine fibronectin in sterile water. After 1 hour, add 4 ml of PBS and store 200 µg/ml fibronectin solution at 4°C. (B) Remove gaskets from plastic permanox 8 well chamber. Cut epoxy mold squares and stick to No. 1.5 gold seal cover glass. Add 125 µl of fibronectin, let sit for 1 hour, rinse once with RPMI culture media, and store in RPMI media until ready to image.
Filamin A is a 280 kD actin-binding protein that induces high-angle cross-linking of actin filaments. Cells lacking filamin A exhibit reduced cytoplasmic viscoelasticity and unstable membranes that constitutively bleb, and the cells are unable to support effective locomotion. Filamin A binds to a region that overlaps the [[CRIB]]/[[PBD]] domain of [[Pak1]] (aa 52–132), and this binding appears to be sufficient to relieve autoinhibition, thereby enhancing intrinsic [[Pak1]] activity. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
The filopodia are slender cytoplasmic projections, similar to lamellipodia, which extend from the leading edge of migrating cells. Cdc42 is not essential for filopodium formation, directed migration, cell polarization, and mitosis in fibroblastoid cells [[Brakebusch 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16014609&query_hl=38&itool=pubmed_docsum]]. Intriguingly, neither is WAVE complex nor Arp2/3 required for filopodia formation [[Stradal 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16597702&query_hl=4&itool=pubmed_DocSum]].
A [[Talin]] homolog that assembles on the leading edge of Dicty cells in response to chemoattractant [[Muller-Tabenberger 1995|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=7698984&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Fimbrin belongs to the calponin homology (CH) domain superfamily of actin cross-linking proteins, which include α-actinin, β-spectrin, dystrophin, ABP-120 and filamin. In humans, three highly homologous, strictly tissue and locale specific isoforms have been identified: I-, T- and L-fimbrin (also known as [[L-plastin]]).
''Buffers''\n\nmHBSS = 150mM NaCl, 4mM KCl, 1mM MgCl2, 10mM glucose, 20mM HEPES, pH 7.2\n\nKprex = 140 mM KCl, 1 mM MgCl2, 1 mM EGTA, 20 mM Hepes, pH 7.5\n\n2X Fixation buffer = Kprex + 0.2% albumin + 500 mM sucrose + 7.4% formaldehyde (add .2% albumin fresh for each use, do not add to stock solution)\n\nStain buffer = Kprex + 0.2% albumin (add .2% albumin fresh for each use, do not add to stock solution, use the cheap stuff in the cold room)+ 0.2% triton (use 10 -20% stock to make this. It’s hard to pipet 100% triton) + 2 µl/ml rhodamine phalloidin OR 2 µl/mL FITC-phalloidin (Add dyes directly before stain.)\nKprex + 0.2% albumin\n\n''Suspension Procedure''\n\n1. Spin cells 1 min 400 x g out of current buffer or by aspirating current buffer for adherent cells.\n2. Resuspend in mHBSS and stimulate cells in suspension.\n3. Add 2x volume Kprex + 7.4% formaldehyde + 500 mM sucrose (prepared the same day). Importance of this buffer is EGTA to chelate calcium (to inhibit calcium-activated severing proteins and proteases) and sucrose to maintain osmolarity (formaldehyde can partially permeabilize cells, allowing water influx due to high internal protein concentration.\n4. Fix for 20 min\n5. Aspirate supernatant or spin down cells (1 min 400 x g) and aspirate supernatant. Discard supernatant in formaldehyde trash (not regular trash).\n6. Permeabilize cells in Kprex buffer + 0.2% triton X-100 (use 10 -20% stock to make this. It’s hard to pipet 100% triton) + 0.2% albumin (use the cheap stuff in the cold room) + 5 µl FITC-phalloidin or 1 µl per 500 ml rhodamine phalloidin (both at – 20C).\n7. Stain for 20 minutes. Wash into above buffer lacking fluorescent phalloidin. Can wash again if background too high. Often put in the same buffer lacking block and detergent for best imaging. However, for many conditions, can image at step 5.\n\n''Adherent Procedure''\n\n1. Fibronectin coat chambered coverglass (refer to //Fibronectin coating// protocol)\n2. Rinse coverglass quickly 2X with PBS and for 3-5 min 1X with mHBSS + .2% albumin\n3. Aspirate off mHBSS\n4. Plate cells and incubate at 37 deg C for 5-20 min\n5. Aspirate off growth media carefully and without touching the bottom of the chamber\n6. Resuspend in mHBSS + .2% albumin\n7. Stimulate cells and add any drugs as desired\n8. Add eqivolume 2X Fixation Buffer. \n9. Carefully aspirate supernatant and discard in formaldehyde waste (not regular trash!).\n10. Add dye to your stain buffer\n11. Add 1X stain to sample\n12. Stain for 20 minutes. \n13. Aspirate and resuspend in stain buffer lacking fluorescent phalloidin. Can wash again if background too high. Often put in the same buffer lacking block and detergent for best imaging.
Mammalian formins include [[mDia1]], [[mDia2]], and [[mDia3]]. All formins contain a conserved formin homology 2 (FH2) domain that mediates interactions with actin, including nucleation, binding barbed ends, inhibiting capping proteins, and filament severing. In the DRFs, the FH2 domain is immediately followed by the DAD. The DAD binds to a 500 residue N-terminal regulatory element, causing autoinhibition of FH2 activity through an unknown mechanism. In the DRF mDia1, binding of the RhoA GTPase to this element causes dissociation of DAD peptides [[Rosen introduction 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15866170&query_hl=6&itool=pubmed_DocSum]].
Includes [[mDia2]] and [[Cappuccino]].
Forskolin increases cAMP levels by activating adenylate cyclase.
Showed that intracellular calcium increases (by fura-2) upon human polymorphonuclear leukocyte spreading [[Maxfield 1986|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=3458251&query_hl=17&itool=pubmed_docsum]].\n\nBuffering of [Ca2+]i (quin-2/AM or BAPTA/AM) or removal of extracellular Ca2+ ([[EGTA]]) reduced or inhibited the migration of neutrophils because they remained anchored at their rear. Cells still exhibited spreading on poly-lysine under these conditions [[Maxfield 1990|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=2295684&query_hl=17&itool=pubmed_docsum]].\n\nBlocking [Ca2+]i transients has little effect on cell spreading, polarization or pseudopod extension. In contrast, cell motility is [Ca2+]i dependent when the cells are examined on physiological substrates such as fibronectin or vitronectin [[Maxfield 1991|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1702443&query_hl=17&itool=pubmed_docsum]].\n\nShowed that cholesterol depletion by [[Methyl-ß-cyclodextrin]] inhibits cell polarity, migration, actin polymerization, and localized Rac (Rac antibody). Showed that inhibition of myosin (myosin light chain kinase inhibitor, ML-7) prevents Rac recruitment to the membrane [[Maxfield 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12522144&query_hl=17&itool=pubmed_docsum]].
Fugetaxis or chemorepulsion, which describes the active movement of leukocytes away from chemokinetic agents including the chemokine, stromal cell derived factor-1, and the HIV-1 envelope protein, gp120 [[Vianello 2005 review|http://www.ncbi.nlm.nih.gov/pubmed/16142473]].
Grb2-associated binding (GAB) proteins include a group of at least four mammalian proteins (GAB1, GAB2, GAB3, and GAB4) that have N-terminal pleckstrin homology ([[PH]]) domains and multiple sites of phosphorylation. These proteins act as adaptors to recruit Src homology 2 ([[SH2]]) and/or phosphotyrosine-binding ([[PTB]]) domain-containing proteins to the membrane in response to stimulation by certain protein-Tyr kinases. The PH domain of GAB1 binds specifically to phosphatidylinositol-3,4,5-trisphosphate [[PI(3,4,5)P3]]. Tyrosine-phosphorylated GAB proteins bind to the regulatory subunit of phosphoinositide 3-kinase (PI3K). Proline-rich sequences in GAB1 and GAB2 bind to SH3 domains of [[Grb2]]. Thus, [[Grb2]] can recruit GAB proteins to receptors by directly binding to Tyr-phosphorylated sites on the receptors or by binding indirectly through SHC [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
See [[GAB]]
See [[GAB]]
See [[GAB]]
GTPase activating protein. Includes [[Arap3]], [[IQGAP]],
GDP Dissociation Inhibitor; GDI proteins slow the rate of dissociation of GDP from GTPases. GDI binds to the switch I and II domains of [[Cdc42]] leading to the inhibition of both GDP dissociation and GTP hydrolysis [[Cerione 2000|http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&dopt=AbstractPlus&list_uids=10676816]].
Guanine nucleotide dissociation stimulators [[Weinberg 1993|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=PubMed&Cmd=ShowDetailView&TermToSearch=8094051]].
GTP exchange factor
Guanine nucleotide exchange factors.
G protein coupled receptor. In chemotactic neutrophils, GPCR's are uniformly distributed on cell surface.
See [[GPCR]].
GSK3 is negatively regulated during insulin signaling and in response to cell-fibronectin interactions by the activation of phosphatidylinositol 3-kinase (PI3K) and integrin-linked kinase (ILK), which leads to activation of protein kinase B ([[Akt]]), which directly phosphorylates GSK3 on Ser9 [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
See [[GSK3]].
GST bead assay\n\nArthur Millius 7/25/07\n\nOverview\n\nTo visualize labeled recombinant WRC or YFP-Hem1 from R6 lysate on GST beads.\n\nBuffers\n\nKprex = 140mM KCl, 1mM MgCl2, 1mM EGTA, 20mM Hepes pH 7.5\nKMEI + 20% glycerol = 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole, 20% glycerol\n\nProcedure\n\nAliquot two tubes of 40 µl GST bead slurry. Spin at 1000 x g for 1 min. Remove supernatant, and replace with 500 µl Kprex. Repeat this wash two more times.\n\nAdd 40 µl Kprex to each tube. Add 4 µl of RacQ61L to one tube. Incubate >1 hour.\n\nSpin down GST beads. Remove superanatant and wash twice with 500 µl Kprex.\n\nResuspend in 40 µl Kprex. Beads are now ready for assay.\n\nAdd 20 µl of R6 lysate or labeled WRC complex to 5 µl of beads. Take 10 µl of that solution and place on a coverlip. Cover and seal with valap and image immediately.\n\nNote: I tried diluting R6 lysate. I was able to dilute the R6 lysate ¼ and still see recruitment. I estimate that the concentration of WAVE complex in this sample was at most 10 nM. When I diluted it 1/10, I no longer saw recruitment.
Molecular switches, which exist in a GTP and GDP state. Rho GTPases, include [[Rac]], RhoA, [[Cdc42]], and RhoG. [[Hall 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12478284&dopt=Abstract]]. Constitutively active GTPases fall into nine distinct classes based on morphology [[Meyer 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12732140&query_hl=20&itool=pubmed_docsum]].
GAP, a protein which aids a GTPase in hydrolysis of GTP to GDP.
RhoGTPases are molecular switches that cycle between two conformational states: a GTP-bound “active” state and a GDP-bound “inactive” state.
Gallein is a small-molecule Gbetagamma inhibitor [[Smrcka 2006|http://www.ncbi.nlm.nih.gov/pubmed/16627746?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=1&log$=relatedarticles&logdbfrom=pubmed]] that suppresses fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. It also blocks fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils [[Smrcka 2008|http://www.ncbi.nlm.nih.gov/pubmed/18006643?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. This drug would potentially make HL-60 cells hyposensitive to chemoattractant - betagamma is tied up in the drug and could not signal. Interestingly, if you knockout the yeast RGS protein SST2, which GAPs the alpha subunit for the pheromone receptor, the cells are hypersensitive to pheromone [[Drubin 2003|http://www.ncbi.nlm.nih.gov/pubmed/12598904?dopt=Abstract]]. The interpretation is that the cells have an overabundance of alphaGTP, which allows betagamma to signal for longer.
Showed that [[Rac2]] regulates the NADPH oxidase of neutrophils [[Bokoch 1991|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1660188&query_hl=3&itool=pubmed_DocSum]] and [[Bokoch 1992|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1331090&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that RhoGDI produced a concentration-dependent inhibition of GTP hydrolysis by [[Rac1]] that paralleled its ability to inhibit GDP dissociation from the [[Rac]] protein [[Bokoch 1993|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8419353&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that membrane binding was correlated with exchange of GTPγS for GDP on [[Rac]], and only GTPγS-bound [[Rac]] became membrane localized [[Bokoch 1994|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7989340&query_hl=3&itool=pubmed_DocSum]].\n\nIdentified [[Pak]] as a [[Rac]] target and showed that Paks phosphorylate the p47phox NADPH oxidase component in a Rac-GTP-dependent manner [[Bokoch 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7618083&query_hl=3&itool=pubmed_DocSum]]\n\nShowed that CA Rac, but not Rho stimulates uncapping of f-actin in permeabilized platelets [[Bokoch 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7664343&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that p67phox interacts preferentially with Rac2 rather than Rac1 [[Bokoch 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8550629&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that Rac GTP binds PI3K in Swiss 3T3 fibroblasts and human neutrophil lysates, and increased PI3K activity became associated with Rac GTP in PDGF stimulated cells [[Bokoch 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8645157&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that activated Rho and Rac inhibit transferrin-receptor-mediated endocytosis when expressed in intact cells [[Bokoch 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8700210&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that two mechanisms can generate PIP3: 1) through the src-related tyrosine kinase Lyn to the "classical" [[p85]]/[[p110]] form of PI3K or 2) through [[Gβγ]]. In this paper, we show that formation of PIP3 in chemoattractant-stimulated neutrophils is substantially attenuated by inhibitors that specifically block the tyrosine kinase activity [[Bokoch 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8810279&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Nck]] binds to [[Pak1]] through its second SH3 domain, while [[Pak1]] interacts with [[Nck]] via the first proline-rich SH3 binding motif at its amino terminus. The interaction of active [[Pak1]] with [[Nck]] leads to the phosphorylation of [[Nck]] at multiple sites [[Bokoch 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8824201&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles [[Bokoch 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9395435&query_hl=3&itool=pubmed_DocSum]].\n\nShowed (along with Zigmond and Devreotes) that CA [[Cdc42]], but not [[Rac]] or [[Rho]] can activate actin assembly in a cell lysate [[Bokoch 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9230078&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that integrin-dependent adhesion led to the rapid activation of [[Pak1]] [[Bokoch 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9658176&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak]] activity can be modulated by physical interaction with [[alpha-Pix]] [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10037684&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak1]] phosphorylates [[MLCK]], resulting in decreased [[MLCK]] activity. [[MLCK]] activity and [[MLC]] phosphorylation were decreased, and cell spreading was inhibited in HeLa cells expressing constitutively active [[Pak1]] [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10092231&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that active [[Cdc42]] and [[Rac]] bind [[Pak1]] in a pull-down assay [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10224076&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak1]] localizes to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10496324&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak1]] is inhibited in trans and that this inhibition is relieved by GTPase binding, which causes [[Pak1]] autophosphorylation [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10551809&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak1]] phosphorylates and activates [[LIMK]] [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10559936&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak1]] is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction [[Bokoch 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10562284&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak1]] is phosphorylated by phosphorylated [[PDK1]] [[Bokoch 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10995762&query_hl=3&itool=pubmed_DocSum]].\n\nDeveloped [[Rac]] [[Pak1]] FRET probe for [[Rac]] activation dynamics [[Bokoch 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11030651&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Pak2]] is regulated by ERK and Ras to phosphorylate [[beta-Pix]] [[Bokoch 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12226077&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that active Rac is at the leading edge, but also in the retracting tail of motile neutrophils [[Bokoch 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12477392&query_hl=3&itool=pubmed_DocSum]].\n\nA nice review of [[Pak]] [[Bokoch review 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12676796&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Cdc42]] acts as a competitive inhibitor of [[Rac1]]- and [[Rac2]]-mediated ROS formation in a recombinant cell-free oxidase system. Transient expression of Cdc42Q61L inhibited ROS formation induced by CA [[Rac1]] in an NADPH oxidase-expressing Cos7 cell line, while sequestration of [[Cdc42]] by the [[CRIB]] domain of [[WASP]] increases ROS formation in stimulated neutrophils [[Bokoch 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15123662&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that phosphorylation of RhoGDI by [[Pak1]] mediates dissociation and that [[Cdc42]]-induced [[Rac1]] activation is inhibited by expression of [[Pak1]] autoinhibitory domain [[Bokoch 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15225553&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that [[Chronophin]] dephosphorylates cofillin [[Bokoch 2005|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15580268&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and colocalizes with [[LIMK]] and [[Cofilin]] in membrane protrusions [[Bokoch 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17500066&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nA nice review of RhoGTPases [[Bokoch 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15752980&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that P-Rex1 translocates from cytoplasm to the leading edge of polarized cells. Showed that activation of [[PKA]], which phosphorylates and inactivates P-Rex1, inhibits its translocation [[Bokoch 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17227822&query_hl=3&itool=pubmed_DocSum]].
Protocol: Gateway LR RXN Protocol + Transformation\n\nI. Note: TE Buffer, pH 8.0 to 4 ul (or use Buffer EB from the Qiagen kits)\nAdd cells to mixture for transformation: dB3 cells for destination vector (which have chloramphenicol & amp resistance, and ccdB that makes them unable to grow in dH5∝ cells) \n\nNote: make a neg and pos control for transformation step: pos w/known vector and neg w/o DNA\n\n\n1. Thaw LR Clonase enzyme on ice from -80C (near DH5 alohas) for about 2 minutes\n2. Add the following to a 1.5mL tube at room temp\na. TOPO entry clone/ pENTR 50-150ng/ .5-5µL\nb. Destination vector (150ng/µL) 1 µL\nc. 5X LR Clonase Reaction Buffer 2 µL\nd. dd H20 to 8 µL\n3. Vortex each tube\n4. Vortex LR Clonase enzyme mix briefly 2x (2 seconds each time)\n5. Add 2 ul LR Clonase to each sample\n6. Vortex and briefly spn down\n7. Vortex each mixture briefly 2x (2 seconds each time)\n8. Incubate @RT for 1+ hrs (overnight for plasmids >10kb)\n9. Add 1 µL Proteinase K at end of incubation 10’ @ 37 C for 10 min ( we don’t do this step)\n10. Proceed to transformation step (i.e. DH5∝ cells, 2YT media!!!)\n\nII. Generic transformation Procedure (for Top 10 [pENTR transformations], Db3.1[Gateway destination vector transformations], and DH5alphas [everything else including gateway LR rxns])\n\nTransforming Competent Cells\nTo avoid contamination, have the Bunsen going and flame any LB that is used. Have a control with no dna. Pre-warm selective plates at 37C Turn on bath\n→ Use 2 negatives (1 has vector + water, & other has air + cells) \n\n1. Thaw on ice one tube of DH5α™ cells. Place 1.5 ml microcentrifuge\ntubes on wet ice.\n2. Gently mix cells with the pipette tip and aliquot 50 μl of cells for\neach transformation into a 1.5 ml microcentrifuge tube.\n3. Refreeze any unused cells in the dry ice/ethanol bath for 5 minutes\nbefore returning to the -80°C freezer. Do not use liquid nitrogen.\n4. Add 1 to 5 μl (1-10 ng)[usually 1:10-1:100 dilution, then use 1ul -keep diluted cells that aren’t used for a day in case of problems] of DNA to the cells and mix gently. Do not mix by pipetting up and down.\n5. Incubate tubes on ice for 30 minutes.\n6. Heat shock cells for 45 seconds in a 42°C water bath without\nshaking.\n7. Place tubes on ice for 2 minutes.\n8. Add 450 μl of pre-warmed medium of choice to each tube. [no antibiotic]\n9. Incubate tubes at 37°C for 1 hour at 225 rpm. [can be less if needed]\n10. Spread 50 μl from each transformation on pre-warmed\nselective plates. We recommend plating two different volumes to\nensure that at least one plate will have well-spaced colonies.\nI use 100uls for a normal cut and paste ligation, but for LR rxns or TOPO rxns or tricky ligations, I spin cells down (5’ @ 4000 rpm) suck off all but 100uls of media and then plate all the cells in the remaining 100uls media\n11. Store the remaining transformation reaction at +4°C. Additional\ncells may be plated out the next day, if desired.\n12. Incubate plates overnight at 37°C.
Showed f-actin flow in a migrating epithelial cell with fluorescence speckle microscopy [[Danuser 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12885672&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that a lamellipodium network is assembled at the leading edge of migrating epithelial cells, but is completely disassembled within 1 to 3 micrometers [[Danuser 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15375270&query_hl=4&itool=pubmed_DocSum]].\n\nDefense of the lamella hypothesis and actin particle tracking [[Danuser 2009|http://www.ncbi.nlm.nih.gov/pubmed/19494124]].
Gelsolin is a prototype for a family of capping proteins, which share modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Family contains at least another six members: villin, adseverin, capG, advillin, supervillin and flightless I. [[Hayoz 2004 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15526166&query_hl=19&itool=pubmed_DocSum]]
Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation [[Crabtree 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7568223&query_hl=23&itool=pubmed_DocSum]].
Granulocytes are a category of white blood cells characterised by the presence of granules in their cytoplasm. They are also called polymorphonuclear leukocytes (PMN or PML) because of the varying shapes of the nucleus, which is usually lobed into three segments. In common parlance, the term polymorphonuclear leukocyte often refers specifically to neutrophil granulocytes, the most abundant of the granulocytes.
There are three types of granulocytes: [[Neutrophil]], Basophil, and Eosinophil, which are named according to their staining properties.
Grb-2 is an adaptor protein with N- and C-terminal Src-homology 3 (SH3) domains and a central SH2 domain. The SH2 domain can bind to phosphoTyr residues of receptors or other adaptor proteins such as SHC. The SH3 domains bind the Ras exchange factor son of sevenless (SOS) but can also bind to other adaptor proteins such as Grb2-associated binding protein GAB1 and GAB2. Thus, Grb-2 is involved in activation of Ras but can also play a role in other signaling pathways in mammalian cells [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
He visualized f-actin waves with LimE-GFP in Dicty when cells recovered after latrunculin treatment [[Gerisch 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15347592&query_hl=1&itool=pubmed_docsum]] and Arp2/3 [[Gerisch 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15894626&query_hl=6&itool=pubmed_docsum]] in motile Dicty cells [[Gerisch 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14711408&query_hl=6&itool=pubmed_docsum]].
Sixteen distinct mammalian Gα protein subunits have been identified and divided into four families based on their sequence similarity: αs, αi, αq, and α12. The q family of heterotrimeric G protein subunits is coupled to phospholipase-C to induce the hydrolysis of phosphatidylinositol bis phosphate, the consequent rise in the intracellular concentration of Ca2+, and the activation of protein kinase C, whereas the s and i families are known to activate and inhibit adenylyl cyclase to increase and decrease the level of cyclic AMP, respectively [[Gutkind 2001 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11313914&query_hl=21&itool=pubmed_DocSum]].
Gα12 and Gα13 elicit divergent cellular responses: phospholipase C activation, phospholipase D activation, cytoskeletal change, oncogenic response, apoptosis, MAP kinase activation and Na/H-exchange activation [[Kurose 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14607242&query_hl=14&itool=pubmed_DocSum]].
Gβγ activates PI3K by direct binding to the [[p110]] subunit [[Nurnberg 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9507010&query_hl=145&itool=pubmed_docsum]]. Gβγ binds Pak1 and (via Gβγ and HA-Pak1 pulldown), via Pak-associated PIXα, activates Cdc42, which in turn activates Pak1 [[Wu 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887923&query_hl=35&itool=pubmed_docsum]].
See HL60
HL-60 (Human promyelocytic leukemia cells) cell line derived from a patient with acute promyelocytic leukemia proliferates continuously in suspension culture in nutrient medium supplemented with fetal bovine serum, L-glutamine, HEPES and antibiotic chemicals. The HL-60 cells continuously proliferate in suspension culture with a doubling time of about 36-48 hours. \n\nOur lab carries the following HL60 stables:\n\n[[pMIG]]\n[[PBD Cherry]]\n[[R6 PBD Cherry]]\n[[R6]]\n[[Hem-1 ΔsiB]]\n[[Actin YFP]]\n[[PH Akt GFP]]\n[[PH Akt YFP Hem1KD]]\n[[Pak PBD YFP Hem1KD]]\n[[Arp3 GFP]]\n[[C5aR GFP]]\n[[NWASP GFP]]
HL60 lysate \nArthur Millius\n\n''Overview''\nPurification of cytosol and membrane on a small scale for use in Rac membrane translocations and other experiments.\n\n''Buffers''\n\nmHBSS (Ca free) + 0.2% albumin = 150mM NaCl, 4mM KCl, 1mM MgCl2, 10mM glucose, 20mM Hepes, pH 7.2, 2 g/L endotoxin-free albumin\n\nSonication buffer + sucrose = 100 mM KCl, 50 mM Hepes, 2 mM EGTA, 2 mM MgC l2, 250 mM sucrose, 1 mM DTT, 0.1 mM PMSF, 3 mM DFP, 1 EDTA-free protease inhibitor tablet (add protease inhibitors just before use)\n\nKprex + 10% glycerol = Kprex + 10% glycerol = 140 mM KCl, 1 mM MgCl2, 1 mM EGTA, 20 mM Hepes pH 7.5, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 EDTA-free protease inhibitor tablet (sterile filter). (add protease inhibitors just before use)\n\nSucrose Gradient buffer = 50 mM Hepes, 1 mM EGTA, 1 mM MgCl2, 1 mM ATP, 60%, 34%, and 15% sucrose, 1 mM DTT, 2 mM PMSF, , 1 EDTA-free protease inhibitor tablet per 50 ml (250 ml, add ATP and proteases before use, store @ -4)\n\n''Procedure''\n\nCollect 5-6 day differentiated cells in 500 ml corning centrifuge tube. Spin at 1500 x g at room temperature for 10 minutes. Remove supernatant and resuspend in 50 ml mHBSS (Ca free). Spin at 400 x g at room temperature for 10 minutes.\n\nAdd protease inhibitors to sonication buffer. Add 3 mM DFP (2 µl 5 M DFP in 3 ml) to sonication buffer under hood. //Caution: DFP is extremely toxic! 300 µl on your skin is lethal!!!!! Keep stocks in 100 µl aliquots, wear gloves, a coat, and safety glasses. Have antidote nearby. Tell someone that you are working with DFP before beginning this step. Put used DFP tips in at least 2 N NaOH to neutralize DFP before discarding. DFP is dead after 90 minutes.//\n\nResuspend cells in cold 3 ml sonication buffer. Do all remaining steps on ice, unless indicated.\n\nSonicate cells 3 x 10 secs at 20% power. Ice 10 – 15 secs between bursts. //Note: I sonicated a trial sample of cells at different power settings until 1% of them remained.//\n\nTransfer to (2) 1 ml tubes and spin 900 x g for 5 min at 4 C to remove cell debris. Remove 200 µl and label “low speed supernatant (LSS)”.\n\nFor remaining LSS, layer on top of 15/34/60 sucrose gradient in the SW 40 compatible tubes. Add 1 mM DTT, 1 mM PMSF, and 1 protease tablet to sucrose gradient buffer. //Note: I forgot to add the protease tablet. Spin for 15 min at 38,000 rpm.//\n\nCollect cytosol (~1 ml). Discard light membrane interface (cytosol/15). Remove plasma membrane (~1 ml). Dilute 1:1 in (2) 1 ml centrifuge tubes. Concentrate plasma membrane by spinning at 95,000 rpm in TLA 120.2 at 4 C for 45 min.\n\nRemove supernatant. Wash once in Kprex + 10 glycerol. Resuspend in 300 µl Kprex + 10% glycerol.
Hemapoietic-specific cortactin [[Daly 2004 review|http://www.ncbi.nlm.nih.gov/pubmed/15186216?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
HT-1080 are adherent epithelial cells derived from a human fibrosarcoma from a 35 year old caucasian male in 1972. These cells contain an activated N-Ras oncogene. \n\nCells should be maintained in 10% FBS in Eagle's MEM (contains Earles Balanced Salt Solution, non-essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate.)
In haptotaxis the gradient of the chemoattractant is expressed or bound on a surface, in contrast to the classical way of chemotaxis when the gradient develops in a soluble space. The main biologically active haptotactic surface is the extracellular matrix (ECM); the presence of bound ligands is responsible for induction of transendothelial migration and angiogenesis.
A HeLa cell (also Hela or hela cell) is an immortal cell line used in medical research. The cell line was derived from cervical cancer cells taken from Henrietta Lacks, who died from her cancer in 1951.
Purified a Rac exchange factor P-Rex1 stimulated by PIP3 and [[Gβγ]] [[Welch 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11955434&query_hl=31&itool=pubmed_docsum]].
Hem-1 complexes (including the [[WAVE complex]]) coordinate diverse regulatory signals at the leading edge of polarized neutrophils, including but not confined to those involving [[WAVE2]]-dependent actin polymerization. [[Weiner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16417406&query_hl=51&itool=pubmed_docsum]]. Hem-1 is only expressed in blood [[Hromas 1995|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=7643388&ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Hem1 KD shRNA fused to CFP. The "B" was the one that gave best KD.
Hem-2 is expressed in brain, heart, liver and testis [[Hromas 1995|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=7643388&ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. [[Hem-2]]/[[Nap1]] homolog [[Kette]] controls axonal pathfinding in Drosophila [[Klambt 2000|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10766742&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Strategy for large-scale purification of Hem1-WAVE complex from pig lysate\nArthur Millius and Andrew Houk 9/22/06\n\n''Overview''\n\nTo immunopurify Hem1 complexes from Pig leukocyte lysate enough for 30 µl 0.1 mg/ml for cryo-EM. Additional Hem1 wave complex will be used for in vitro biochemistry reconsitution of actin assembly from Cdc42 and Rac beads. Maybe an input into permeabized cells by adding active WAVE complex IPed from LatB treated membrane fractions.\n\n''Buffers''\n\nKprex = 140mM KCl, 1mM MgCl2, 1mM EGTA, 20mM Hepes pH 7.5, 1mM DTT, 1 EDTA-free protease inhibitor table per 50 ml (Make 50 ml; add protease inhibitors and DTT just prior to use).\n\nKprex + 400 mM KCl = 9mL Kprex + 3mL 1M KCl for Kprex 400 mM KCl (mild ionic wash)\n\nKprex-T = 10 mL Kprex + 0.1% Tween-20 (mild hydrophobic wash)\n\nTBS = 50 mM Tris, 0.9% NaCl, pH 7.5 (TBS-T and TBS + 500 mM KCl made as above)\n\n''Antibodies''\n\n646 antibody = antibody raised against an internal region of Hem1 (646-659)\n1114 antibody = antibody raised against the c-terminus of Hem1 (1114-1127)\n\n''Procedure''\n\nAll spins at 3000 rpm x 1 min on table centrifuge in cold room (harder spins might crush the porous beads). Spin after each wash.\n\nMake 1 tube of protein A beads, 100 µl packed beads in 500 µl tube \n\nWash protein A beads (each wash is with 400 µl, don’t suck in the beads as the beads will stick to the pipet tip, the washes are instantaneous). \n2x TBS-T (TBS + 0.1% Tween 20)\n2x TBS + 500mM KCl\n1x TBS-T\n\nAdd 100 µl Hem1 (646) antibody to 100 µl TBS-T. Add 200 µl antibody solution to 100 µl of protein A beads. \n\nStick the antibodies to the beads\nIncubate 1hr 4C with inversion (on the nutator), discard sup.\n\nWash off non antibody?\nWash 2x TBS + 500 mM KCl\nWash 2x TBS + 1% NP40\nWash 1x TBS-T\nWash 1x TBS\n\nIncubate 2 ml lysate 1-2hrs at 4C with inversion (keep 60 µl of load – Add 10 µl 6x loading buffer (LB) to 50 µl lysate snap freeze and store in -80 C).\n\nSpin down the tubes and keep 60 µl of supernatant in LB at -80 C\n\nAdd DTT to the Kprex. (Protease inhibitors may be added earlier because they last longer.)\n\nWash off non-specific binding to the antibody\nWash beads 2x in KprexT (0.1% tween 20)\nWash 3x Kprex + 400 mM KCl\nWash 2x Kprex\n\nAfter last wash remove supernatant. Spin again and remove last drop of supernatant. At this step it’s important to keep the molarity of beads to eluting peptide at the right ratio.\n\nElution 1\n\nElute with 400ug/mL 646 peptide in 100 µl Kprex. Add 4.4 µl 1 mg/ml 646 peptide to 106 µl Kprex. Take 100 µl of peptide solution and add to antibody beads.\n\nElute overnight at 4C, keep eluate at 4C for dialysis. Otherwise, eluate may be snap frozen.\n\nWash the beads\nWash beads 2x Kprex\n\nElution 2\nEluted again as above.\n\nWash the beads\nWash beads 2x Kprex\n\nSave 10 µl of beads for analysis in protein gel.\n\nAnalysis\n\nRun a gradient protein gel NuPage 4% - 12% at constant 200 V for 1 hour.\n\nFix in 50% MeOH 7% Acetic acid (Add 70 ml 50% MeOH 12% acetic acid to 50 ml MeOH) for 20 min. Note: Wear lab coat, gloves, and glasses when dealing with acids.\n\nWash 3x in ddH2O for 5 min each wash.\n\nStain in gel code blue up to 1 hour.\n\nDestain in ddH2O.\n\nAnalyze on the Odyssey (password = lance) in the Weissman lab.\n\n
Showed that PH-Akt translocation in latrunculin-treated cells is transient, in contrast to persistent PH-Akt localization in control cells. Showed that PIP3 is important for deadhesion of the uropod by treating neutrophils with intermediate concentrations of PI3K inhibitors (see supplemental movie 4a) [[Bourne 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12080345&query_hl=23&itool=pubmed_docsum]].\n\nShowed that latrunculin treatment block polarization and fMLP and Rac-induced PIP3 accumulation; Rac, but not Cdc42 stimulates PIP3 accumulation; DN Cdc42 does not block active Rac localization; and DN Cdc42 prevents consolidation of a single leading edge [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12551955&query_hl=1&itool=pubmed_docsum]].\n\nShowed that fMLP induces multiple pseudopods (presumably through deactivation of RhoA) in the presence of dominant-negative variants of Gα12 and Gα13 [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887922&query_hl=19&itool=pubmed_docsum]].\n\nShowed that PIP3 (with PI3K inhibitors) stabilizes polarity in two ways: first, by locally enhancing Rac activity to stabilize frontness at the leading edge; and second, by stimulating the activation of Cdc42, which promotes RhoA-dependent backness at the trailing edge, thereby preventing the formation of multiple pseudopods [[Bourne 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16864657&query_hl=1&itool=pubmed_docsum]].\n\nShowed that RhoA activation occurs at the back edge of the cell and at the beginning of the pseudopod with a RhoA FRET biosensor [[Bourne 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16537448&query_hl=6&itool=pubmed_docsum]]\n\nShowed that nocodazole (i) stimulates backness by increasing Rho- and actomyosin-dependent contractility, as reported by Niggli, and also (ii) impairs fMLP-dependent frontness: pseudopods are flatter, contain less F-actin, and show decreased membrane translocation of PH-Akt-GFP, a fluorescent marker for 3'-phosphoinositide lipids [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15860582&query_hl=1&itool=pubmed_docsum]].
Updated by Arthur 8/31/11\n\nPutting together the thesis pdf \n\nI used open office to set margins and spacing. I imported .eps files (an option of illustrator) directly into the open office document and zotero to manage references. I built chapters first and then combined them into a single .odt file. Separately, I built a preliminary pages (those with roman numerals) document which contains abstract, acknowledgement, table of contents, etc. Remember to have two extra preliminary pages (i.e. i and ii) for title page and copyright page. At the end of the manuscript, and an extra page for the library release, add arabic numerals, and print out the library release page. Export these your manuscript (minus the last page - the library release) and preliminary pages (minus the first page - the title page) as PDFs. Print and sign the library release (with the appropriate arabic numeral); print and have signed the title page. Scan each and use automator -> combine pdf function to append PDFs.\n\nWeiner Lab publishing guidelines\n\nOrion pays for all publishing costs, this includes:\n-Two copies hard-bound 8 1/2 x 11 in copies - one for the lab and one for your to keep.\n-Open access, web-searchable, etc.\n-The copyright charge\n\nYou have the option to upload movies and you should!\n-There is a 250 MB limit for all files. Note: Add file description names after all movies have been uploaded. The description resets after you upload each movie.
Showed that the direction of flagellar rotation depends linearly on the amount of CheY bound [[Berg 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9419353&query_hl=52&itool=pubmed_DocSum]].\n\nShowed that assemblies of bacterial chemoreceptors work in a highly cooperative manner, mimicking the behaviour of allosteric proteins [[Berg 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15042093&query_hl=52&itool=pubmed_DocSum]].
See [[Hspc300]].
Hspc300 or Brick1 is the smallest member of the [[WAVE complex]]. Free Brick1 is composed of three homodimers and incorporated into only newly translated [[WAVE complex]] [[Gautreau 2008|http://www.ncbi.nlm.nih.gov/pubmed/18560548?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Cytokine for CXCR2, CCR1. IL8 is produced by stimulated monocytes, endothelial cells, fibroblasts. The Kd of IL8 for its receptor is around 2 nM [[Besemer 1994|http://www.ncbi.nlm.nih.gov/pubmed/8195702?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
The name IQGAP is derived from the next two motifs, the IQ and the Ras ~GTPase-activating protein ([[GAP]])-related domains. The IQ domain is a tandem repeat of four IQ motifs in human IQGAP1, which binds calmodulin, myosin essential light chain and ~S100B. The [[GAP]]-related domain (GRD) mediates the binding of the RhoGTPases, [[Cdc42]] and [[Rac1]], but not RhoA or [[Ras]]. [[Sacks 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12776176&query_hl=63&itool=pubmed_DocSum]]
IQGAP1 is a scaffolding protein that binds to a diverse array of signalling and structural molecules. IQGAP1 participates in multiple cellular functions, including Ca2+/calmodulin signalling, cytoskeletal architecture, [[Cdc42]] and [[Rac]] signalling, E-cadherin-mediated cell–cell adhesion and β-catenin-mediated transcription. [[Sacks 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12776176&query_hl=63&itool=pubmed_DocSum]]. IQGAP1 is a large (350 kDa) effector of Rac1 and Cdc42 required for lamellipodial assembly and uses its calponin homology domain (CHD) to directly bind the EVH1 domain of N-WASP and stimulate Arp2/3-dependent actin assembly [[Goode 2009 review|http://www.ncbi.nlm.nih.gov/pubmed/19168341]].
Insulin receptor substrate or BAIAP. IRSp53 has two other mammalian homologs including IRTKS, which when expressed in Cos-7 cells gives a more "Rac-like" phenotype than IRSp53 [[Machesky 2007|http://www.ncbi.nlm.nih.gov/pubmed/17430976?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] Activated [[Rac]] binds to the amino terminus of IRSp53 (in the IMD actin-bundling domain), and carboxy-terminal Src-homology-3 domain of IRSp53 binds to [[WAVE]] to form a trimolecular complex. Importantly, ~GTP-loaded [[Rac]] on IRSp53–WAVE2 complex does not have any positive/negative effect on the actin polymerization kinetics [[Takenawa 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11130076&query_hl=16&itool=pubmed_DocSum]]. The interaction of [[Cdc42]] with the partial [[CRIB]] motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. IRSp53 could be coprecipitated from cells with [[Cdc42]], but not with [[Rac]]. IRSp53 interacts with [[Mena]] to synergestically induce filpodia, and [[Mena]] when coexpressed with full-length IRSp53 containing two amino acid substitutions (~F428A, ~P429A) in the SH3 domain interacts poorly with [[Mena]], and, when expressed alone, it does not induce filopodia. [[Hall 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11696321&query_hl=16&itool=pubmed_DocSum]]. The IMD domain of IRSp53 is sufficient to induce membrane curvature in liposomes [[Lappalainen 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17371834&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and in cells in a Rac-dependent manner [[Takenawa 2006|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17003044&ordinalpos=14&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. Recombinant WAVE complex can be further activated by IRSp53, but, oddly, is not activated by RacGTPgS [[Takenawa 2006|http://www.ncbi.nlm.nih.gov/pubmed/16702231?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Integrins are cell surface receptors that interact with the extracellular matrix and mediate various intracellular signals. Integrins have a critical role in anchoring cells to extracellular matrices and alter cell function by activating intracellular signaling pathways after ligand binding ("outside-in" signaling). Integrins can shift between high- and low-affinity conformations for ligand binding ("inside-out" signaling) [[Fassler 2009 review|http://www.ncbi.nlm.nih.gov/pubmed/19443776?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. See also [[Harburger 2009 review|http://www.ncbi.nlm.nih.gov/pubmed/19118207?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
See [[Integrin]].
Stimulation of [[Cdc42]] by Intersectin-l accelerates actin assembly via N-WASP and the [[Arp2/3]] complex. N-WASP binds directly to Intersectin-l and upregulates its GEF activity, thereby generating ~GTP-bound [[Cdc42]], a critical activator of N-WASP. [[McPherson 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11584276&query_hl=14&itool=pubmed_docsum]]
JMY combines two nucleating activities by both activating [[Arp2/3]] and assembling filaments directly using a [[Spire]]-like mechanism. Increased levels of JMY expression enhance motility, whereas loss of JMY slows cell migration. When slowly migrating HL-60 cells are differentiated into highly motile neutrophil-like cells, JMY moves from the nucleus to the cytoplasm and is concentrated at the leading edge [[Zuchero and Mullins 2009|http://www.ncbi.nlm.nih.gov/pubmed/19287377?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Showed that endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly via NWASP and moved in the Xenopus egg extract system [[Taunton 2000|http://www.ncbi.nlm.nih.gov/pubmed/10662777]].\n\nA nice review of actin-driven organelle movement [[Taunton 2001|http://www.ncbi.nlm.nih.gov/pubmed/11163138]].\n\nShowed that PIP2 activation of N-WASP-mediated actin polymerization in vitro and in extracts is ultrasensitive: above a certain threshold, N-WASP responds in a switch-like manner to a small increase in the density of PIP2 and that this activation threshold can be tuned by varying the length of the polybasic motif [[Taunton 2005|http://www.ncbi.nlm.nih.gov/pubmed/15664188]].\n\nReconstituted NWASP-based actin assembly on lipid-coated glass beads, using purified soluble proteins [[Taunton 2007|http://www.ncbi.nlm.nih.gov/pubmed/17350575]].\n\n
Jasplakinolide stabilizes actin filaments.
Showed that in goldfish epithelial keratocytes, the actin microfilaments in the lamellipodium remain approximately fixed relative to the substrate as the cell moves over them, regardless of cell speed [[Theriot 1991|http://www.ncbi.nlm.nih.gov/pubmed/2067574]].\n\nShowed that actin polymerization drives Listeria [[Theriot 1992|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1589024&query_hl=15&itool=pubmed_DocSum]].\n\nShowed that keratocyte shape correlated with Ena/Vasp levels and developed mathematical models to show this [[Theriot 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17760506&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
One web definition said:\n\nA fibroblastic stromal cell of the cornea. They produce keratan-sulfate proteoglycans (KSPG) such as lumican and keratocan to form and sustain proper collagen interfibrillar spacing and fibril diameter of the cornea. \n\nThe keratocytes that people in the motility field use are from fish scales, not corneas, and they are highly migratory (moving 0.1-1 microns per second).\n\nObtaining keratocytes:\nPlace fish scales in a dish with media (DMEM and 10% FBS) and the keratocytes will crawl out of the scale into the media.\n\nMorphology:\nKeratocytes are shaped like a semicircle or a scimitar. They are about 10-20um x 50um. They are basically a lamellipod surrounding a tall cell body. The lamellipod is 10 or more microns in length and is about 150-200nm tall.\n\nMotility:\nKeratocytes are spontaneously motile and move at speeds from 0.1-1 microns per second.
Another name for [[Nap1]] and [[Hem-1]].
Includes [[LY294002]], [[Wortmannin]], and [[Y-27632]]. A good review on the specificity of these inhibitors and other kinases inhibitors [[Cohen 2000|http://www.ncbi.nlm.nih.gov/pubmed/10998351?ordinalpos=22&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Developed a method [[Hahn 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11036621&query_hl=19&itool=pubmed_docsum]] for imaging Rac activation with FRET [[Hahn 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11030651&query_hl=21&itool=pubmed_docsum]].\n\nShowed that active Rac is at the leading edge, but also in the retracting tail of motile neutrophils [[Hahn 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12477392&query_hl=21&itool=pubmed_docsum]].\n\nShowed that a genetically encoded photoactivatable Rac could control the motility of living cells [[Hahn 2009|http://www.ncbi.nlm.nih.gov/pubmed/19693014]].
Leukocyte specific plastin. [[L-plastin]] is unique in the fimbrin family because it can become phosphorylated on serine [[Mercurio 1993|http://www.ncbi.nlm.nih.gov/pubmed/8221900?dopt=Abstract]] in the headpiece region in response to fMLP [[Dekker 2004|http://www.ncbi.nlm.nih.gov/pubmed/14556648?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. L-plastin-derived peptides that included the phosphorylation site (Ser-5) rapidly induced leukocyte integrin-mediated adhesion when introduced into the cytosol of freshly isolated primary human PMN and monocytes. Substitution of Ala for Ser-5 abolished the ability of the peptide to induce adhesion [[Brown 1998|http://www.ncbi.nlm.nih.gov/pubmed/9689080?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
LIM kinases-1 and -2 are serine kinases implicated in the regulation of actin cytoskeletal dynamics through their ability to specifically phosphorylate members of the cofilin/actin depolymerizing factor (ADF) family, which inactivate cofilin. The 70-kDa LIM kinases are characterized by the presence of two N-terminal LIM domains followed by a PDZ domain and a C-terminal kinase domain. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
LY294002 is a less potent inhibitor of PI3K than wortmannin. It is an ATP analog, which reversibly attaches to PI3K preventing ATP hydrolysis. Importantly, LY294002 also targets mTOR because the putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases [[Abraham 1996|http://www.ncbi.nlm.nih.gov/pubmed/8895571?dopt=Abstract]]. LY294002 also inhibits [[Casein kinase2]] [[Cohen 2000|http://www.ncbi.nlm.nih.gov/pubmed/10998351]], which has been shown to phosphorylate WAVE2 [[Cory 2009|http://www.ncbi.nlm.nih.gov/pubmed/19012317]]. LY294002 has also been shown to directly bind GSK3 [[Waterfield 2007|http://www.ncbi.nlm.nih.gov/pubmed/17302559]].
The lamella is network behind the lamellipodia, where actomyosin contraction is integrated with substrate adhesion [[Danuser 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15375270]].
The lamellipodium (pl. Lamellipodia) is a cytoskeletal actin projection on the mobile edge of the cell. It contains a two-dimensional actin mesh; the whole structure pulls the cell across a substrate. Within the lamellipodia are ribs of actin called microspikes, which, when they spread beyond the lamellipodium frontier, are called filopodia. A lamellipodium network assembles at the leading edge but completely disassembles within 1 to 3 micrometers. It was weakly coupled to the rest of the cytoskeleton and promoted the random protrusion and retraction of the leading edge [[Danuser 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15375270]].
Latrunculin binds actin monomers near the nucleotide binding cleft with 1:1 stoichiometry and prevents them from polymerizing. WAVE complex can still be recruited and amazingly Rac activation still occurs [[Weiner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16417406&query_hl=43&itool=pubmed_docsum]]. Latrunculin does not prevent asymmetric PIP3 establishment in neutrophilis [[Weiner 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10669415&query_hl=11&itool=pubmed_docsum]], but blocks PIP3 maintenance [[Weiner 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12080345&query_hl=11&itool=pubmed_docsum]].
The front of the cell where the lamellipodia forms and productive protrusion occurs.
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Purified a Rac exchange factor P-Rex1 stimulated by PIP3 and [[Gβγ]] [[Stephens 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11955434&query_hl=31&itool=pubmed_docsum]].\n\nShowed that P-Rex1 is regulated by PIP3 through its PH domain and by [[Gβγ]] through its DH domain [[Stephens 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15545267&query_hl=31&itool=pubmed_docsum]].\n\nShowed that P-Rex1 null mice have defects in superoxide production, but not in chemotaxis [[Stephens 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16243035&query_hl=31&itool=pubmed_docsum]].
lentivirus is usually most infectable right after harvest. virus can be stored at 4 degrees, but the transduction efficiency will be compromised after prolonged periods. //be sure to take proper biosafety precautions when handling virus.//\n\nthe procedure is the same for adherent and suspension cells, with minor variations. aim for ~ 50% confluency for adherent cells, and use 1 million cells for undifferentiated HL-60s in suspension at the time of infection.\n\n//Day 0.//(the day before infection)\n- for adherent cells: seed cells in a 6-well plate to achieve ~ 50-60% confluency the following day. alternatively, you can seed cells in the morning, and infect in the evening.\n\n//Day 1.//(day of infection)\n- for infecting undifferentiated HL-60 cells with some non-perturbing, easy-to-work-with gene, follow the protocol below. if your gene of interest is annoying and problematic, follow the protocol for [[The Bourne Infection]]:\n- prepare cells to achieve 1 million cells/mL in growth media in one well of a 6-well plate.\n- prepare viral supe mixtures in a total of 1 mL. for new constructs, i will typically infect with three volumes of virus-- 1 mL, 0.5 mL, and 0.25 mL (make up the rest with normal growth media) and then assay all three to see which population looks the best. each 6-well of cells will then contain 1 mL of growth media + 1 mL of viral supe:media mixture. allow transduction for 24 h.\n\n//Day 2.//\n- for adherent cells: replace media with fresh growth media. ideally, the adherent cells are still subconfluent.\n- for suspension cells: spin out viral media and replace with fresh growth media.\n\n//Day 3.//\npassage adherent cells or dilute suspension cells and assay for transduction efficiency.\n
lentiviruses are useful for transducing your gene of interest into cells that are normally difficult to transfect. for safety reasons, the HIV genome has been broken up into different vectors. we use these standard vectors with 293T cells to produce virus:\n\npHRSIN (HIV genome + gene of interest)\npCMV8.91 Ex QV (gag, pol, rev, tat)\npMD2.G (VSV-G)\n\n''Protocol''\n//Day 0.//\nplate out 293T cells in a 6-well plate to achieve ~70-80% confluency for transfection the next day. a for starters, 400,000 - 600,000 cells should be in the ballpark.\n\n//Day 1.//\n- check the confluency of the cells. too low or too high will reduce transduction efficiency. \n- for transfection, we use Trans-IT 293 (MirusBio, cat. no. MIR 2704) according to manufacturer's instructions. use a 1:1:1 ratio of DNA. i use 0.5 micrograms of each of the three vectors. (update for 2012: i've been using 1.5 ug transfer vector, 1.3 ug p8.91, and 0.167 ug pMD2.G with 8 uL of transfection reagent).\n- apply transfection mix to cells and let incubate for 2 days.\n\n//note.// from this point on, viral production will have started. take proper precautions and use ''personal protective equipment and biosafety procedures.'' do not open dishes or tubes of virus unless in the tissue culture hood.\n\n//Day 3.//\nthe best time to collect virus is around 2-3 days since: (i) 293T cells remain attached, (ii) this is enough time for the viral particles to reach a decent concentration, and (iii) media is not too acidic to negatively affect the cells to be transduced. \n\n- collect virus into 2 mL tubes and discard the cells (bleach plates for 30 min).\n- remove particulate matter/extraneous cells by centrifugation (full speed for a few min in a tabletop microcentrifuge) or filtration through a 0.45 micron filter. \n- at this point, virus does not need to be concentrated prior to use, but can be done if you choose.\n- use immediately, or store at 4 degrees for up to two weeks. transduction efficiency is reduced after about a week and a half. viral supes can also be frozen, but i am not sure how much the transduction efficiency drops after a freeze/thaw. \n\n\n \n//many thanks to John R. James in the Vale lab for helpful hints and tips for successful lentiviral production.//\n\n\n\n
White blood cells. They comprise part of the immune system and include [[Granulocytes]] (polymorphonuclear leukocytes) and [[Agranulocytes]] (mononuclear leukocytes).
Mass Spec Guru from Havard...check out the T-Rex paper in Science.
Lipid bead Rac translocation 9.28.08\n\nBuffers\n\nVesicle Buffer = 20 mM Hepes (pH ~7.5), 100 mM NaCl, 320 mM sucrose\nKMEI + 20% glycerol + 1 mM DTT = 100 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole\n\nLipid coated beads\n\nTook 10 µl frozen CCS2 (25% cholesterol, 45% PC, 25% PS, and 5% PIP2) and added to 10 µl 1.5 µm beads and 20 µl of vesicle buffer. Sonicate for 30 sec, rest and repeat twice. Rotate at RT for 20 min. Briefly spin using tabletop centrifuge. Wash 1x with 100 µl KMEI + 20% glycerol + 1 mM DTT .\n\nEDTA-mediated Rac Translocation \n\nMake a master mix:\n\n50 µl KMEI buffer\n10 µl 10 µM GTPgS\n10 µl OG lipid bead slurry\n10 µl 100 mM EDTA\n\nNote: Keep covvered and rotating at RT. \n\nAdd 0.5 µl 20µM RacGDI. Add 4 µl master mix to a tube without Rac and one containing Rac. \n\nRotate at RT for 5 min. Add 0.5 µl 130 mM MgCl2. Rotate at RT for 5 min. Add 0.5 – 1 µl of desired concentration of PBD. Rotate at RT for 5 min. Place 4 µl on a coverslip and image.\n\nTrio-mediated Rac Translocation\n\nMake a master mix:\n\n50 µl KMEI buffer\n10 µl 10 µM GTPgS\n10 µl OG lipid bead slurry\n10 µl 43 µM Trio\n\nAdd 0.5 µl 20µM RacGDI. Add 4 µl master mix to a tube without Rac and one containing Rac. \n\nRotate at RT for 1 min. Add 0.5 – 1 µl of desired concentration of PBD (10 µM for a final concentration of 1 µM works best). Rotate at RT for 1 min. Place 4 µl on a coverslip and image.
Lipofectamine 2000 Transfection\n\nArthur Millius 11/27/06\n\nOverview\n\nWe will transfect plasmids into different cell lines for a transient transfection with the reagent lipofectamine 2000.\n\nBuffers\n\nOpti-Mem I Reduced Serum Medium\n\nCell’s Growth Media + 12% FBS\n\nProcedure\n\n1. Plate cells on 8-well lab tek dish for 3 hours at a dilution of ~1:10 or 1:20. After 3 hours remove media and add warmed growth media with 12% serum, but without antibiotics.\n2. Determine the concentration of the plasmid DNA.\n3. Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) of 1:2 or 1:3. We used 1:2.5, which means 0.4 µg of DNA to 1 µl of Lipofectamine.\n4. Add 1 µl of Lipofectamine to 15 µl of Opti-Mem1. Incubate for 5 minutes at room temperature. \n5. Separately, dilute appropriate amount of DNA in 15 µl of Opti-Mem 1.\n6. Combine the diluted DNA with the diluted Lipofectamine and incubate for 20 minutes at room temperature. Note: solution may appear cloudy.\n7. Add 30 µl of solution to each well containing 150 µl of plated cells. Mix cells gently by rocking plate back and forth.\n8. Incubate at 37 ºC for 4-6 hours.\n9. Replace media with lipofectamine-free growth media.\n10. Check for expression the next day.
A theory proposed by Meinhardt and Geier to generate patterns. This pattern formation could be extended to generate polarity in cells [[Meinhardt 1972|http://organic.usc.edu:8376/reference/04Gierer72.pdf]]. An excellent paper summarizes left-right asymmetry in mouse using a reaction-diffusion model [[Hamada 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17011489&query_hl=43&itool=pubmed_docsum]]. Another paper summarizes the range of inhibition in hydra [[Holstein 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11050241&query_hl=46&itool=pubmed_docsum]].
Local stimulation by microfluidics approaches:\n1) 2-micropipette systems: one small pipette injecting liquid, another nearby, larger pipette removing fluid. Can create gradients (probably not as sharp as Charras device), but perhaps with less shear stress applied to cell. Requires double-pipette holder (available from Narishige). [[Popov 1993|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7678471]], [[O'Connell 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11369234]]\n2) PARTCELL: approach from Takayama, Whitesides. Consists PDMS chamber, with 3 inlets, 1 outlet. Stream from inlets flow laminarly, only diffusive mixing between them. Loading of cells far downstream from inlets, seems difficult to change location of stream on fly, usually used with large, slow/non-motile cells. Theoretical calculation of intracellular gradient generated near boundary of locally perfused cell [[Takayama 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12618184]] \n3) Charras device: pipette in conjuction with chamber generating rapid, laminar flow under a bridge where cells are plated. Large volumes of liquid flowing in open-topped chamber, requires care to operate (dangerous on expensive microscopes). Sharp gradient generated, quickly movable. [[Charras 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15902261]] \n4) Irimia device – Series of small tube chambers through which cells migrate, cells occlude chambers fully to seal them. Drug/agonist can be locally applied to either front/back of cells. Good for getting repeatable population measurements, b/c cells adopt morphology of the channel, which may aid in quantitative modeling. [[Irimia 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18030401]]\n\n
Showed that expression of [[Arno]] in [[MDCK]] cells led to activation of [[Arf6]] and [[Rac1]], which resulted the formation of lamellipodia. Arno-induced activation of [[Arf6]] also results in increased activation of phospholipase D ([[PLD]]), and inhibition of [[PLD]] activity also inhibits motility without inhibiting [[Rac1]] activation [[Santy 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11481345&query_hl=7&itool=pubmed_docsum]].\n\nShowed that Arno-dependent activation of [[Rac]] is mediated by a bipartite Rac GEF, the [[DOCK180]]-[[Elmo]] complex. [[DOCK180]] and [[Elmo]] colocalize extensively with [[Arno]] in migrating [[MDCK]] cells and a catalytically inactive mutant of [[DOCK180]] prevents [[Arno]]-induced [[Rac]] activation [[Santy 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16213822&query_hl=7&itool=pubmed_docsum]].
A lymphocyte is a type of white blood cell involved in the vertebrate immune system. There are two broad categories of lymphocytes, namely the large granular lymphocytes and the small lymphocytes. The large granular lymphocytes are more commonly known as the natural killer cells (NK cells). The small lymphocytes are the T cells and B cells.
Madin Darby Canine Kidney epithelial cells.
Mouse embryonic fibroblasts
Myosin is activated by phosphorylation on it regulatory light chain by [[MLCK]].
The phosphorylation of the regulatory myosin light chain ([[MLC]]) at Ser19 (and Thr18) by Ca2+-dependent myosin light chain kinase (MLCK) has been shown to be a critical regulatory step for physiological modulation of myosin contractility. Phosphorylation of MLCK by [[Pak1]] in vitro decreases MLCK activity toward [[MLC]] by more than 50%. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
Rat mammary adenocarcinoma cell line. These cells grow easily in tissue culture and, and form first a primary tumor and then lymph node and lung metastases, when injected into rats.
Another name for [[Myosin phosphatase]].
Macrophages (Greek: "big eaters", makros = large, phagein = eat) are cells within the tissues that originate from specific white blood cells called monocytes. Monocytes and macrophages are phagocytes, acting in both nonspecific defence (or innate immunity) as well as specific defence (or cell-mediated immunity) of vertebrate animals. Their role is to phagocytize (engulf and then digest) cellular debris and pathogens either as stationary or mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.
[[Proteins]]\n[[Drugs]]\n[[Protocols]]\n[[Vocabulary]]\n[[People]]\n[[Cell Lines]]
Reconstituted Cdc42-induced actin polymerization in Xenopus egg extracts and purified the [[Arp2/3]] complex from this assay [[Kirschner 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9860974&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that full-length N-WASP is less effective at initiating actin assembly, its activity can be greatly enhanced by Cdc42 and PIP2 [[Kirschner 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10219243&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells [[Kirschner 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10898977&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that the amino and carboxy termini interactin of N-WASP must be intramolecular because in solution N-WASP is a monomer [[Kirschner 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10995436&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that [[Nck]] SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of [[Arp2/3]] in vitro. [[Arp2/3]] is further stimulated by PIP2, but not by GTP-Cdc42, suggesting that [[Nck]] and [[Cdc42]] activate N-WASP by redundant mechanisms [[Kirschner 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11340081&query_hl=63&itool=pubmed_DocSum]].\n\nShowed (but not reproducibly) that GTP-[[Rac1]] and [[Nck]] cause dissociation of the [[WAVE1]] complex, which releases active [[WAVE1]]-[[Hspc300]] and leads to actin nucleation [[Kirschner 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12181570&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization [[Kirschner 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12853475&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that [[Sra]], [[Nap]], [[WAVE2]], [[Abi]], and [[Hspc]] co-purify with [[WAVE2]]. The complex is organized around a core of [[Nap]] and [[Abi]]. [[Sra]] is a peripheral subunit recruited on the [[Nap]] side, whereas the [[WAVE]] and [[Hspc]] subunits are recruited on the [[Abi]] side of the core [[Kirschner 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15070726&query_hl=63&itool=pubmed_DocSum]].\n\nShowed that [[Toca-1]] binds both N-WASP and [[Cdc42]] and is a member of the evolutionarily conserved PCH protein family. [[Toca-1]] promotes actin nucleation by activating the N-WASP-WIP complex [[Kirschner 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260990&query_hl=63&itool=pubmed_DocSum]].\n\nTwo papers on Cdc42-mediated actin assembly in vitro [[Kirschner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16472657&query_hl=63&itool=pubmed_DocSum]] and [[Kirschner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16472657&query_hl=63&itool=pubmed_DocSum]].
Showed that [[Rho]] physically interacts with and stimulates PIP5K [[Schwartz 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8868471&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that DN Rac and DN Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate [[Cdc42]], which leads to the subsequent activation of [[Rac]] [[Schwartz 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9658176&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that suspended cells have higher [[Rho]] activity than adherent cells in the presence of serum indicating the existence of an adhesion-dependent negative-feedback loop [[Schwartz 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9927417&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that an activated [[Rac]] mutant lacking a membrane-targeting sequence did not activate [[Pak]] in adherent cells, while mutations that forced membrane targeting restored [[Pak]] activation in suspended cells [[Schwartz 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10790367&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that [[Rac]] recruits integrins to the lamellipodia in endothelial cell migration [[Schwartz 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11231584&query_hl=3&itool=pubmed_DocSum]]. \n\nReview on integrins [[Schwartz 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11719050&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that the translocation of GTP Rac to membranes is independent of effector interactions, but instead requires the polybasic sequence near the carboxyl terminus [[Schwartz 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11862216&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that equibiaxial stretch inhibited lamellipodia formation through deactivation of [[Rac]]. Showed that treatment with [[Y-27632]] or [[ML-7]] that inhibits myosin phosphorylation and contractility increased lamellipodia through [[Rac]] activation and decreased cell polarization [[Schwartz 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12105187&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that zizimin-1 is a new GEF for [[Cdc42]] [[Schwartz 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12172552&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that disruption of dynamin(-2) function alters [[Rac]] localization and inhibits cell spreading and lamellipodia formation even though [[Rac]] is activated [[Schwartz 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14617821&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed that active Rac1 binds preferentially to low-density, cholesterol-rich membranes. Showed that cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells [[Schwartz 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14764880&query_hl=3&itool=pubmed_DocSum]]. \n\nShowed Rac membrane dynamics with photobleaching [[Schwartz 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16597700&query_hl=3&itool=pubmed_DocSum]].\n\nShowed that RhoG depletion did not substantially inhibit cell adhesion, spreading, migration or Rac activation [[Schwartz 2008|http://www.ncbi.nlm.nih.gov/pubmed/18505794?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Membrane Hem1-WAVE complex immunoprecipitation\n\nArthur Millius 10/05/06\n\nOverview\n\nTo immunopurify Hem1 complexes from Pig leukocyte lysate and membrane. Immunoprecipitates will be compared to determine differences in Hem1-associated proteins at the membrane and in the cytosol. Maybe also be an input into permeabilized cells by adding active WAVE complex IPed from LatB treated membrane fractions.\n\nBuffers\n\nKprex = 140mM KCl, 1mM MgCl2, 1mM EGTA, 20mM Hepes pH 7.5, 1mM DTT, 1 EDTA-free protease inhibitor table per 50 ml (Make 50 ml; add protease inhibitors and DTT just prior to use).\n\nKprex + 400 mM KCl = 9mL Kprex + 3mL 1M KCl for Kprex 400 mM KCl (mild ionic wash)\n\nKprex-T = 10 mL Kprex + 0.1% Tween-20 (mild hydrophobic wash)\n\nTBS = 50 mM Tris, 0.9% NaCl, pH 7.5 (TBS-T and TBS + 500 mM KCl made as above)\n\nAntibodies\n\n646 antibody = antibody raised against an internal region of Hem1 (646-659)\n1114 antibody = antibody raised against the c-terminus of Hem1 (1114-1127)\ncrude = contains a mixture of antibodies\n\nProcedure\n\nAll spins at 3000 rpm x 1 min on table centrifuge in cold room (harder spins might crush the porous beads). Spin after each wash.\n\nMake 1 tube of protein A beads, 100 µl packed beads in 500 µl tube \n\nWash protein A beads (each wash is with 400 µl, don’t suck in the beads as the beads will stick to the pipet tip, the washes are instantaneous). \n2x TBS-T (TBS + 0.1% Tween 20)\n2x TBS + 500mM KCl\n1x TBS-T\n\nAdd 100 µl Hem1 (646) antibody to 100 µl TBS-T. Add 200 µl antibody solution to 100 µl of protein A beads. \n\nStick the antibodies to the beads\nIncubate 1hr 4C with inversion (on the nutator), discard sup.\n\nWash off non antibody\nWash 2x TBS + 500 mM KCl\nWash 2x TBS + 1% NP40\nWash 1x TBS-T\nWash 1x TBS\n\nIncubate 2 ml lysate 1-2hrs at 4C with inversion (keep 60 µl of load – Add 10 µl 6x loading buffer (LB) to 50 µl lysate snap freeze and store in -80 C).\n\nSpin down the tubes and keep 60 µl of supernatant in LB at -80 C\n\nAdd DTT to the Kprex. (Protease inhibitors may be added earlier because they last longer.)\n\nWash off non-specific binding to the antibody\nWash beads 2x in KprexT (0.1% tween 20)\nWash 3x Kprex + 400 mM KCl\nWash 2x Kprex\n\nAfter last wash remove supernatant. Spin again and remove last drop of supernatant. At this step it’s important to keep the molarity of beads to eluting peptide at the right ratio.\n\nElution 1\n\nElute with 400ug/mL 646 peptide in 100 µl Kprex. Add 4.4 µl 1 mg/ml 646 peptide to 106 µl Kprex. Take 100 µl of peptide solution and add to antibody beads.\n\nElute overnight at 4C, keep eluate at 4C for dialysis. Otherwise, eluate may be snap frozen.\n\nWash the beads\nWash beads 2x Kprex\n\nElution 2\nEluted again as above.\n\nWash the beads\nWash beads 2x Kprex\n\nSave 10 µl of beads for analysis in protein gel.\n\nAnalysis\n\nRun a gradient protein gel NuPage 4% - 12% at constant 200 V for 50 minutes.\n\nFix in 50% MeOH 7% Acetic acid (Add 70 ml 50% MeOH 12% acetic acid to 50 ml MeOH) for 20 min. Note: Wear lab coat, gloves, and glasses when dealing with acids.\n\nWash 3x in ddH2O for 5 min each wash.\n\nStain in gel code blue up to 1 hour.\n\nDestain in ddH2O.\n\nAnalyze on the Odyssey in the Weissman lab.
Membrane translocation of Rac\nArthur Millius (2/18/07)\n\nOverview\nTo induce Rac to translocate from the cytosol to the plasma membrane. May also be used to check whether Rac(GTPgS)RhoGDI is appropriately charged.\n\nBuffers\n\nmHBSS (Ca free) + 0.2% albumin = 150mM NaCl, 4mM KCl, 1mM MgCl2, 10mM glucose, 20mM Hepes, pH 7.2, 2 g/L endotoxin-free albumin\n\nSonication buffer + sucrose = 100 mM KCl, 50 mM Hepes, 2 mM EGTA, 2 mM MgC l2, 250 mM sucrose, 1 mM DTT, 0.1 mM PMSF, 3 mM DFP, 1 EDTA-free protease inhibitor tablet (add protease inhibitors just before use)\n\nKprex + 10% glycerol = Kprex + 10% glycerol = 140 mM KCl, 1 mM MgCl2, 1 mM EGTA, 20 mM Hepes pH 7.5, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 EDTA-free protease inhibitor tablet (sterile filter). (add protease inhibitors just before use)\n\nSucrose Gradient buffer = 50 mM Hepes, 1 mM EGTA, 1 mM MgCl2, 1 mM ATP, 60%, 34%, and 15% sucrose, 1 mM DTT, 2 mM PMSF, , 1 EDTA-free protease inhibitor tablet per 50 ml (250 ml, add ATP and proteases before use, store @ -4)\n\nProcedure\n\nCollect 5 day differentiated cells in 500 ml corning centrifuge tube. Spin at 1500 x g at room temperature for 10 minutes. Remove supernatant and resuspend in 50 ml mHBSS (Ca free). Spin at 400 x g at room temperature for 10 minutes.\n\nAdd protease inhibitors to sonication buffer. Add 3 mM DFP (2 µl 5 M DFP in 3 ml) to sonication buffer under hood. Caution: DFP is extremely toxic! 300 µl on your skin is lethal!!!!! Keep stocks in 100 µl aliquots, wear gloves, a coat, and safety glasses. Have antidote nearby. Tell someone that you are working with DFP before beginning this step. Put used DFP tips in at least 2 N NaOH to neutralize DFP before discarding. DFP is dead after 90 minutes.\n\nResuspend cells in cold 3 ml sonication buffer. Do all remaining steps on ice, unless indicated.\n\nSonicate cells 3 x 10 secs at 20% power. Ice 10 – 15 secs between bursts. Note: I sonicated a trial sample of cells until 1% of them remaining.\n\nTransfer to (2) 1 ml tubes and spin 900 x g for 5 min at 4 C to remove cell debris. Remove 400 µl and label “low speed supernatant (LSS)”.\n\nFor remaining LSS, layer on top of 15/34/60 sucrose gradient in the SW 40 compatible tubes. Add 1 mM DTT, 1 mM PMSF, and 1 protease tablet to sucrose gradient buffer. Spin for 15 min at 38,000 rpm.\n\nCollect cytosol (~1 ml). Collect ~1 ml light membrane interface (cytosol/15). Remove plasma membrane (~1 ml). Concentrate plasma membrane by spinning at 95,000 rpm in TLA 120.2 at 4 C for 45 min. Note: I forgot to dilute 1:1 in (2) 1 ml centrifuge tubes. Remove supernatant. Wash once in Kprex + 10 glycerol. Resuspend in 300 µl Kprex + 10% glycerol.\n\nMeasure the amount of protein in the cytosol, plasma membrane, light membrane, and low speed supernatant. Want a cytosol to membrane ratio of 1:3.\n\nMix in the following ratios (x µl):\n\nCondition Membrane Cytosol RacGTP or RacGDP Kprex + 10% glycerol 500 mM EDTA 1 mM GTPgS\nPlasma membrane \n-GTPgS, -EDTA 40 8 0 40 0 0\n+GTPgS, -EDTA 40 8 0 40 0 8.8\n+GTPgS, +EDTA 40 8 0 40 1.6 9.1\nLight membrane \n-GTPgS, -EDTA 32 16 0 32 0 0\n+GTPgS, -EDTA 32 16 0 32 0 8.8\n+GTPgS, +EDTA 32 16 0 32 1.6 9.1\nLSS \n-GTPgS, -EDTA 0 32 0 48 0 0\n+GTPgS, -EDTA 0 32 0 48 0 8.8\n+GTPgS, +EDTA 0 32 0 48 1.6 9.1\nRacGTPgSRhoGDI (PM) \n-GTPgS, -EDTA 32 0 5 45 0 0\n+GTPgS, -EDTA 32 0 5 45 0 9.1\n+GTPgS, +EDTA 32 0 5 45 1.6 9.3\nRacGTPgSRhoGDI (LM) \n-GTPgS, -EDTA 32 0 5 50 0 0\n+GTPgS, -EDTA 32 0 5 50 0 9.1\n+GTPgS, +EDTA 32 0 2 50 0 9.3\nRacGDP (PM) \n+GTPgS, -EDTA 32 0 5 45 0 9.7\n+GTPgS, +EDTA 32 0 5 45 1.6 9.8\nRacGDP (LM) \n-GTPgS, -EDTA 32 0 5 50 0 9.7\n+GTPgS, -EDTA 32 0 5 50 0 9.7\n+GTPgS, +EDTA 32 0 5 50 1.6 9.8\n\nIncubate the following reactions for 15 min at 30 C. Spin for 30 min at 95,000 rpm in TLA 100. Remove 50 µl of supernatant for “cytosol”. Remove remaining cytosol and wash once in Kprex + 10% glycerol. Resuspend in 50 µl Kprex + 10% glycerol. Spin for 30 min at 95,000 rpm in TLA 100. Resuspend in 50 µl Kprex + 10% glycerol for “membrane”. Add 10 µl 6x LB and run on gel. Transfer to western, block for 30 min, and incubate for one hour with a 1/2000 Rac or GDI antibody at room temperature.\n\nGel 1 – Cytosol from the following reactions (blot anti-Rac1)\nWell Membrane RacGTP or RacGDP 500 mM EDTA 1 mM GTPgS\n1 Molecular weight\n2 PM Cytosol No No\n3 PM Cytosol No Yes\n4 PM Cytosol Yes Yes\n5 PM GTP No No\n6 PM GTP No Yes\n7 PM GTP Yes Yes\n8 PM GDP No Yes\n9 PM GDP Yes Yes\n10 LM Cytosol No No\n11 LM Cytosol No Yes\n12 LM Cytosol Yes Yes\n13 LM GTP No No\n14 LM GTP No Yes\n15 LM GTP Yes Yes\n16 LM GDP No Yes\n17 LM GDP Yes Yes\n\nGel 2 – Membrane from the following reactions (blot anti-Rac1)\nWell Membrane Cytosol or RacGTP or RacGDP 500 mM EDTA 1 mM GTPgS\n1 Molecular weight\n2 PM Cytosol No No\n3 PM Cytosol No Yes\n4 PM Cytosol Yes Yes\n5 PM GTP No No\n6 PM GTP No Yes\n7 PM GTP Yes Yes\n8 PM GDP No Yes\n9 PM GDP Yes Yes\n10 LM Cytosol No No\n11 LM Cytosol No Yes\n12 LM Cytosol Yes Yes\n13 LM GTP No No\n14 LM GTP No Yes\n15 LM GTP Yes Yes\n16 LM GDP No Yes\n17 LM GDP Yes Yes\n\nGel 3 – First half blotted for Rac1, second half for GDI\nWell Membrane Cytosol or RacGTP or RacGDP 500 mM EDTA 1 mM GTPgS\n1 Molecular weight (Rac1)\n2 LSS (cytosol) - No No\n3 LSS (cytosol) - No Yes\n4 LSS (cytosol) - Yes Yes\n5 LSS (membrane) - No No\n6 LSS (membrane) - No Yes\n7 LSS (membrane) - Yes Yes\n8 LSS (cytosol) - Yes No\n9 LSS (membrane) - Yes No\n10 Molecular Weight (GDI)\n11 PM (cytosol) Cytosol No No\n12 PM (cytosol) Cytosol No Yes\n13 PM (cytosol) Cytosol Yes Yes\n14 PM (membrane) Cytosol No No\n15 PM (membrane) Cytosol No Yes\n16 PM (membrane) Cytosol Yes Yes\n17 LM GDP Yes No
See also [[VASP]]. [[Cdc42]] binds to IRSp53 and stimulates filopodium formation through Mena [[Hall 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11696321&query_hl=24&itool=pubmed_docsum]].
Merlin is homologous to the members of the ERM (ezrin, radixin, moesin) family of cell membrane–cytoskeletal linker proteins and localizes to cortical actin structures. The activity of Merlin is suppressed by [[Rac]] and [[Cdc42]] through phosphorylation at Ser518 mediated by [[Pak]], perhaps specifically by [[Pak2]]. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
Methyl-ß-cyclodextrin disrupts lipid rafts by depleting the membrane of cholesterol. Methyl-ß-cyclodextrin inhibits Rac from translocating to the membrane and prevents Pak1 activation. Trapping lipid rafts on beads (with cholera toxin B antibody, which binds GM1-containing domains, a component of lipid raft proteins) in cells that have been suspended causes retention of Pak1 activity [[Schwartz 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14764880&query_hl=34&itool=pubmed_docsum]].
Showed that CA Cdc42 and CA Rac1 can activate actin assembly in permeabilized cells. DN Cdc42 inhibits actin assembly stimulated by Cdc42, but DN Cdc42 does not inhibit [[Rac]] mediated actin assembly. fMLP stimulated actin assembly can be inhibited by GDPbS [[Glogauer 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10953003&query_hl=22&itool=pubmed_DocSum]].\n\nShowed that Rac1-deficient neutrophils have profound defects in inflammatory recruitment in vivo, migration to chemotactic stimuli, and chemoattractant-mediated actin assembly, but unlike [[Rac2]] null neutrophils have normal superoxide production [[Glogauer 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12759446&query_hl=22&itool=pubmed_DocSum]].\n\nShowed that [[Rac1]] null neutrophils have a defect in chemotaxis, while [[Rac2]] null neutrophils have a defect in chemokinesis. Showed that [[Cdc42]] activation is abrogated in [[Rac2]] null neutrophils, but not [[Rac1]] null neutrophils. Showed that PIP3 localization is disrupted in [[Rac1]] null neutrophils, but not in [[Rac2]] null neutrophils [[Glogauer 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15308574&query_hl=22&itool=pubmed_DocSum]].\n\nShowed that [[Rac1]] null neutrophils have a defect in [[Rho]] activation [[Glogauer 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16809619&query_hl=22&itool=pubmed_DocSum]].\n\nShowed that [[Rac2]] null neutrophils have a more severe defect than [[Rac1]] null neutrophils in actin nucleation. Showed that [[Rac1]] null neutrophils has a defect in capping protein release indicating that [[Rac1]] plays a role in uncapping. Showed that [[Rac2]] null neutrophils have dfect in cofilin dephosphorylation [[Glogauer 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17954607&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Showed that [[Dock2]] is expressed specifically in leukocytes, while [[Dock180]] is expressed ubiquitously with an exception in leukocytes [[Matsuda 1999| http://www.ncbi.nlm.nih.gov/pubmed/10559471?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n\nShowed that after addition of NGF, [[Rac1]] and [[Cdc42]] were transiently activated in broad areas of the cell periphery in [[PC12]] cells and then more localized to protruding neurite tips. Also, showed that Rac1-V12 and Cdc42-V12 produced membrane ruffles and microspikes, respectively [[Matsuda 2004|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14570905&ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that [[Vav]] KD prevents [[Rac1]] and [[Cdc42]] accumulation at neurite tips and sustained PIP3 accumulation (although there is an initial burst of PIP3), whereas KD of [[SOS]] has no effect. Both [[Vav]] and [[SOS]] KD prevent neurite outgrowth [[Matsuda 2005|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15728722&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nDeveloped a FRET probe for PIP3 based on the Akt PH domain and for PI(3,4)P based on the TAPP1 PH domain [[Matsuda 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17079732&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. \n\nSee also [[Umezawa 2003|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14528311&ordinalpos=19&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] for a PIP3 probe based on the GRP PH domain.\n\nShowed that [[Rac1]] can produce PIP3, but only in the presence of NGF using Tobias' inducible system and [[Rac]] has a sustained effect on PI(3,4)P2 levels. Showed that PTEN and SHIP KD have an effect on PIP2 (increase for PTEN, decrease for SHIP) levels and modeled this with [[Rac]] promoting [[SHIP]] activation. Quantified the amount of protein in a cell using eGFP microspheres as a standard. [[Matsuda 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17535963&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that [[Dock180]] binds sorting nexin5 and localizes to endosomes where it's thought to mediate endosome to Golgi retrograde transport [[Matsuda 2008|http://www.ncbi.nlm.nih.gov/pubmed/18596235?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that knockdown of the [[Rac]] GEF [[Asef]] blocks EGF induced ruffle formation and [[Rac]] activation through the Raichu biosensor. Knockdown also blocks [[Cdc42]] activation and phosphorylated [[Asef]] localizes to the lamellipodia in A431 cells [[Matsuda 2008|http://www.ncbi.nlm.nih.gov/pubmed/18653540?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n\nShowed that human Mena associates with Rac1 small GTPase in glioblastoma cell lines [[Matsuda 2008|http://www.ncbi.nlm.nih.gov/pubmed/19277120]].\n\nShowed that PAK1 acquires an intermediate semi-open conformational state upon recruitment to the plasma membrane, this intermediate PAK1 state is hypersensitive to stimulation by Cdc42 and Rac1, and PIX proteins contribute to PAK1 stimulation at membrane protrusions, in a GTPase-independent way [[Matsuda 2009|http://www.ncbi.nlm.nih.gov/pubmed/19574218]].
Materials:\n1. Glass capillary with filament (TW100-4, World Precision Instruments) (see Note 4).\n2. Alexa Fluor 594 hydrazide (A-10438, Invitrogen) 1 mg sodium salt dissolved in 132 µl DMSO to give a 10 mM working stock; store at 4°C and protect from light.\n3. Chemoattractant solution: prepare 200 nM fMLP and 10 µM Alexa594 in RPMI culture media; protect from light.\n\nMethods:\n1. Pull glass filaments on Sutter Model P-87 (Program: heat = 750, pull = 0, velocity = 20, time = 250, pressure = 100, loops 2 or 3 times) to achieve ~2-3 µm needle diameter.\n2. Backfill needles with chemoattractant solution and connect to a needle holder (MINJ4, Tritech). Flick to remove air bubbles at the tip. Needle holder is held by a micromanipulator (MM-89, Narishige) and agonist flow controlled by adjusting balance pressure between 0-3 psi on an IM-300 injection system (Narishige) (see Note 16).\n3. Place cells seeded on a coverslip on microscope and find focus of cells in brightfield.\n4. Orient needle in light path and switch to the back focal plane of the objective (usually labeled “B” for Bertrand lens). Find the needle and lower it until just above the cells, then switch back to the normal focal plane for viewing.\n5. Image dye in fluorescence or Total Internal Reflection Fluorescence (Nikon Eclipse TE2000-E inverted microscope with a 60x, PlanApo TIRF, 1.49 NA objective) microscopy using appropriate filter sets (Chroma) with ~20 ms exposures and near maximal multiplication on an EM-CCD camera (Cascade II 512, Photometrics) (see Note 17).
A nice review of microscopy as applied to cell migration with an Dicty as an example [[Weijer 2006|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16900100&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Microtubule disassembly does not impair receptor-linked chemotaxis, instead it induces development of polarity and chemokinesis in neutrophils concomitant with polarized distribution of [[alpha-actinin]] and F-actin. [[Colchicine]]-induced development of polarity was insensitive to treatment with [[Pertussis toxin]] and [[Wortmannin]], but is inhibited by the Rho kinase inhibitor [[Y-27632]] [[Niggli 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12571279]].\n\n[[Nocodazole]] (i) stimulates backness by increasing Rho- and actomyosin-dependent contractility, as reported by Niggli, and also (ii) impairs fMLP-dependent frontness: pseudopods are flatter, contain less F-actin, and show decreased membrane translocation of ~PH-Akt-GFP, a fluorescent marker for 3'-phosphoinositide lipids [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15860582&query_hl=1&itool=pubmed_docsum]].\n\nImportantly, cytokineoplasts, which lack microtubules altogether, can still polarize and migrate [[Malawista 1982|http://www.ncbi.nlm.nih.gov/pubmed/6891383?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Monocytes have two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (approx. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response.
See [[Monocyte]]
Myosins are actin-activated Mg-ATPases that convert the energy of ATP hydrolysis into force between actin and myosin filaments, leading to either contraction or tension. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
Myosin I functions as monomer in vesicle transport. The ability of yeast extracts to nucleate actin assembly in a permeabilized cell system requires myosin I ATPase activity; dense clusters of endosomes colocalized with myosin Iα and actin, especially at the cell's leading edge [[Taunton 2001 review|http://www.ncbi.nlm.nih.gov/pubmed/11163138]].
Myosin II is a motor protein for actin consisting of a heavy and light chain. It's activated by phosphorylation on its regulatory light chain ([[MLC]]). Several kinases phosphorylated MLC including [[MLCK]], [[ROCK]]/ROK/Rho-kinase, [[Pak]] (p21-activated kinase), citron kinase, ILK (integrin-linked kinase), MRCK (myotonic dystrophy protein kinase-related, cdc42-binding kinase) and ~DAPKs (death-associated protein kinases including ZIPK). Myosin II is activated by phosphorylation of its regulatory light chain (MLC) at Ser19/Thr18 [8]. The major phosphorylation site is Ser19, which allows myosin II to interact with actin, [[Matsumura 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15935670&dopt=Abstract]]
Myosin IIA negatively regulates cell migration. Ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which is dependent on the small G-protein [[Rac1]], its activator [[Tiam1]] and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles [[Yamada 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17310241&query_hl=3&itool=pubmed_docsum]]. Depletion of myosin-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period [[Sheetz 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16920834&query_hl=6&itool=pubmed_docsum]].
[[Pak1]] regulates the phosphorylation and subcellular localization of Myosin IIB [[Ravid 2005|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15993754&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
The activity of myosin phosphatase is inhibited by phosphorylation at Thr695 by kinases including [[ROCK]], ZIP kinase, ILK, [[Pak]] and myotonic dystrophy protein kinase and its related kinases (myotonic dystrophy protein kinase-related, ~RhoA-binding kinase and MRCK). In addition, [[ROCK]] phosphorylates MYPT1 at Thr850, which induces myosin dissociation from myosin phosphatase. [[Matsumura 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15935670&dopt=Abstract]]. RhoGAP4 is the regulatory subunit of mysoin phophatase.
Myosin VI is a pointed-end actin based motor [[Sweeney 1999|http://www.ncbi.nlm.nih.gov/pubmed/10519557]]. Mutation is responsible for a form of deafness in mice [[Jenkins 1995|http://www.ncbi.nlm.nih.gov/pubmed/7493015]] and humans [[Gasparini 2001|http://www.ncbi.nlm.nih.gov/pubmed/11468689]]. MyosinVI walks in a hand over hand mechanism taking 72 nm steps [[Spudich 2004|http://www.ncbi.nlm.nih.gov/pubmed/15286724]] mediated by a flexible tail [[Sweeney 2005|http://www.ncbi.nlm.nih.gov/pubmed/15721263]], but also switches to smaller step depending on ADP concentration [[Yanagida 2010|http://www.ncbi.nlm.nih.gov/pubmed/20850010]]. \n\nOptineurin links MyosinVI to the golgi [[Buss 2005|http://www.ncbi.nlm.nih.gov/pubmed/15837803]] and MyosinVI can bind liposomes via PIP2 [[Kendrick-Jones 2007|http://www.ncbi.nlm.nih.gov/pubmed/17187061]]. Inhibition of myosin VI activity by small interfering RNA (siRNA)-mediated knockdown or by overexpression of dominant-negative myosin VI tail leads to a delay in metaphase progression and a defect in cytokinesis [[Buss 2007|http://www.ncbi.nlm.nih.gov/pubmed/17881731]]. Coupled motors can walk on an extracted keratocyte network [[Spudich 2009|http://www.ncbi.nlm.nih.gov/pubmed/19786577]]. \n\nDepletion of MyosinVI inhibits fly border cell migration [[Montell 2002|http://www.ncbi.nlm.nih.gov/pubmed/12134162]] and is required for the accumulation cortactin, arp2/3 complex and actin structures during spermatogenesis [[Miller 2002|http://www.ncbi.nlm.nih.gov/pubmed/12432073]]. Down-regulation of myosin VI by siRNA significantly reduced the migratory activity of leukemic cells [[Entschladen 2010|http://www.ncbi.nlm.nih.gov/pubmed/20493527]]. In migratory cells ablation of myosin VI or optineurin inhibits the polarized delivery of the epidermal growth factor receptor (EGFR) into the leading edge and leads to profound defects in lamellipodia formation. Depletion of either myosin VI or optineurin, however, does not impair the overall ability of cells to migrate in a random migration assay, but it dramatically reduces directed migration towards a growth factor stimulus [[Buss 2010|http://www.ncbi.nlm.nih.gov/pubmed/20604900]].
Neuronal WASP expressed ubiquitously. N-WASP contains a WH1 domain, [[CRIB]] domain, a basic domain, and a [[VCA]] domain. [[Cdc42]] activates N-WASP through [[Toca-1]]. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. ~N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia [[Alt 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11584271&query_hl=40&itool=pubmed_docsum]]. [[Rac1]] is a significantly more potent N-WASP activator than [[Cdc42]], but has no effects on WASP [[Sakowicz 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17302440&query_hl=4&itool=pubmed_DocSum]].
Neuronal WASP, originally thought to be regulated similarly to WASP. However, Sackowicz showed that Rac1 may slightly activate N-WASP [[Sackowicz 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17302440&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
See [[N-WASP]]
~Na-H(+) exchanger 1 (NHE1) is reported to be necessary at the front of migrating cells for polarity and directional motility [[Barber 2007|http://www.ncbi.nlm.nih.gov/pubmed/17984318?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
NWASP fused to GFP.
NanoEnabler Protocol\n\nPreparation.\n\nBring DI water, pipet and tips, solution to be stamped, and silanized slides.\n\nMix PSB solution and your solution (streptavidin) in a one to one mixture. (Usually 1 µl of each).\n\nTo handle the SPT wafer use SPT forceps. Check that the cantilever has not broken from the SPT wafer under the dissecting scope.\n\nTo remover current SPT wafer, hold sides of wafer, which extends from the edge, with forceps and lift carefully.\n\nPlace SPT wafer in UV cleaner to clean.\n\nRemove currect sticky with metal forceps. Place a piece of sticky tape on holder.\n\nPlace SPT wafer a 1/3 the way extending on holder.\n\nTurn on both computer and enabler by the power switch on the unit in the lower right. Fill humidifier to line on outside of cup with DI water. Remember to leave empty after each use.\n\nLoad SPT wafer with ~0.5 µl of solution. Bring pipet vertically to reservoir and expel liquid. Do not touch cantilever. DO NOT use a kim wipe to suck up extra liquid. Fiber could clog cantilever channel.\n\nLoad holder upside down with cantilever facing toward you. The holder should align under Enabler with magnets holding the holder.\n\nTake out sample holder from stage. Use large tab (less sticky) to hold sample to stage. Best to not use too many sticky tabs or else impossible to remove slider from stage holder.\n\nManipulating the Enabler.\n\nTurn up illumination.\n\nUse fast focus control and zoom control to find cantilever. Use two thumbscrews on left to find cantilever. Start with the grey knob.\n\nTip of SPT should be in the center. Zoom in and focus. If you can see the channel of the cantilever clearly than it is blocked. Rinse SPT wafer with DI water and reapply 0.5 µl of solution. Or, can front load cantilever. (To front load, add a drop of liquid to sample on stage and push cantilever into droplet using computer controls).\n\nFocus on SPT and set SPT focus by pushing “Set” button under SPT focus for focus presets.\n\nZoom all the way out.\n\nPosition laser on cantilever (just behind narrow tip) by adjusting two thumbscrews on left side of Enabler. Turn laser intensity up by moving laser slider. Ideally, want to sum between 8 and 9. If it reaches 10, then it is maxed out on intensity. When you adjust height of wafer and try to test drop, laser has a tendency to change intensity. Adjust intensity accordingly.\n\nAdjust the difference on the detector to between ±0.5. This difference also change rapidly with enabler height.\n\nMove head down using coarse z to 2 – 3 mm above the surface.\n\nFocus on surface with focus control fast. Set focus with focus presets. Switch between SPL focus and surface focus to verify that each is set properly.\n\nFind surface using “find surface” button.\n\nTest spot. Increase dwell time if no spot appears. Can check z distance by raising fine z to find surface.\n\nTo measure spot size check ruler (first button under image).\n\nWhen arraying multiple spots, increase wait time to give a more consistent flow.\s\n\nTo take a picture, turn laser off and save image.\n\nCan import an image to be arrayed as long as it is a bit file.\n\nChip location saves location on chip surface.
Showed that basal polymerization and depolymerization occurred throughout lamellipodia with largely constant kinetics, and polymerization was promoted within one micron of the lamellipodium tip [[Watanabe 2002|http://www.ncbi.nlm.nih.gov/pubmed/11834838]]\n\nShowed by single-molecule imaging revealed fast directional movement of mDia1 FH1-FH2 for tens of microns in living cells [[Watanabe 2004|http://www.ncbi.nlm.nih.gov/pubmed/15044801]].\n\nShowed that low dose latB treatment paradoxically increases G-actin content and induces processive movement of both mDia1 and FRL1 (formin-related gene in leukocytes) [[Watanabe 2008|http://www.ncbi.nlm.nih.gov/pubmed/18827014]]\n\nShowed that within the lamellipodia of fibroblast cells, STI571 (gleevec) induces rapid translocation of abl to the lamellipodium tip [[Watanabe 2008|http://www.ncbi.nlm.nih.gov/pubmed/188270142009|http://www.ncbi.nlm.nih.gov/pubmed/18835981]].
See [[Nap1]] or [[Hem-1]].
Another name for [[Hem-1]] and [[Kette]]. A tagged version of [[Nck]] will pull down Nap1 from Cos7 cells [[Kasuga 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8605018&query_hl=11&itool=pubmed_docsum]].
Natural killer (NK) cells are a form of cytotoxic lymphocyte which constitute a major component of the innate immune system. NK cells play a major role in the host-rejection of both tumours and virally infected cells. They were named "natural killer" because of the initial notion that they do not require activation in order to kill cells which are "missing self" ("missing-self" recognition is a term used to describe cells with low levels of MHC (major histocompatibility complex) class I cell surface marker molecules—a situation which could arise due to viral infection, or in tumors under strong selection pressure of killer T cells).
Nck is an adaptor protein composed of a single SH2 domain and three SH3 domains. The Nck binds via its SH3 domains to a proline-rich region on [[WASP]] and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. Nck is bound in living cells to the serine-threonine kinase [[Pak1]]. The association between Nck and [[Pak1]] is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of [[Pak1]] [[Schlessinger 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=8798379]]. Nck may bind the [[WAVE complex]] [[Kirschner 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12181570&query_hl=5&itool=pubmed_DocSum]] and is a more potent activator of [[WASP]] and N-WASP than [[Rac]] and [[Cdc42]] [[Sakowicz 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17302440&query_hl=4&itool=pubmed_DocSum]]. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network [[Pawson 2003|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=12808099&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. Clustering of all the SH3 domains on Nck triggers N-WASP, but not [[WAVE]]-dependent, actin polymerization [[Mayer 2004|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14711409&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlus]].
Neutrophil granulocytes, generally referred to as neutrophils, are the most abundant type of white blood cells and form an integral part of the immune system. Their name arrives from staining characteristics on hematoxylin and eosin (H&E) histological preparations. These phagocytes are normally found in the blood stream. However, during the acute phase of inflammation, particularly as a result of bacterial infection, neutrophils leave the vasculature and migrate toward the site of inflammation in a process called chemotaxis.
Depolymerizes microtubules. Nocodazole impairs chemotaxis, but not migration. It decreases PhAkt accumulation in the membrane, PhAkt phosphorylation, and slightly affects actin accumulation [[Bourne 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15860582&query_hl=11&itool=pubmed_docsum]].
Showed that systems of genes regulate different aspects of cell morphology in a huge drosophila cell screen [[Perrimon 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17588932&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and published a complete methods paper [[Perrimon 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17853882&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
10x = 1.6um/pixel , 1.06um/pixel\n20x = 0.8um/pixel, 0.53um/pixel\n40x = 0.4um/pixel, 0.26um/pixel\n60x = 0.26um/pixel, 0.18um/pixel
Showed that chemoattractant receptor [[C5a]] is uniformly distributed in response to chemoattractant in [[PLB-985]] cells [[Weiner 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10198064&query_hl=23&itool=pubmed_docsum]].\n\nShowed that new actin polymerization and the [[Arp2/3]] complex localize to the front of a protruding cell [[Weiner 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10559877&query_hl=23&itool=pubmed_docsum]].\n\nShowed that PH-Akt (a readout for PIP3) asymmetrically localizes to the leading edge, and this localization can be blocked by [[C. Difficile Toxin]], but not [[Latrunculin]] [[Weiner 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10669415&query_hl=23&itool=pubmed_docsum]].\n\nShowed that PH-Akt asymmetrically localizes to the leading edge with exogenous PIP3 as an input. This localization could be blocked with either PI3K inhibitors (LY294002) or RhoGTPase inhibitors ([[C. Difficile Toxin]]). Oddly enough, in this paper exogenous PIP3 localizes to the rear of the cell [[Weiner 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12080346&query_hl=23&itool=pubmed_docsum]].\n\nThe important paragraph for the chemical compass review:\n\n"Negative regulators of PIP3 accumulation include the 3′ lipid phosphatase PTEN and the 5′ lipid phosphatase SHIP. In mouse fibroblasts, a loss of PTEN activity results in spontaneous cell motility and activation of Cdc42 and Rac [17], suggesting that PTEN is important in setting an appropriate level of PIP3 degradation to ensure that cells do not spontaneously activate in the absence of stimulation. However, levels of stimulated AKT activation (AKT is an effector of PIP3) are normal in PTEN-null cells, suggesting that stimulation of PTEN activity is not necessary for setting levels of PIP3 production following receptor activation [18]. SHIP or other 5′ phosphatases account for the bulk (>90%) of the phosphatase activity toward PIP3 in neutrophil lysates [19], suggesting that this may represent the major pathway for degrading PIP3 in neutrophils. SHIP-null cells exhibit a normal baseline-level PIP3 but potentiated PIP3 accumulation (both in extent and duration), suggesting that SHIP is an important stimulated degrader of PIP3 [20]. However, SHIP-null cells exhibit no defect in chemotaxis [21], indicating either that there is redundancy in the system (there are multiple SHIP isoforms) or that more sensitive assays are necessary to uncover the chemotactic defect."\n\n[[Weiner 2002 review|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=11891119&ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that Hem-1 (360 nm in pig lysate) localizes to the leading edge and exists in complexes independent of WAVE2 (130 nm in pig lysate) with gel filtration. Hem-1 cells have a reduction in polarity, actin polymerization (rhodamine phalloidin), Rac activation (Pak phosphorylation and pull-down), and PIP3 production (Akt phosphorylation). Importantly, Rac activation (Pak phosphorylation and pull-down) is NOT impaired in latrunculin treated cells, which indicates that the WAVE complex has other activities (i.e. Rac activation) independent of actin assembly. Superoxide production in Hem-1 KD cells is normal, but have impaired inhibition of MLC phosphorylation [[Weiner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16417406&query_hl=23&itool=pubmed_docsum]].\n\nShowed that Hem-1 displays dynamic wave-like patterns with TIRF microscopy. Showed that latrunculin "freezes" these waves, and that these waves get brighter indicating that actin polymer is both required to propagate waves, but also to remove Hem-1 from the membrane [[Weiner 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17696648&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
A [[Rac]] GEF synergistically activated by [[PI(3,4,5)P3]] and Gbetagammas both in vitro and in vivo. Purified on the basis of [[Rac]] activation [[Stephens 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11955434&query_hl=5&itool=pubmed_DocSum]]. P-Rex1 KO mice show defects in superoxide production and only have mild chemotaxis defects. [[Stephens 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16243035&query_hl=5&itool=pubmed_DocSum]]\n\n
Protein kinase C and casein kinase 2 substrate in neurons’ proteins contain [[BAR]] domains.
p21 binding domain
Rac binding domain of Pak fused to mCherry.
PBD labeling\n\nArthur Millius 11/04/07\n\nOverview\n\nTo label Klaus N-cys PBD with Alexa 594 and purify away from dye.\n\nBuffers\n\nBuffer A = 100 mM NaCl, 50 mM Na2PO4, 2 mM MgCl2, 1 mM DTT, 10% glycerol\nTCEP = 1 M TCEP\n\nProcedure\n\nThaw 1 ml recombinant PBD at 200 µM (~2 mg/ml). Divide into two tubes: one with 400 µl and one with 600 µl.\n\nAdd 280 µl 2 mM alexa 594 to 400 µl tube. This gives 823 µM alexa 594 to 117 µM PBD final (a seven fold excess of PBD).\n\nAdd 140 µl 2mM alexa 594 to the 600 µl tube. This is approximately twice the concentration of alexa 594 to PBD.\n\nMix at room temperature in the dark for two hour. Take a 30 µl aliquot every 30 min for a gel.\n\nDialyze extensively (5 changes over 72 hours). Note: I lost half of the 600 µl sample because I broke the dialysis bag. I diluted the sample in half and added it to the dialysis bag.\n\nTake 30 µl post dialysis aliquot.\n\nSpin remaining for 30 min at 95,000 rpm at 4C. Remove supernatant, save 30 µl aliquot and snap freeze the rest in 10 µl (36% labeled) and 50 µl (23% labeled) aliquots.
Pombe Cdc15 homology, onsisting of an N-terminal Fes/CIP4 homology (FCH) or [[BAR]] domain, followed by a coiled coil (CC) region and by one or two C-terminal Src homology 3 (SH3) domains.
Phosphoinositide-dependent kinase 1 (PDK1) localizes to the plasma membrane by binding to the lipid products of phosphoinositide 3-kinase (PI3K). This enzyme can phosphorylate a critical Thr residue in the activation loop of a variety of protein kinases in the protein kinase C (PKC) and protein kinase B (Akt) superfamilies [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]]. TORC2 directly phosphorylates a conserved phosphorylation site within the hydrophobic carboxy-terminal motif (HM) of Akt [[Hay 2005 review|http://www.ncbi.nlm.nih.gov/pubmed/17419990?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=8]]. PDK1 knockout Dicty (pdkA-pdkB-) cannot phosphorylate Akt (PKB) and have impaired chemotactic index, but normal motility [[Devreotes 2008|http://www.ncbi.nlm.nih.gov/pubmed/20075071?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1]].
N-terminal pleckstrin homology (PH) domain binds to the lipid products of phosphoinositide 3-kinase (PI3K). The PH domain fold appears to comprise a large but diverse superfamily with at least three phylogenetically distinguishable branches: (1) canonical PH domains, (2) [[PTB]] domains, and (3) WH1 domains [[Lim 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10338211&query_hl=30&itool=pubmed_docsum]].
The PH domain of Akt fused to GFP reports on PIP3 levels.
The PH domain of Akt in Hem1KD line.
The PH domain of Akt is used as a readout for PIP3.
Phosphatidylinositol-3,4-bisphosphate PI(3,4)P2 is a lipid product of phosphoinositide 3-kinase ([[PI3K]]) produced by phosphorylation of phosphatidylinositol-4-phosphate (PI4P); it is also produced by hydrolysis of phosphatidylinositol-3,4,5-trisphosphate [[PI(3,4,5)P3]] or phosphorylation of PI3P by a 4-kinase [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract.]].
Phosphatidylinositol-3,4,5-trisphosphate PI(3,4,5)P3 is a lipid product of phosphoinositide 3-kinase ([[PI3K]]) produced by phosphorylation of phosphatidylinositol-4,5-bisphosphate [[PI(4,5)P2]] at the 3' position of the inositol ring [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
See [[PIP2]].
See [[PI3K]].
Phosphoinositide 3-kinase (PI3K) catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Class Ia enzymes (alpha, beta, delta) are primarily responsible for production of D-3 phosphoinositides in response to growth factors. Class Ia contains the catalytic subunit [[p110]] and the regulatory subunit [[p85]] [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]]. Class Ib are activated by the beta-gamma subunit of a heterotrimeric G protein. Class Ib contain p101 and p110gamma subunits. Drugs, which inhibit PI3K include ~IC87114 (selectively inhibits gamma), ~TGX-115 (beta and gamma), ~PI-103 (alpha> beta and gamma), PIK-90 (alpha, gamma, and delta), and ~PIK-93 (alpha and gamma> delta).\n\nPI3Kdelta is hemopoietic-specific and PI3Kdelta inhibition blocks directionality, but not random movement in neutrophils [[Staunton 2003|http://www.ncbi.nlm.nih.gov/pubmed/12594293?dopt=Abstract&holding=npg]].\n\nPI3Kgamma is the dominant class 1 PI3K in neutrophils and PI3Kgamma inhibition determines the proportion of neutrophils that can move, but does not affect chemotaxis or speed [[Stephens 2006|http://www.ncbi.nlm.nih.gov/pubmed/17173040?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. \n\n''Class 1a'': includes &alpha; &beta; &delta;\n##primarily responsible for production of D-3 phosphoinositides in response to growth factors\n##contains the catalytic subunit [[p110]] and the regulatory subunit [[p85]] [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]]\n\n''Class 1b'': includes &gamma;\n##
See [[PI3K]].
Inhibits PI3K alpha, beta, and gamma. PIK-90 treated cells are impaired in chemotaxis and are unable to activate Rac, although Pak-PBD can still localize to the multiple leading edges produces by PIK-90 treatment. PIK-90 also reduces RhoA activation [[Bourne 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16864657&query_hl=1&itool=pubmed_docsum]].
Normally refers to PtdIns(4,5)P2, but could also be PtdIns(3,4)P2.
Phosphatidylinositol-3,4,5-trisphosphate. In Dicty PIP3 is not required for chemotaxis [[Kay 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17462897&query_hl=2&itool=pubmed_docsum]].
p21-activated kinase-interacting exchange factor. PIX/COOL are guanine nucleotide exchange factors. PIX/COOL binds to a nonconventional proline-enriched motif in [[Pak]]. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
cAMP-dependent protein kinase A, the main intracellular target of cAMP.
Another name for [[Akt]], because of homology with protein kinase A and protein kinase C family members.
Protein kinase C; serine/threonine kinase activated by calcium. See wikipedia [[PKC|http://en.wikipedia.org/wiki/Protein_kinase_C]].
See [[PKCz]].
See [[PKC]]. Requires DAG, but do not require Ca2+ for activation.
Pronounced PKC-zeta. PKCz binds in a complex with Par3 and Par6. Overexpression of PKCz in a normally non-migratory breast cancer cell line enables migration. Knockdown of PKCz causes ruffling everywhere, cells look overadhered and multinucleate, and prevents directional migration (they migrate in circles). Perhaps PKCz is positively regulating back genes (Rho, ROCK, myosin), such that when you knock-out PKCz you lose the ability to form a back (Suha Naffar-Abu Amara personal communication).
Loss of PLA does not alter PI(3,4,5)P(3) regulation, but chemotaxis becomes sensitive to reductions in PI3K activity. This defect can be rescued by global application of arachidonic acid, indicating that PLA does not spatially direct chemotaxis [[Devereotes 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17419997&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Retrovirus system experiment protocol\n\n1. Seed Phoenix packaging cells in 100 mm plate (cell concentration: 3-5*105/ml). \n2. After 24 hrs, co-transfect the packaging cells with pMSCVneo *Venus and pVSV-G plasmids using lipofectamine in 5 ml opti-MEM serum-free media (pMSCV*Venus : pVSV-G = 3 : 1).\n3. After 4-6 hrs, remove the serum-free medium and add 10 ml DMEM + 10%FBS complete medium.\n4. After 24-48 hrs, check the transfection efficiency by visualizing the expression of GFP in the cells. You want most of the cells to be expressing GFP so you get high enough titers.\n5. After 48-72 hrs, collect the virus-containing medium and transfer into 15 ml tube. Centrifuge at 500Xg for 5 min remove dead cell and cell debris. Transfer the medium into a new tube or a 10 ml syringe. \n6. Add 15 mg/ml polybrene into medium (dilution 1:1000), gently mix the solution and filter with 0.45 um filter.\n7. Add 2-4 ml filtered virus medium to 2 ml HL-60 or PLB985 cells (cell concentration: 1-2*106)\n8. After 48-72 hrs, centrifuge the cells and culture them in G418 selection medium (G418 concentration: HL-60 = 1.6mg/ml; PLB-985 = 0.6mg/ml)\n9. Change selection medium every 2-3 days. If there is lot of dead cells, separate the live cells and continue selection with G418 medium for two weeks or more. \n\n\nLentivirus system experiment protocol\n\n1. Seed HEK-293T cells in 100 mm plate (cell concentration: 3-5*105/ml). \n2. After 24 hrs, when cell reach 80-90% confluency, co-transfect the HEK-293T cells with pGIPZ shRNA cloning vectors and the packaging plasmids (from Open Biosystems) using lipofectamine in 5 ml opti-MEM serum-free media. After 4-6 hrs, remove the serum-free medium and add 10 ml DMEM + 10% FBS complete medium.\n3. After 24 hrs, check the transfection efficiency by visualizing the expression of GFP in the cells. You want most of the cells to be expressing GFP so you get high enough titers.\n4. After 48 hrs, most of the transfected cell should be dead and viruses are released to medium. Collect the virus-containing medium and transfer it into 15 ml tubes. Centrifuge at 500Xg for 5 min to eliminate of dead cells and cell debris. Transfer the medium into a new tube or 10 ml syringe. Add 15 mg/ml polybrene into medium (dilution 1:1000), gently mix the solution and filter through a 0.45 um filter.\n5. Add 4-6 ml filtered virus medium to 2 ml HL-60 or PLB985 cells (cell concentration: 1-2*106)\n6. After 48-72 hrs, centrifuge the cells and culture them in puromycin selection medium (puromycin concentration: HL-60 = 0.4-0.6 ug/ml; PLB-985 = 0.4-0.6ug/ml)\n7. Change medium every 2 to 3 days. If there is lot of dead cells, separate the live cells and continue selection with puromycin containing medium for two weeks or more.\n8. We obtained consistent knock down results with PLB-985 cells. For unknown reasons, we have only been successful with HL-60 cells on a few occasions.\n\nDead and Viable HL-60/PLB-985 cell separation\n\n1. Put 5 ml of warm (or RT) Sigma-Aldrich's Histopaque 1077 (catalog # 10771-500 ml) on the bottom of a 15 ml tube (or 15ml for 50 ml tube).\n2. Slowly layer up to 2/3 tube volume of suspended cells on the top of the Histopaque 1077. Avoid mixing the layers.\n3. Centrifuge at 700Xg for 15 min at RT without acceleration and de-acceleration (with swinging buckets).\n4. The living cells will be at the interface of the media and Histopaque 1077. The dead cells will be in the bottom of the tube. Take out the living cell layer and add 5 ml of warm PBS.\n5. Centrifuge cells at 500Xg for 5 min. Wash the cells with warm PBS twice. Place cells in G418 or puromycin containing RPMI medium as before.\n\n
A myeloid cell that can be differentiated into a neutrophil.
An enzyme that catalyses the hydrolysis of the phosphodiester bond of phosphatidyl choline to generate phosphatidic acid (PA) and free choline. PA has signalling properties. In mammals, there are two PLD isoforms, ~PLD1 and ~PLD2, both of which are subject to complex regulation by lipid cofactors, protein kinases and ~GTP-binding proteins of the [[Arf]] and [[Rho]] subfamilies. In HL-60 cells both ~PLD1 and ~PLD2 localize to the front of the cell in response to chemoattractant [[Gomez-Cambronero 2006|http://www.ncbi.nlm.nih.gov/pubmed/16873675]]. ~PLD2 is found on the plasma membrane and ~PLD1 in a peri-nuclear region. While ~PLD1 activity is upregulated by Rac1 and other Rho family ~GTPases, ~PLD2 has a high basal activity that is not affected [[Frohman 1997|http://www.ncbi.nlm.nih.gov/pubmed/9395408]].
Phorbol 12-myristate 13-acetate, causes translocation of Rac at 37C after stimulation with 1 ug/ml [[Bokoch 1993|http://www.ncbi.nlm.nih.gov/pubmed/8407934?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]
POR1 binds directly to Rac1, and the interaction of POR1 with [[Rac1]] is GTP dependent. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of [[Rac1]], ~V12Rac1, in quiescent rat embryonic fibroblast cells. Furthermore, POR1 synergizes with an activated mutant of [[Ras]], ~V12Ras, in the induction of membrane ruffling [[Bar-Sagi 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8670882&query_hl=36&itool=pubmed_DocSum]]. [[Arf6]](~Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery, but may be inhibited by deletion mutants of POR1. [[Arf6]] interacts directly with POR1 and this interaction is GTP dependent [[Van Aelst 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9312003&query_hl=36&itool=pubmed_DocSum]].
Inhibits mTORC1 and mTORC2, without hitting PI3K [[Feldman 2009|http://www.ncbi.nlm.nih.gov/pubmed/19209957?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1]]
Protein domain, which binds phosphorylated tyrosine residues.
"Tensin homology protein". The phosphatase PTEN dephosphorylates the 3 position of [[PI(3,4,5)P3]] to produce [[PI(4,5)P2]]. Loss of PTEN protein or function has been found in a large fraction of advanced human cancers, indicating that uncontrolled signaling through PI3K contributes to metastatic cancers [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]]. Loss of the ~PtdIns(3,4,5)P(3) phosphatase PTEN has no impact on neutrophil chemotaxis [[Sasaki 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17173042&query_hl=3&itool=pubmed_docsum]]. In opposing gradients, PTEN becomes distributed throughout the cell. PTEN KO mice have no defect in chemotaxis to fMLP, but show a reduced chemotactic index to CXCR2 ligands [[Kubes 2008|http://www.ncbi.nlm.nih.gov/pubmed/18536720?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
The p21-activated kinases (Paks) 1-3 are serine/threonine protein kinases that form self-inhibited dimers whose activity is stimulated by the binding of active [[Rac]] and [[Cdc42]] GTPases, which causes [[Pak1]] autophosphorylation and dissociation. Canonical PXXP ~SH3-binding motifs interact in [[Pak1]] with the ~SH3-containing adapter protein [[Nck]], and the nonclassical (PXP) SH3 binding site interacts with the adapter protein [[Grb2]], and the noncanonical site with the PIX family of proteins have been described. [[Rac]] and [[Cdc42]] minimally bind to the so-called CRIB (for [[Cdc42]] and [[Rac]] interactive binding) domain (aa 75–90 in [[Pak1]]). The more inclusive p21-binding domain ([[PBD]]) (aa 67–113 in [[Pak1]]) contributes to overall binding affinity. Pak regulates [[Stathmin]], [[MLCK]], [[LIMK]], and [[Filamin A]]. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]\n\nPakC in Dicty shows a uniform distribution [[Firtel 2004|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15483055&ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]], whereas PakA in Dicty localizes to the back of the cell and controls myosin regulation [[Firtel 1999|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10545500&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
The Rac binding domain of Pak in Hem1KD line.
The GTPase binding domain of Pak.
Pak1 colocalizes with F-actin in membrane ruffles. These membrane ruffles and Pak1 localization is inhibited by disruption of PI3K (wortmannin) or F-actin (cytochalasin D) [[Bokoch 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9298982&query_hl=33&itool=pubmed_docsum]]. In contrast, F-actin disruption in stimulated neutrophils, which do not depend on adhesion for polarization, does not decrease Pak1 phosphorylation [[Weiner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16417406&query_hl=15&itool=pubmed_docsum]]. This could be interpreted as [[Rac]] translocation to membranes (readout by Pak activity) is dependent on adhesion in adherent cells and not dependent on adhesion in non-adherent cells. [[Cdc42]] activation, but not [[Rac]] activation is affected in Pak1 null macrophages [[Wu 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887923&query_hl=35&itool=pubmed_docsum]].
PakA null cells are defective in [[Myosin II]] assembly, as the [[Myosin II]] cap in the posterior of chemotaxing cells and [[Myosin II]] assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PakA leads to an upregulation of [[Myosin II]] assembly [[Firtel 1999|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=10545500&ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
PakC null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient [[Firtel 2004|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15483055&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
[[Par6]], [[Par3]], [[aPKC]], and [[Cdc42]] form a complex that is essential for polarization in the C. elegans embryo [[Hall 2003 review|http://www.ncbi.nlm.nih.gov/pubmed/12517706?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and although to date there is no evidence that specialized migrators—the slime mold, Dictyostelium, and mammalian neutrophils—use PAR signaling at all, even though they harness phosphoinositides to polarize cell movment [[Goldstein 2007 review|http://www.ncbi.nlm.nih.gov/pubmed/17981131?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. [[Par6]] localizes to Rac-induced membrane ruffles in HeLa cells [[Sumimoto 2001|http://www.ncbi.nlm.nih.gov/pubmed/11260256?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
IP approach for determining bound proteins to beta-adenergic receptor. Suggested papers from him:\n\n[[Ott 2005|http://peds.oxfordjournals.org/cgi/content/full/18/3/153]]\n[[Sigma|http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Biological_Detergents/Key_Resources/Detergent_Theory.html]]
A marker of focal adhesion sites.
[[Alan Hall]]\n[[Akihiro Kusumi]]\n[[Ann Richmond]] \n[[Anna Huttenlocher]]\n[[Anne Ridley]]\n[[Clare Waterman-Storer]]\n[[Daniel Fletcher]]\n[[Daniel Lew]]\n[[David Soll]]\n[[Denise Montell]]\n[[Dyche Mullins]]\n[[Frederick Maxfield]]\n[[Gaudenz Danuser]]\n[[Gary Bokoch]]\n[[Gerald Crabtree]]\n[[Gunther Gerisch]]\n[[Heidi Welch]]\n[[Henry Bourne]]\n[[Howard Berg]]\n[[Jack Taunton]]\n[[Julie Theriot]]\n[[Klaus Hahn]]\n[[Len Stephens]]\n[[Lew Cantley]]\n[[Lorraine Santy]]\n[[Marc Kirschner]]\n[[Martin Schwartz]]\n[[Michael Glogauer]]\n[[Michiyuki Matsuda]]\n[[Naoki Watanabe]]\n[[Norbert Perrimon]]\n[[Orion Weiner]]\n[[Paul Temkin]]\n[[Peter Devreotes]]\n[[Peter Hordijk]]\n[[Phillipe Chavrier]]\n[[Richard Cerione]]\n[[Rick Firtel]]\n[[Robert Insall]]\n[[Ron Vale]]\n[[Rong Li]]\n[[Sally Zigmond]]\n[[Scott Simon]]\n[[Sergio Grinstein]]\n[[Shamshad Cockcroft]]\n[[Shiro Suetsugu]]\n[[Steven Altshuler]]\n[[Tadaomi Takenawa]]\n[[Theresia Stradal]]\n[[Tian Jin]]\n[[Tim Mitchison]]\n[[Tobias Meyer]]\n[[Torstein Wittman]]\n[[Uri Alon]]\n[[Wendell Lim]]\n[[Verena Niggli]]\n[[Victor Small]]\n[[Yoshinori Fukui]]\n
Permeabilized cells can be used for biochemical dissection of signal transduction cascades that are impossible to study in cell lysates. This strategy has been applied to study secretion [[Gomperts 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8868468&query_hl=67&itool=pubmed_DocSum]], superoxide generation [[Cockcroft 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10548486&query_hl=70&itool=pubmed_docsum]] [[Yaffe 2003|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=12535519&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]], and nuclear import [[Blobel 1993|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8413630&query_hl=72&itool=pubmed_DocSum]]. Implementation of this technique to polarity will allow exchange of circuit components, temporal control of activators and inhibitors, and a means to biochemically identify components in [[Rac]]-induced [[Actin]] polymerization and [[WAVE]] complex recruitment.
Pertussis Toxin, first purified by [[Bokoch 1983|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=6296122&query_hl=14&itool=pubmed_docsum]] ,catalyzes the ADP-ribosylation of the α subunits of the heterotrimeric guanine nucleotide regulatory proteins Gi, Go, and Gt. This prevents the G-proteins from interacting with cell membrane receptors, thus interfering with intracellular communication. Pertussin toxin causes uropod formation closest to site of the drug when delivered with a micropipette [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887922&query_hl=11&itool=pubmed_docsum]].
Pertussis toxin catalyzes the ~ADP-ribosylation of the α subunits of the heterotrimeric G proteins Gi, Go, and Gt. This prevents the G proteins from interacting with G protein-coupled receptors on the cell membrane, thus interfering with intracellular communication.
Showed that [[cAMP]] waves correlated with Dicty waves [[Devreotes 1981|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=6259734&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that [[cAR1]] is the [[cAMP]] receptor [[Devreotes 1988|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=3047871&query_hl=4&itool=pubmed_DocSum]] and designated it [[cAR1]] [[Devreotes 1991|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1989903&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that another receptor besides cAR1 is linked to [[ACA]] activation, but both receptors couple to [[Gα]]. Showed that GTPγS can couple directly to [[ACA]] activation [[Devreotes 1992|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1333842&query_hl=4&itool=pubmed_DocSum]].\n\nPurified [[CRAC]] [[Devreotes 1994|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8188693&query_hl=4&itool=pubmed_DocSum]] and showed that chemoattractants or GTPγS promote translocation of [[CRAC]] from the cytosolic to the membrane fraction.\n\nShowed that Gβ is required for GTPγS stimulation of [[ACA]] activity, suggesting that the βγ dimer activates the enzyme directly. Additionally, β null cells display normal motility but do not move towards chemattractants [[Devreotes 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7790362&query_hl=4&itool=pubmed_DocSum]].\n\nDiscovered a CA [[ACA]] in a suppressor screen in [[CRAC]]-null phenotype [[Devreotes 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8702473&query_hl=4&itool=pubmed_DocSum]].\n\nShowed (along with Zigmond and Bokoch) that CA [[Cdc42]], but not [[Rac]] or [[Rho]] can activate actin assembly in a cell lysate [[Devreotes 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9230078&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that GFP cAR1 remains evenly distributed on the cell surface during chemotaxis [[Devreotes 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9334341&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that elimination of phosphorylation of a chemoattractant receptor in Dicty did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation [[Devreotes 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9341180&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that [[CRAC]] localizes to the leading edge of a cell given a gradient and when treated with latrunculin [[Devreotes 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9778249&query_hl=4&itool=pubmed_DocSum]].\n\nSuggested that [[CRAC]] responses depend on the relative level of chemoattractant and not absolute concentrations [[Devreotes review 1999|http://www.ncbi.nlm.nih.gov/pubmed/10221901?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that GFP-Gβ localizes to the leading edge of a Dicty cell in a gradient. Interestingly, in contrast to the PH domain of [[CRAC]], Gβ fails to localize to the up gradient edge of a latrunculin-treated cell [[Devreotes 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10669414&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that PTEN-GFP localized to the surface membrane at the rear of the cell and that disruption of [[PTEN]] broadens the PH domain relocation and actin polymerization [[Devreotes 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12062103&query_hl=13&itool=pubmed_docsum]].\n\nShowed that Gβ-null cells has an affect electrotaxis, and, in contrast to chemotaxis, there is no PH-CRAC gradient [[Devreotes 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12045182&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that the localization of [[PTEN]] does not depend on its phosphatase activity, the actin cytoskeleton, or the intracellular level of PIP3. Showed that the phosphatase activity of [[PTEN]] mediates chemotaxis and that the sharp localization of PIP3 requires localization of [[PTEN]] to the rear of the cell [[Devreotes 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14764604&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that the [[PH]] domain of [[CRAC]] localizes to the membrane for approximately 120 ms [[Devreotes 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16507590&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that PLA2 and PI3K act in parallel to mediate chemotaxis in Dicty [[Devreotes 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=17419997&dopt=AbstractPlus]].\n\nShowed that Tor2C can mediate chemotaxis independently of PIP3 through activation of PKB, which phorsphorylates and activates [[Talin]], Ras GEFs, and a RhoGAP. Active Tor2C as visualized by phosphorylated PKB is localized to the leading edge of Dicty [[Devreotes 2008|http://www.ncbi.nlm.nih.gov/pubmed/18635356?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Showed that increased [Ca2+]i and [[PKC]] activation induce phosphorylation of RhoGDI and induce the translocation of cytosolic Rac to the plasma membrane. Showed that [Ca2+]i potentiates [[Rac]] activation [[Hordijk 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12888567&query_hl=14&itool=pubmed_DocSum]].\n\nShowed that [[Rac1]], via a proline stretch in its carboxy-terminus, binds directly to the SH3 domain of the Cdc42/Rac GEF [[beta-Pix]]. Showed that the interaction with [[beta-Pix]] is nucleotide independent and is necessary and sufficient for [[Rac1]] recruitment to membrane ruffles and to focal adhesions [[Hordijk 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16492808&query_hl=14&itool=pubmed_DocSum]].\n\nShowed that [[Tiam-1]] interacts with a component of the [[Arp2/3]] complex. Deletion of this [[Arp2/3]] binding impairs the ability of [[Tiam-1]] to activate [[Rac]]. [[Tiam-1]] also promotes [[Arp2/3]] localization, thus suggesting an actin-dependent Rac activation feedback loop [[Hordijk 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16599904&query_hl=14&itool=pubmed_DocSum]].\n\nShowed that Rac1 through its c-terminus binds SET, an inhibitor of the serine/threonine phosphatase PP2A. Membrane targeting of SET stimulates cell migration in a Rac1-dependent manner [[Hordijk 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17245428&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
The [[PH]] domain of Akt. It is used as a read-out for PI(3,4,5)P and PI(3,4)P lipids.
Showed that inducible recruitment of Cdc42 and WASP to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions [[Chavrier 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10209117&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that recruitment of an activated Rac1 triggers phagocytosis of a latex bead [[Chavrier 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10934035&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that the recruited VCA domain of WASP is not sufficient to induce actin assembly, but requires the proline-rich region (which binds VASP) [[Chavrier 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11598004&query_hl=4&itool=pubmed_DocSum]].\n\nShowed that Rac activation persists after actin disassembly during phagocytosis. This indicates that inactivation of Rac is not required for actin disassembly [[Chavrier 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15809313&query_hl=4&itool=pubmed_DocSum]].
Pig Leukocyte Membrane Extraction\nArthur Millius, Scott Foster, and Scott Hansen (12/20/06)\n\n''Overview''\nTo trap the WAVE complex at the membrane, and obtain cytosol and plasma membrane fractions from leukocytes.\n\n''Buffers''\nAnticoagulant buffer = 80 mM sodium citrate, 15 mM NaH2PO4, 160 mM glucose, 17 mM citric acid, 2 mM adenine (9 L, store @ -4).\n\nCoagulant buffer = 3% polyvinylpyrrolidone (PVP; w/v), 154 mM NaCl (10 L, store @ 23)\n\n10x mHBSS (Ca free) = 1.5 M NaCl, 40 mM KCl, 10 mM MgCl2, 100 mM glucose, 200 mM Hepes, pH 7.2 (1.5 L, sterile-filtered, store @ -4)\n\nmHBSS (Ca free) + 0.2% albumin = 150mM NaCl, 4mM KCl, 1mM MgCl2, 10mM glucose, 20mM Hepes, pH 7.2, 2 g/L endotoxin-free albumin (make from 10x and store @ 23)\n\nCavitation buffer = 100 mM KCl, 50 mM Hepes (pH 7.2), 3.5 mM MgCl2, 1 mM DTT, 2 mM PMSF, 1 EDTA-free protease inhibitor tablet per 50 ml (1 L, add DTT, PMSF, and protease tablet just before use, store @ -4)\n\n10x EGTA (500 mM into 200 mM in cavitation buffer, store @ -4)\n\nSucrose Gradient buffer = 50 mM Hepes, 1 mM EGTA, 1 mM MgCl2, 1 mM ATP, 60%, 34%, and 15% sucrose, 1 mM DTT, 2 mM PMSF, , 1 EDTA-free protease inhibitor tablet per 50 ml (250 ml, add ATP and proteases before use, store @ -4)\n\n''Pre-cavitation preparation''\nObtain (4) 2.5 gallon bottles of fresh pig blood. We obtained the blood from the Stagnos Meat Co. in Modesto, CA. Their phone number is 209-578-1748.\n\nDistribute 4 L (1 gallon) of pig blood into 5 L beakers. Add 1000 ml of coagulant buffer to 5 L beakers on top of the blood. Mix thoroughly and allow to settle into two phases for ~ 20 - 40 minutes.\n\nTo clean up unused blood, use a 1:10 solution of bleach and fill the 2.5 gallon container with the bleach solution. You will also do this for the 5L beakers that hold the blood.\n\nTransfer upper phase into another 5 L beaker. Initially, pour quickly and then slowly, the top phase into another bucket. We designated this bucket with a cross. This maximizes the amount of liquid from the first phase you can obtain. Do this by pouring off whiter (clear) liquid until you see a denser red blood cell layer coming off. Every 2 L, transfer to 1 L centrifuge bottles.\n\nSpin at 1500 x g, which turns out to be 2270 rpm on Beckman Allegra 6R for 15 min in 1 L containers and room temperature. Remember to have the brake on low. //Note: Supernatant should be clear at this point. A cloudy, red supernatant would indicate either the brake was on too high or red blood cell lysis.//\n\nAdd fresh blood to bottles with pellets (i.e. you can spin more than one time in the same bottle without removing the pellet). //Note: You get ~ 20 L to spin. We used three centrifuges and 12 bottles, spinning one set 3 times and the other sets twice.//\n\nTransfer last bit of upper layer with contaminating red blood cells into another bucket. Do this for each bucket, in order to pool the top layers.\n\nResuspend pellets in minimal mHBSS (without Ca) + 0.2% albumin. Combine into two 500 ml Corning centrifuge tube. //Note: We had 300 ml in each tube.// \n\nLow speed (1500 x g) spin for 15min at RT. Note: We got a ~100 ml pellet for each centrifuge tube. You should see 3 layers, the bottom red layer is the red blood cells, the middle layer is mostly leukocytes plus a little red blood cells, and the clear layer on top is platelets. The white line between the platelets and leukocytes are platelets.\n\nWe scraped the sides of the pellet with a metal spatula. Resuspend in appropriate volume of mHBSS. //Note: We added up 100 ml mHBSS to each pellet. We then split this volume into (4) 2 L buckets, so we had 100 ml of 1:1 pellet:buffer slurry in each bucket.//\n\nResuspend in 20x volume ddH2O. Bring to 1x Ca-free mHBSS with 1.1 vol 10x mHBSS.\n(For a 50 ml pellet, 1 L). //Note: We added 1575 ml of ddH2O for exactly 20 secs. Then we brought the volume up to 1750 L with 175 ml 10x mHBSS to return the salt concentration to 1x mHBSS. We did not count the pellet toward amount of 10x mHBSS we need to add because it was resuspended in 1x mHBSS.//\n\nLow speed (1500 x g) spin in (8) 1 L bottles for 15min at RT. \n\nPour off supernatant. Resuspend cells in minimal volume 1x mHBSS (Ca-free with 0.2% albumin), pool, and transfer to a 500 ml Corning centrifuge tube.\n\nLow speed (1500 x g) spin for 15min at RT. This is the rinse step for the “ghost” red blood cells. //Note: We got ~110 ml white pellet. The supernatant will be red from the lysed blood cells. There will still be some red streaks in the white pellet from the ghost (the plasma membrane) red blood cells.//\n\nAll steps following will be done at 4°C.\n\nResuspend in ~4x volume of cold mHBSS (Ca-free with 0.2% albumin). //Note: we just brought the pellet up to a final volume of 500 ml.//\n\nIn a chemical hood with extreme caution and protection, add DFP to a final concentration of 2 mM. We added ~180 µl to 500 ml. //''Caution: DFP is extremely toxic! 300 µl on your skin is lethal!!!!! Keep stocks in 100 µl aliquots, wear gloves, a coat, and safety glasses. Have antidote nearby. Tell someone that you are working with DFP before beginning this step. Put used DFP tips in at least 2 N NaOH to neutralize DFP before discarding. DFP is dead after 90 minutes.''//\n\nKeep cold for at least 20 min.\n\nSplit into two tubes. Put 120 ml into a 250 ml corning centrifuge tube and kept the remaining ~400 ml cell slurry in the 500 ml tube. The 400 ml (~80 ml of leukocyte pellet) cell slurry is “wild type”. The 120 ml (~30 ml of leukocyte pellet) cell slurry is for “latrunculin” treatment.\n\n''Experimental Conditions''\n\n''Wild-type''\nTake 400 ml cell slurry to cold centrifuge. Low speed (1500 x g) spin for 15min at RT..\n\nPour supernatant into a bucket with 2 N NaOH to inactivate DFP.\n\nResuspend in 4x volume of cavitation buffer (with fresh DTT, PMSF, protease inhibitor tablets). We added less than 4x volume of cavitation buffer (to bring to approx. 320 ml total volume), so that we would only have to cavitate twice. We scraped side of centrifuge tube to completely resuspend pellet. //Note: Do not add DTT, PMSF, and protease inhibitors until ready to cavitate. Check off when you add each to the cavitation buffer.//\n\nCavitate at ~350 psi for ~30 min.\n\nCavitation:\n-Get nitrogen tank from the hallway on the 3rd floor (make sure it’s nitrogen and not some other gas).\n-Make sure all valves are closed and the pressure is off.\n-Take off nitrogen regulator, and replace with parr bomb regulator. Make sure threads are closed.\n-Tighten seal with wrench.\n-Place ~160 ml of cells in decapitated 250 ml centrifuge tube inside cavitator.\n-Carefully place top of parr bomb on cavitator (make sure black, rubber gasket is in place).\n-Put ring collar on bottom of cavitator. Place half ring on each side and tighten with thread.\n-Release pressure from nitrogen valve to regulator valve. Release pressure from regulator valve to cavitator inject valve. You should see a jump in the tube. Slowly open inject valve to inject nitrogen into the cavitator.\n-Add pressure up to 350 psi.\n-Let pressurize for ~30 minutes.\n-After 30 minutes, add 1.5 ml of 10x EGTA diluted in cavitation buffer (200 mM final EGTA conc.) into chilled 250 ml conical centrifuge tube for collection. If you cavitate in the absence of calcium (that is, with cavitation buffer containing EGTA, it will cause the nuclei to burst). No one knows why, it is just empirical evidence.\n-One person will drive, the other person will collect.\n-The driver slowly opens the cavitator release valve to get a steady drip of lysate. Simultaneously, the driver opens the inject valve to maintain pressure at 350 psi.\n-The collector (wearing gloves) collects the lysate into the conical using an inverted funnel to block splatter. Remember the lysate is full of super-toxic DFP.\n-After 150 ml, a burst of air will come out. Close the release valve immediately. Put the conical on ice, replace with another conical.\n-Release the remaining pressure keeping the inject valve closed. Close the regulator valve and release the pressure in the connecting tube by opening the inject valve.\n-Rinse the cavitator by repeating these steps with DI water\n\nCavitate the first 160 ml of wild-type.\n\nMeanwhile, we poured 6 sucrose gradients. The low salt in the sucrose gradient buffer promotes protein-protein interactions during the spin.\n\nAdd fresh 1 mM DTT, 2 mM PMSF, and 1 protease tablet per 50 ml to all sucrose solutions just before use.\n\nLayer with serological pipets 3ml of 60%, then 9 ml of 34%, then 10 ml of 15% sucrose solutions in Beckman 25x89 mm centrifuge tubes. These tubes hold a total volume of 31 ml and can be spun in the SW28 rotor at 28,000 rpm (~100,000 x g).\n\nCollect first cavitant and low speed (1500 x g) spin for 15min at at 4C. Note: There should be 3 layers after this spin. The top layer is foam, the middle layer is liberated cell contents, and the bottom layer is debris. If your cavitation did not succeed, you will see a clear layer and a larger pellet. We did not see much of a foam layer because we avoided bubbling our sample.\n\nStart second wild-type cavitation.\n\nTake liberated cell contents (supernatant) from first cavitation and pool into chilled 50 ml falcon tubes (total yield of ~110ml). The nuclei pellet was ~50 ml.\n\nLayer 12 ml of cell contents on each sucrose gradient.\n\nSpin in SW28 at 28K for 1 hr in chilled Beckman centrifuge on 3rd floor (ended up spinning for longer due to time conflicts)\n\nCollect cavitant from second cavitation and low speed spin (1500 x g) for 15min at 4C.\n\nPool liberated cell contents with contents from first cavitation and place in (3) ~60 ml Ti-45 centrifuge tubes. Spin at 35,000 rpm (~96,000 x g) for 1 hr in chilled Beckman centrifuge on 3rd floor. //Note: We had ~110 ml of total liberated cell contents from each cavitation.//\n\nFrom the sucrose gradient spin, the cytosol will stay above the 15% sucrose layer. Plasma membrane is found at the 15/34 interface. Granules and other stuff are found at the 34/60 interface.\n\nRemove and pool cytosol till just above cytosol/15 interface. Add 10% glycerol final. For instance, if there was 45 ml of cytosol, I would add 5 ml or enough to bring the total liquid level to 50 ml.\n\nRemove and discard cytosol/15 interface as much as possible until ~5 ml above 15/34. There may be still be some contaminating cytosol at this interface.\n\nWith a 1 ml pipet remove 3 – 4 ml of the plasma membrane layer. Cross the cytosol/15 interface and expel air bubbles into the 15% layer to ensure that no contaminating cytosol has touched the tip. Do this every time you cross this interface.\n\nPool the plasma membrane and label it “control plasma membrane”.\n\nFrom the Ti-45 high speed spin, remove the “blush or rose” supernatant. This is the pure cytosol. We got ~175 ml of this cytosol and froze as is without adding any glycerol or albumin.\n\n''Latrunculin Treated''\nLow speed spin (1500 x g) for 15min at 4C. \n\nWe had a 30 ml pellet to which we added up to 160 ml of RT mHBSS (Ca-free with 0.2% albumin).\n\nAdd 2 µM fMLP (32 µl of 10 mM fMLP) for 10 secs to 160 ml pellet slurry.\n\nAdd 10 µM latrunculin (160 µl of 10 mM latrunculin) for 3 minutes at room temperature.\n\nFill the rest of the centrifuge tube (340 ml) with cold mHBSS (Ca-free with 0.2% albumin) with 5 µM latrunculin and 2 µM fMLP and place on ice.\n\nLow speed spin (1500 x g). Resuspend in 160 ml of cold cavitation buffer + 5 µM latrunculin + protease inhibitors and fresh DTT and PMSF. \n\nCavitate for ~30 min.\n\nMeanwhile, set-up (9) sucrose gradients in Beckman 25x89 centrifuge tubes as before.\n\nCollect cavitant and low speed spin (1500 x g) for 15min at 4C.\n\nPool equal volumes of supernant from low speed spin of cavitant (low speed sup) into (4) 50 ml falcon tubes. We had ~35 ml of liberated cell contents in each falcon tube.\n\nWe labeled two “latrunculin” and the other two “GTPγS”.\n\n''Latrunculin''\nLayer ~12 ml on top of 5 sucrose gradients.\n\nSpin in SW28 for 1 hr at 28,000 rpm in chilled upstairs, Beckman centrifuge. Note: The rotor on the 3rd floor gave an error twice. Therefore, these samples were spun twice and then stopped. Furthermore, they had to be moved upstairs, so it was probably 30 min before they actually begun their hour long spin.\n\nCollect cytosol and plasma membrane and pool in separate tubes labeled “latrunculin cytosol” and “latrunculin plasma membrane”. Freeze cytosol from these fractions in 10% glycerol. Note: we took 3-4 mls of the latrunculin treated plasma membrane from each tube. \n\n''GTPγS''\nAdd 20 µM GTPγS (6 µl of 100 mM GTPγS to 30 ml of cell contents). \n\nBring to room temperature in the 37 degree bath. Wait a minute and then put on ice.\n\nLayer on top of (4) sucrose gradients and spin in SW28 at 28,000 rpm for 1 hr.\n\nCollect cytosol and plasma membrane and pool in separate tubes labeled “GTPγS cytosol” and “GTPγS plasma membrane”. Freeze cytosol from these fractions in 10% glycerol. Note: we took 3-4 mls of the GTPγS plasma membrane from each tube.\n\nRemove 2 ml of plasma membrane from “control”, “latrunculin”, and “GTPγS”. Add 200 µl of 50 mg/ml albumin and 550 µl of 50% glycerol for 4 mg/ml and 10% glycerol final for freezing.\n \nPlace remaining plasma membrane in Ti-45 centrifuge tubes. Add 4x volume cavitation buffer with protease inhibitors and fresh DTT and PMSF. Note: We filled the tubes up to 60 ml.\n\nSpin for 1 hr at 40,000 rpm (125,000 x g) in chilled Beckman centrifuge upstairs.\n\nRemove supernatant. The pellet is concentrated, pure plasma membrane. Scrape pellet with cut 1 ml tip, and resupend in 2 ml cavitation buffer containing 4mg/ml albumin, 10% glycerol (1 ml of 40 mg/ml + 1 ml 100% glycerol + 8 ml cavitation buffer), mini protease inhibitor tablet, and fresh DTT and PMSF\n\n''Freezing''\nCytosol\n1) high speed supernatant (cytosol) from wild-type preparation (175 ml)\n2) wild-type sucrose gradient (60 ml, 10% glycerol)\n3) latrunculin treated sucrose gradient (60 ml, 10% glycerol)\n4) GTPγS treated sucrose gradient (50 ml, 10% glycerol)\n\nPlasma Membrane\n5) before high speed spin post-supernatant (HS3) from control (2 ml, 4 mg/ml BSA, 10% glycerol)\n6) before HS3 from latrunculin (2 ml, 4 mg/ml BSA, 10% glycerol)\n7) before HS3 from GTPγS (2 ml, 4 mg/ml BSA, 10% glycerol)\n8) after HS3 from control (2 ml, 4 mg/ml BSA, 10% glycerol)\n9) after HS3 from latrunculin (2 ml, 4 mg/ml BSA, 10% glycerol)\n10) after HS3 from GTPγS (2 ml, 4 mg/ml BSA, 10% glycerol)\n\nGranules\n11) from control sucrose gradient 34/60 layer\n\nBoxes:\nHigh speed supernant = 1\nCytosol from sucrose gradient = 2,3,4\nNon-concentrated plasma membrane = 5,6,7,11\nConcentrated plasma membrane = 8,9,10\n\nLabels for tubes:\n1 = Leuk HSS 12/06 (pink)\n2 = HS3 Leuk 12/06 (yellow)\n3 = Lat HS3 12/06 (orange)\n4 = GTPgS HS3 12/06 (violet)\n5 = CT PM 12/06 (yellow)\n6 = Lat PM 12/06 (orange)\n7 = GTPγS PM 12/06 (violet)\n8 = CT PMC 12/06 (clear)\n9 = Lat PMC 12/06 (clear)\n10 = GTPγS PMC 12/06 (clear)\n11 = granules 12/06 (amber)
p53-inducible mRNA 121. Pir121 is the direct link between the [[WAVE complex]] and [[Rac]]. Pir121 null mutants are extremely large, contain an exceptionally high proportion of F-actin and extend protrusions at an uncontrolled rate all over their surface [[Insall 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=12956949&dopt=Abstract]]
Plastin is a member of the [[Fimbrin]] family of actin-binding proteins characterized by two actin-binding domains and a headpiece region containing two EF hand-type calcium-binding domains. [[Fimbrin]] did not give any phenotype when knocked out in S2 cells [[Rogers 2003|http://www.ncbi.nlm.nih.gov/pubmed/12975351?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Calcium binding inhibits actin-bundling activity of plastins in vitro, but the role of calcium in regulating [[L-plastin]] function in cells is not known [[Derancourt 2003|http://www.ncbi.nlm.nih.gov/pubmed/8461306?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. On VCA coated beads, T-plastin protects the actin network from being depolymerized in HeLa extracts [[Friederich 2005|http://www.ncbi.nlm.nih.gov/pubmed/15741236?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Polarization defines the propensity of the cell to assume an asymmetric shape with a defined anterior and posterior [[Janetopoulos 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12672811&dopt=Abstract]]
The effect enhances the perturbation in the direction of the perturbation. This can generate ultrasensitivity. Cell polarity can arise solely through positive feedback given limiting components [[Altshuler 2008|http://www.nature.com/nature/journal/v454/n7206/full/nature07119.html]].
Profilin is an actin binding protein that acts to reverse the effects of [[Cofilin]] by promoting reassembly of actin monomers. Profilin binds poly-proline domains.
Protein Gel Procedure\n\nArthur Millius and Andrew Houk 9/28/06\n\nOverview\n\nTo analyze proteins from biochemistry purifications.\n\nBuffers\n\n1x MOPS \n\nAntioxidant\n\nProcedure\n\nRun a gel and stain with gel code blue.\n\nClean the gel box and all dishes extensively with water and scrubbing with gloves (keratin will kill your silver stain since the stain is so sensitive).\n\nUse a 4-12% BT NuPage Gradient gel, rinse wells with water, then rinse wells with running buffer.\n\nLoad the gel into gel chambers.\n\nAdd 200 ml running buffer + 500 µl antioxidant to the inner chamber only. Check for leakage between the outer and inner chambers.\n\nAdd running buffer without antioxidant to outer chamber just above bottom of gel.\n\nMake samples up in 1x LB\n\nPut your frozen samples on 90C heat block for 5 minutes, pulse spin the samples (for an IP, immediately take the supernatant off the beads; you will have boiled the antibody and any remaining protein bound to the antibody off of the beads).\n\nLoad 10 µl sample into appropriate wells.\n\nRun for 50 minutes at 200 V.\n\nRinse with ddH20. Fix with 50% methanol and 7% acetic acid for 15 minutes. Rinse with ddH20, and stain with gel code blue for 1 hour. Wash with ddH20.
[[Abelson kinase]]\n[[Abi1]] \n[[Actin]] \n[[Adducin]]\n[[Aip1]]\n[[Akt]]\n[[alpha-actinin]] \n[[alpha-Pix]] \n[[Arap3]] \n[[Arf]] \n[[Arf1]]\n[[Arf6]] \n[[ArfGEFs]] \n[[Arl4]] \n[[Arno]] \n[[Arp2/3]]\n[[Asef2]]\n \n[[Bem1]] \n[[beta-Pix]]\n\n[[Capping protein]]\n[[Cdc24]]\n[[Cdc42]]\n[[CheY]]\n[[Chronophin]]\n[[Cofilin]]\n[[Coronin]]\n[[Cortactin]]\n[[CRAC]]\n[[Crk]]\n[[Cyfip1/2]]\n\n[[Dab2]]\n[[Dock]]\n[[Dock2]]\n[[Dock180]]\n[[Dynamin]]\n\n[[E3B1]]\n[[Elmo]]\n[[Ena]]\n[[Eps8]]\n[[EVL]]\n\n[[Filamin A]]\n[[Filopodin]]\n[[Formin]]\n[[FPCR]]\n\n[[Gα]]\n[[Gαi]]\n[[Gαq]]\n[[Gαs]]\n[[Gα12/13]]\n[[Gβγ]]\n[[GAB]]\n[[Gelsolin]]\n[[GDI]]\n[[GDS]]\n[[GPCR]]\n[[Grb2]]\n[[GSK3]]\n[[GTPase]]\n\n[[Hem-1]]\n[[Hem-2]]\n[[HS1]]\n\n[[Integrin]]\n[[Intersectin]]\n[[IQGAP]]\n[[IQGAP1]]\n[[IRSp53]]\n\n[[JMY]]\n\n[[Kette]]\n\n[[LIMK]]\n\n[[mDia]]\n[[Mena]]\n[[Merlin]]\n[[Microtubules]]\n[[MLCK]]\n[[Myosin]]\n[[Myosin I]]\n[[Myosin II]]\n[[Myosin IIA]]\n[[Myosin IIB]]\n[[Myosin VI]]\n[[Myosin phosphatase]]\n\n[[Nap1]]\n[[n-Chimaerin]]\n[[Nck]]\n[[N-WASP]]\n\n[[p190B]]\n[[Pak]]\n[[Pak1]]\n[[Par]]\n[[Paxillin]]\n[[PDK1]]\n[[Pir121]]\n[[PI3K]]\n[[PIX]]\n[[PKA]]\n[[PKB]]\n[[PKCz]]\n[[PLA]]\n[[Plastin]]\n[[PLD]]\n[[POR1]]\n[[P-Rex1]]\n[[PTEN]]\n\n[[Rac]]\n[[Rac1]]\n[[Rac2]]\n[[Rack1]]\n[[Ras]]\n[[RasC]]\n[[Rho]]\n[[RhoA]]\n[[RhoG]]\n[[RhoGAP4]]\n[[RhoGAPs]]\n[[RhoGDI]]\n[[RhoGEF]]\n[[RhoGEFs]]\n[[RhoGTPases]]\n[[RICS]]\n[[Rnd]]\n[[ROCK]]\n[[RPTK]]\n\n[[SCAR]]\n[[Shank]]\n[[SHIP]]\n[[Slingshot]]\n[[SLO]]\n[[SOS]]\n[[Spike1]]\n[[Sra1]]\n[[Stathmin]]\n\n[[Talin]]\n[[Tetraspanin]]\n[[Tiam-1]]\n[[Toca-1]]\n[[TorC2]]\n[[Trio]]\n[[Tropomyosin]]\n[[Tuba]]\n\n[[VASP]]\n[[Vav]]\n[[Vps34]]\n\n[[WASP]]\n[[WASP3]]\n[[WAVE]]\n[[WAVE complex]]\n[[WAVE1]]\n[[WAVE2]]\n[[WIP]]\n[[WRP]]
[[Actin Staining]]\n[[Amaxa Electroporation]]\n[[Competent Cells, how to make them]]\n[[Denoising software]]\n[[EDTA charging bacterial Rac]]\n[[Electroporation of HL60 cells]]\n[[Enzymatic assembly of DNA molecules]]\n[[EZ-Taxis]]\n[[Fibronectin coating]]\n[[FPLC Protocol]] \n[[Gateway LR Reaction]] \n[[GST Bead Assay]]\n[[Hem1 IP]]\n[[HL60 lysate]]\n[[How to submit your thesis in the Weiner Lab]]\n[[Lentiviral production using 293T cells]]\n[[Lentiviral infection of cultured cells]]\n[[Lipid Bead Rac Translocation]]\n[[Lipofectamine Transfection]]\n[[Membrane Hem1 IP]]\n[[Membrane Translocation]]\n[[Micropipette]]\n[[Objective Lens Distance Calibration]]\n[[NanoEnabler]]\n[[PBD Labeling]]\n[[Permeabilized Cells]]\n[[Pig Leukocyte Preparation]]\n[[PLB shRNA]]\n[[Protein Gel Procedure]]\n[[Purification of GSTRac/HisGDI]]\n[[Purification of Trio]]\n[[Quiescent HL60s]]\n[[Retrovirus production using GPG-293 cells]]\n[[Retroviral Infection of Adherent Fibroblasts]]\n[[Retroviral infection of HL-60 cells]]\n[[Splitting HL60 cells]]\n[[Splitting Adherent Cells, plus media recipes]]\n[[Streptolysin Purification]]\n[[The Bourne Infection]]\n[[Western Blot]]\n[[WRC Labeling]]\n[[XCelligence Assays]]\n\n
See [[Gierschik 2000|http://www.nature.com/nsmb/journal/v7/n2/full/nsb0200_122.html]]\n\nPurification of GST-Rac/His-GDI\nArthur Millius (3/27/10)\n\nOverview\nTo purify a Rac/RhoGDI complex for use in permeabilized cell experiments and in vitro assays.\n\nBuffers\nNickel Lysis Buffer = 300 mM NaCl, 50 mM NaH2PO4 pH 7.5, 5 mM MgCl2, 2 mM PMSF, 1 EDTA-free protease inhibitor per 50 ml, 0.4% CHAPS, 10 mM imidazole\n\nNickel Wash Buffer = 300 mM NaCl, 50 mM NaH2PO4 pH 7.5, 5 mM MgCl2, 20 mM imidazole\n\nNickel Elution Buffer = 300 mM NaCl, 50 mM NaH2PO4 pH 7.5, 5 mM MgCl2, 300 mM imidazole\n\nGSH Wash Buffer = 200 mM NaCl, 50 mM HEPES pH 7.5, 5 mM MgCl2, 2 mM DTT\n\nGSH Elute Buffer = 200 mM NaCl, 50 mM HEPES pH 7.5, 5 mM MgCl2, 20 mM GSH, 2 mM DTT\n\nDialysis Buffer = 100 mM NaCl, 20 mM HEPES pH 7.5, 5 mM MgCl2, 2 mM DTT\n\nProcedure\nSf9 Cell Growth\nVirus Amplication – Add 250 ul virus to 25 ml of Sf9. Harvest (spin cells and filter) after 28 – 72 hrs. Do not go longer because you do not want cells to lyse when amping virus. Add 25 ul P1 to 25 ml Sf9. Add 25 ul P2 to 200 ml Sf9. After small scale test production to check for the appropriate MOI, amplify Sf9 cultures to between 4 and 6 liters. Note: You should inject at a MOI between 1 and 5. You would like to balance protein production against cell death. Alternatively, you can infect at a low MOI have the cells amplify the virus, so that they subsequently infect the remaining culture to produce protein. \n \nDay 1 - Infect cells at a density of 1.8 – 2.2 million cells per ml at appropriate MOI. Note: I used the innova44R shaker in the Lim Lab TC room at 27 degrees, 110 rpm, 2.8 L glass culture flasks, and infected at a density of 2.2 million cells per ml.\n\nDay 2 – Count cells, they should not have increased in number if all the cells are infected and they will swell on the outside. Note: The cells were at a density of 2.2 million per ml.\n\nDay 3 – Again count cells, they now should have swelled considerably with the nucleus occupying most of the volume.\n\nDay 4 –Harvest cells. Spin cells 1500 x g for 15 minutes. Remove supernatant and save ~500 ml.\n\nResuspend cells in 200 mM NaCl 50 mM Hepes and transfer to 50 ml falcon and spin to packed cell pellet. Can save for later at -80 (snap freeze).\n\nPurification\nGet equal numbers (and size) of bacterial and Sf9 pellets from -80. Note: I usually do (2) 30 ml pellets of bacteria and (2) 30 ml pellets of Sf9.\n\nResuspend bacterial cells in 3-5 volumes 300 mM NaCl, 50 mM NaH2PO4 pH 7.5, 5 mM MgCl2, 2 mM PMSF, 1 EDTA-free protease inhibitor per 50 ml, 10 mM imidazole and disrupt by Lim sonication program 9 (0.5 sec on, 0.5 sec off for 20 secs) for three rounds cooling 1 min on ice between rounds. Rotate in cold room while…\n\nResuspend insect cells in 3 – 5 volumes nickel lysis buffer. Homogenize them using 40mL homogenizer (glass). 40 strokes for each 30mL.\n\nDivide the lysates into four-six tubes (each containing approximately 25mL of GST-Rac SF9 lysate and 25mL of His-GDI E. coli lysate).\n\nRotate the tubes at 4C for 1 hour.\n\nSpin the tubes at 15000 rpm, 20 minutes at 4C in Ti45. Collect supernatant on 50 ml falcon.\n\nIncubate each tube with 2mL Ni-NTA Superflow resin slurry.\n\nBatch wash with Ni2+ wash buffer two or three times (combine into 1 tube).\n\nPour through large column – should have around 10 ml of Ni resin total.\n\nElute in 30 mL Ni2+ elution buffer. Bradford fractions for eluted protein – combine fractions. Spike combined Ni eluate with 2 mM DTT.\n\nEquilibrate GST beads in GSH wash buffer\n\nBind to 5 ml GSH-agarose for 1 hour at 4 C. Pour into column.\n\nWash 50 ml GSH wash buffer. Elute with 15 ml GSH elution buffer. Dialyze fractions that look good by bradford into dialysis buffer.\n\nCentrifuge amicon concentration.\n\nSpec with nanodrop. Dope with glycerol to 10%. Recovered ~5.5 mg/ml Rac/GDI complex. \n
Purification of Trio DH PH\nArthur Millius (9/29/07)\n\nOverview\nTo purify the DH PH domain of trio complex for use in in vitro assays.\n\nBuffers\nTrio DH PH Lysis Buffer = 300 mM NaCl, 50 mM Na2PO4 pH 8, 10 mM imidazole, 1 mM PMSF, 1 tablet protease inhibitor per 50 ml\n\nTrio DH PH Wash Buffer = 300 mM NaCl, 50 mM Na2PO4 pH 8, 20 mM imidazole, 1 mM PMSF\n\nTrio DH PH Elution Buffer = 300 mM NaCl, 50 mM Na2PO4 pH 8, 250 mM imidazole, 1 mM PMSF\n\nDialysis Buffer = 300 mM NaCl, 50 mM Na2PO4 pH 8\n\nFreezing Buffer = 300 mM NaCl, 50 mM Na2PO4 pH 8, 10% glycerol\n\nProcedure\nE. Coli Cell Growth\nDay #1,2\nStreak fresh plate from glycerol. Note: It’s important that colonies are less than 48 hours old on the plate. Otherwise, you can get satellite colonies without your protein expressed.\n\nMake (2) 5 ml starter cultures in the morning. In the evening, start two 100 ml cultures overnight.\n\nDay #3\nPrepare (2) 1 L flasks and grow to O.D. 0.7 – 0.8 (~4 hours). Induce with 1 mM IPTG for another 4 – 5 hours. . Save both 1 ml of uninduced and induced bacteria. Spin 1 ml at 8000 rpm for 3 minutes, add 100 µl of water and 30 µl of 4x LB, and freeze at – 20 C. Spin at ~3000 rpm (~2000 g) for 30 min at 4 C in 500 ml conical bottles Note: It’s okay to spin faster, but this is the fastest speed for the centrifuges we currently have.\n\nScrap pellet into 50 ml falcon. Resuspend remaining debris with Trio DH PH lysis buffer and add to 50 ml falcon. Add PMSF and protease inhibitor. Sprinkle with lysozyme and immediately snap freeze.\n\nPurification\nSonicate 3 cycles of 20 s (0.5 on/ 0.5 off) at 50% amplitude (program 9) with the microtip. Place on ice and mix between each cycle. Save 100 µl as “lysate”.\n\nSpin at 35 K for 30 minutes in ultracentrifuge (3rd floor) at 4 C. Meanwhile wash 2 ml (4 ml slurry) of talon resin beads three times in Trio DH PH lysis buffer in a 50 ml falcon.\n\nAdd supernatant to beads in 50 ml falcon. Take a 100 µl aliquot of the high speed supernatant and label as “HSS”.\n \nIncubate while mixing at 4 C for >1 hour.\n\nSpin the beads at <1000 x g (I did 200 g) for 3 minutes.\nRemove supernatant and take a 100 µl aliquot of “unbound”.\n\nAdd 30 ml of Trio DH PH wash buffer and incubate while mixing for 10 minutes. Spin the beads at <1000 x g (I did 200 g) for 3 minutes and take an aliquot labeled “wash”. Repeat.\n\nResuspend in 5 ml Trio DH PH wash buffer and add to empty column. Resuspend remaining beads and layer on top. Let beads settle for 10 minutes. Allow column to drain and collect an aliquot as the last “wash” (usually wash 3).\n\nAdd 10 ml of Trio DH PH elute buffer and collect 700 µl fractions (~ 15 drops).\n\nRun PAGE to determine which fractions contain Trio. Can also do by Bradford.\n\nDialyze into Trio DH PH lysis buffer overnight. Resuspend with 10 % glycerol and snap freeze aliquots. (Note: I probably should have picked a more appropriate buffer to dialyze into. I also should have dialyzed in 10% glycerol.)
1. Fibronectin-coat wells w/ .1 mg/mL fibronectin. Fibronectin should be at 4C just prior to plating. \n2. Allow wells to plate 1 hour at room temp before aspirating fibronectin. Keep in humidified chamber.\n3. While fibronectin is plating, make 1X mHBSS + Ca + 2% BSA and blank RPMI (serum free) + 2% BSA (use low-endotoxin from Sigma for both solutions). Let BSA passively dissolve on top of solution in 37C bath.\n4. When BSA fully dissolved, sterile filter both solutions (this step might not be necessary but doesn’t hurt).\n5. At the end of 1 hour, wash wells 2X w/ D-PBS (Ca/Mg free) – use 300 ul/well if using 8-well plates.\n6. Store well in 300 uL D-PBS until using wells.\n7. Spin cells out of media, wash 1x w/RPMI + BSA, resuspend at normal density in RPMI + BSA and starve 1 hour in incubator (37 C, 5% CO2) in TC-treated dish. 5-day differentiated cells seems to give highest % of “off” cells but haven’t tested extensively.\n8. At end of 1 hour, concentrate 1.5 ml of cells -> 250 uL and plate in same media (RPMI + BSA). These #’s are based on having an initial density of ~ 1e6 cells/mL.\n9. Plate 5 min in incubator, remove and wash 5X with 600 uL of mHBSS + Ca + BSA, to remove almost all floating cells. Resuspend in 250 uL mHBSS + Ca + BSA. \n10. Image at RT, and perform experiment shortly after plating cells. Cells should be quiescent at least 5 min., haven’t carefully tested how long they stay off.\n\nDebugging: \nDifferent cell lines have different adhesivities, so it is possible that even lower fibronectin conc. is needed. Lower fibronectin concentration if there are obvious retraction fibers or tail retraction defects. Also drop fibro conc. if cells look initially off but reanimate before adding fMLP. \n\nBetter long term solution may be to silanize coverslip, and then add fibronectin on top of this – may give more control over adhesivity of surface. This is because cells stick even to uncoated glass. Theriot lab has protocol for doing this.\n\n
Hem1 KD shRNA fused to CFP and brought back with Hem1 YFP.
Rac binding domain of Pak fused to mCherry in a R6 line.
A [[Rac]] and [[Cdc42]] [[GAP]] involved in neurite outgrowth [[Akiyama 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16716191&query_hl=2&itool=pubmed_docsum]] is inhibited after phosphorylation by Ca/calmodulin-dependent kinase II.
ROCK, a Rho-effector kinase activated by RhoA, enhances cell contractility. ~ROCKs phosphorylate various substrates, including myosin light chain phosphatase, myosin light chain, ezrin–radixin–moesin proteins and LIM (for Lin11, Isl1 and Mec3) kinases, and mediate the formation of actin stress fibres and focal adhesions in various cell types. [[Ridley 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12778124&query_hl=33&itool=pubmed_DocSum]].\n\nIn fission yeast there is a related kinase called Orb6. Orb6 a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle [[Nurse 1998|http://www.ncbi.nlm.nih.gov/pubmed/9636183?ordinalpos=15&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Transmembrane protein-Tyr kinases act as receptors for growth factors such as platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, or the hormone insulin. Binding of growth factors to the extracellular domain results in multimerization and trans-autophosphorylation on Tyr residues and consequent activation of the catalytic domain. Often cytosolic proteins with Src-homology 2 ([[SH2]]) domains or phosphotyrosine binding ([[PTB]]) domains bind to the autophosphorylation sites of the activated receptors. The [[SH2]]- and [[PTB]]-containing proteins may be enzymes such as phosphoinositide 3-kinase ([[PI3K]]) or phospholipase C-gamma, or they may be adaptor proteins such as Shc, IRS1, [[Grb2]] or GAB1 that recruit other enzymes involved in signal transduction. In some cases, the activated receptor phosphorylates the associated signaling protein to modify its activity or create binding sites for additional [[SH2]]- or [[PTB]]-containing proteins [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
Constitutively active Rac injected into Swiss 3T3 cells induces lamellipodia [[Hall 1992|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1643658&query_hl=10&itool=pubmed_DocSum]], but fails to induce actin assembly in cell lysates [[Zigmond 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9230078&query_hl=8&itool=pubmed_DocSum]]. Constitutively active Rac in permeabilized cells induces free barbed ends and cannot be inhibited by DN [[Cdc42]] [[Glogauer 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10953003&query_hl=27&itool=pubmed_docsum]]. Constitutively active Rac injected into cells with WAVE2 knocked down reduces invasion of B16F10 cells [[Takenawa 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15608687&query_hl=18&itool=pubmed_docsum]]. Rac knock-out mice have defects in chemotaxis [[Rac1]] and chemokinesis [[Rac2]] [[Glogauer 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15308574&query_hl=13&itool=pubmed_DocSum]]. [[Sra1]], [[Nap1]], WAVE2 and [[Abi1]] translocate to the tips of membrane protrusions after microinjection of constitutively active Rac. Removal of [[Sra1]] or [[Nap1]] by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation [[Stradal 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14765121&query_hl=12&itool=pubmed_docsum]]. Active Rac read out by FRET localizes to both the front and back of a migrating neutrophil (but this could be an artifact of FRET with a moving cell) and depends on both stimulus and adhesion [[Bokoch 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12477392&query_hl=10&itool=pubmed_DocSum]]. Active Rac examined by fluorescent Pak-PBD only localizes to the front (Weiner, unpublished). Rac, but not other small GTPases, stimulates [[cGMP]] activity through [[Pak]] [[Xin-Yun Chang 2007|http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4MWYF5C-K&_user=4430&_coverDate=01%2F26%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059594&_version=1&_urlVersion=0&_userid=4430&md5=23bbb2e4474ec943e29656a7e646d13c]]. \n\nTuning the level of Rac1 may change the modality of migration between chemotaxis and random migration [[Yamada 2005|http://www.ncbi.nlm.nih.gov/pubmed/16129786?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n\nRac may be involved in memory loss in Drosophila [[Zhong 2010|http://www.ncbi.nlm.nih.gov/pubmed/20178749?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1]].\n\nAndrew's summary\n\nRac is the signal that I have spent the most time thinking about during my time in graduate school. After the collapse of the PIP3 empire, Rac emerged as the most likely internal signal to convey the spatial information that directs protrusion and guides the cell. \n\nGeneral Rac Biochemistry\n\nRac is a small GTPase. It is active when bound to GTP. Over time it hydrolyzes the GTP into GDP. When this happens, Rac becomes inactive. Rac will become active again only when it exchanges the GDP for another GTP. Both exchange and GTP hydrolysis are regulated by other proteins in the cell. Guanosine nucleotide exchange factors (GEFs) accelerate the exchange of GDP for GTP and thus activate Rac. GTPase Activatin Proteins (GAPs) accelerate GTP hydrolysis and thus inactivate Rac. Alleles of Rac which cannot hydrolyze GTP are constitutively active. Alleles of Rac that cannot bind GTP and instead sequester GEFs are dominant negative. Importantly, since any particular GEF may activate multiple different G-proteins, dominant negative Rac may actually inhibit other G-proteins besides Rac.\n\nAfter Rac is translated, hydrocarbon chains get added to a couple of residues near the C-terminus. In our lab, we refer to these hydrocarbon chains as “prenyl” groups, although I am not sure if that is technically the correct term for them. “Prenylated” Rac likes to bind the membrane, since the prenyl groups can directly intercalate into the membrane. Nevertheless, in mammalian cells, most? of the Rac is not bound to the membrane and instead is in the cytosol. This is possible because Rac binds a protein called Guanosine Dissociation Inhibitor (GDI) – which covers up the prenyl group and allows prenylated Rac to be soluble in water. As its name suggests, GDI dramatically slows down the basal exchange of GDP for GTP, which is quite fast otherwise (2-5 minute time constant in the absence of GDI). GDI does not bind Rac so tightly that Rac is completely unable to sample the membrane. Rather, Rac/GDI complexes are always coming apart and reforming and there is always a pool of Rac that is transiently sampling the membrane before being whisked away again by GDI. This membrane sampling is important because GEFs appear to only act at the membrane. Thus, exchange occurs exclusively at the membrane. GTP bound Rac cannot be extracted from the membrane by GDI and instead remains at the membrane and activates its effectors until the GTP is hydrolyzed. One of Rac’s primary effectors is the WAVE complex, which, in turn, activates the Arp2/3 complex to produce lamellipodia/pseudopodia. Other effectors include the kinase PAK and the machinery for reactive oxygen species production.\n\nI cannot emphasize the importance of the membrane enough in Rac biochemistry. Rac must sample the membrane for GTP exchange to occur. Rac must also be localized to the membrane for it to activate its downstream effectors. That’s right: a constitutively GTP-bound allele of Rac (Q61L or G12V) will not activate its effectors unless it is also localized to the membrane. Thus, if you mutate the prenylation sites on RacQ61L, and add a domain that can be inducibly dragged to the membrane you now have the means to turn on Rac signaling in an inducible manner. This has already been done and Rac mutants can be dragged around by light or drugs (rapamycin). Endogenous Rac activity can be artificially controlled by dragging catalytic fragments of GEFs or GAPs to the membrane using an identical scheme.\n\nThe Role of Rac during migration\n\nOne of the principles that a beginning biologist must adapt to is that different cells do things in different ways. A particular intracellular signal may have very different roles between cell types, even in the same organism. In the next few paragraphs I will illustrate this concept by discussing how Rac regulates motility/morphology in different cell types.\n\n* In addition to its role in motility, Rac has other important roles within the cell such as regulation of proliferation and reactive oxygen species production. I am not as interested in these other roles, so I know little about them and will not discuss them further. *\n\nRac in neutrophils\n\nThe experimental data in this system are the easiest to understand so we will start here. \n\nRac is critically important for migration in neutrophils. Neutrophils express Rac1 and Rac2. Glogauer made Rac1 and Rac2 knockout mice (tissue specific) as well as the combo deletion and analyzed the properties of the neutrophils. His findings:\n\n1. Rac2 deletion causes neutrophils to migrate 6 times more slowly than WT cells. Despite the reduction in speed, these cells still bias their movement up the gradient. They make a single front, but they have less F-actin than WT cells.\n\n2. Rac1 deletion causes neutrophils to ignore the gradient yet still move at the same speed as WT cells (they also have normal amounts of F-actin). Rac1 deletion also causes the cells to have multiple leading edges and tail retraction problems. Subsequent work from this lab demonstrated that Rac1 deletion prevents neutrophils from activating Rho effectively.\n\n3. The combined deletion of Rac1 and Rac2 leaves the cells with almost no motility at all, it is probably something like 0.1 – 0.2 micron/minute, whereas WT cells move at 6 micron/minute. These combo delete cells are also completely unpolarized morphologically.\n\nThus, Rac is clearly required for both morphological polarization and cell movement in the neutrophil. Unpublished studies from our lab have shown that Rac is active at the front of migrating neutrophils. Gary Bokoch’s lab has found Rac activity at the rear of migrating neutrophils using FRET probes, but this is probably a movement-induced artifact of the probes themselves.\n\nTobias Meyer’s lab has provided sufficiency data in the neutrophil by showing that the uniform recruitment of Rac to the neutrophil membrane causes uniform ruffling and uniform F-actin assembly (and no net migration). In addition to showing that Rac is sufficient to produce F-actin assembly in the neutrophil, this study shows that the polarized spatial distribution of Rac activity is important as well.\n\nThus, we know that Rac is required for migration and polarity in the neutrophil. We know that the spatial distribution of Rac is consistent with it being the signal that directs migration of the cell. We know that Rac is sufficient to induce ruffling and F-actin assembly in the neutrophil. The only thing that is missing is to locally activate Rac in the neutrophil and show that this local activation guides the neutrophil. As we shall see, this last experiment has been done in fibroblasts. Even without this last piece of data, I believe that Rac is the key intracellular signal that guides the neutrophil.\n\n------------------------------------------------------------------------------------------------------------\n\nRac in macrophages\n\nAs it turns out, neutrophils and macrophages are not as similar as I had once believed. Macrophages are much slower in vitro than neutrophils. The role of Rac also appears to be fairly different between the two cells.\n\nEarly studies by the Ridley lab in macrophages relied on dominant negative and constitutively active alleles of Rac (as well as Cdc42 and Rho) to distinguish the roles of these GTPases in morphology/migration. The injection of constitutively active Rac into macrophages causes uniform ruffling and F-actin and abolishes cell migration. Meanwhile, dominant negative alleles of Rac prevent ruffling/lamellipodia formation and abolish cell migration. Thus, it was concluded that Rac has a very similar role in macrophages and neutrophils.\n\nHowever, Ridley’s lab then knocked out both Rac alleles in macrophages. Surprisingly, they found that while Rac deletion profoundly affected cell morphology and reduced F-actin, these aberrantly shaped cells migrate with the same speed as wild-type cells and chemotax just fine. The combined deletion of both Rac alleles in macrophages reduces their invasiveness through matrigel, but migration on 2D surfaces is astonishingly normal – especially given their considerable morphological changes. The morphological changes are as follows: the deletion of either Rac1 or Rac2 causes the cells to be highly elongated with vary narrow pseudopodia at their ends and the combined deletion of both Rac isoforms causes the cells to adopt a stellate morphology, reminiscent of that seen in WAVE-knockdown S2 cells. Rac elimination also prevents macrophages from spreading in response to CSF.\n\nThus, Rac appears to be sufficient to induce ruffling/F-actin assembly in the macrophage. Rac is also required for the normal polarized morphology of macrophages as well as CSF-induced spreading. Aberrant, uniform spatial distributions of constitutively active Rac are able to prevent migration. Despite this, Rac is apparently not required for migration in macrophages. It is thus clear that morphology and migration are not always directly correlated. Rac is probably redundant with other signals in this cell type.\n\n\n\nRac in fibroblasts\n\nThe mode of migration in fibroblasts is quite different than in neutrophils. MEFs are much slower than neutrophils and they adhere to their substrate much more tightly than neutrophils – which is probably one of the reasons they are so much slower. Fibroblasts also generally lack an obviously polarized morphology like neutrophils, at least to my eyes. Signals like Rac and Rho are not polarized to the front and back of MEFs as they are in neutrophils. In MEFs, both Rac and Rho are active at the membrane in regions of cell protrusion. Also, work by Gaudenz Danuser’s lab has shown that both of these signals appear to become active AFTER membrane protrusion with Rho preceding Rac by about 30-40 seconds. This result has to be taken with a grain of salt because protrusion/retraction is often periodic in the fibroblast, and so it is hard to say that something happens before something else. \n\nThe first data implicating Rac in cytoskeletal regulation came from fibroblasts (more specifically 3T3 cells) in the early 90s. These early studies found that overexpression or microinjection of constitutively active Rac induced ruffling while dominant negative Rac prevented the ruffling that is normally induced by PDGF stimulation. Dominant negative Rac has also been shown to prevent chemotaxis towards PDGF and wound closure as well as reduce spreading on fibronectin. \n\nFibroblasts only express Rac1. Rac1 was finally knocked out of fibroblasts in 2006 by the Kwiatkowski lab. Their study found that Rac deletion causes cells to lack lamellipodia and instead become highly elongated and somewhat blebby. In addition to regulating fibroblast morphology, Rac also regulates focal adhesions in MEFs. There was also less PDGF-induced F-actin assembly in Rac knockout MEFs. Despite the major morphological/F-actin defects, the Rac knockout MEFs were able to migrate at 70% of the speed of WT MEFs and were also able to chemotax through Boyden chambers with 70% of WT efficiency. Once again, a severe perturbation to morphology (in this case, the destruction of lamellipodia) does not cripple migration. Intriguingly, knockout of WAVE2 almost completely abolishes chemotaxis through Boyden chambers in MEFs (Alt Lab 2003), although this difference could be due to different protocols between labs. Still, it appears that WAVE has additional inputs besides Rac that are able to mediate chemotaxis in the absence of Rac. WAVE is thus completely essential for chemotaxis in MEFs while Rac is not.\n\nIt is also worth noting that some people (John Condeelis, and his disciples) think cofilin generates a lot of the free barbed ends during MEF migration. This is a fundamentally hard question to address because cofilin is essential for monomer recycling, and it is hard to distinguish its essential role in recycling from additional roles in actin-based motility.\n\nWhile Rac is not required for the migration of fibroblasts, Rac is sufficient to produce F-actin ruffles and lamellipodia in fibroblasts. Recent work from Klaus Hahn’s lab using photouncaged Rac has demonstrated that the internal spatial distribution of Rac is capable of guiding fibroblast migration. This result was strengthened further by locally activating a dominant negative Rac allele and showing that it induces migration in the opposite direction. Thus, Rac appears to be an important signal for migration in fibroblasts but it is not an essential one. At present, nobody really knows what the essential internal migration signals in fibroblasts are.\n------------------------------------------------------------------------------------------------------------\n\nSummary:\n\nRac is clearly an important signal. It is sufficient to induce actin assembly and membrane protrusion in every mammalian cell-type that I am aware of. As far as I know, Rac is always required for the proper morphology of mammalian cells. Nevertheless, in both macrophages and fibroblasts, Rac does not appear to be required for cell migration. It could be redundant with other signals in those cell types. Interestingly, the WAVE complex does appear to be essential for migration in fibroblasts (nobody has looked in macrophages, to my knowledge) so perhaps WAVE is receiving other inputs besides Rac in fibroblasts and those other inputs compensate for a loss of Rac. The spatial distribution of Rac is capable of guiding fibroblast migration although it would be interesting to see whether uniform Rac prevents fibroblast migration. In neutrophils, Rac is required for migration and its spatial distribution is also clearly important since uniform Rac activation prevents migration. Thus, Rac appears to play a central role in neutrophil migration but is probably redundant with other signals in fibroblasts and macrophages.\n
Rac1 is expressed ubiquitously. Rac1 knock-out mice have a defect in chemotaxis and PIP3 localization [[Glogauer 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15308574&query_hl=13&itool=pubmed_DocSum]], Rho activation, and MLC phosphorylation [[Glogauer 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16809619&query_hl=3&itool=pubmed_docsum]]. Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of molecular weight 85 (catalytic subunit of PI3K), 110 (regulatory subunit of PI3K), 125 ([[Hem-1]]), 140 ([[Pir121]]) and 170 ([[IQGAP]]) kDa were detected, all of which were associated exclusively with guanosine 5'-[gamma-thio]triphosphate-bound Rac1, not with ~GDP-bound Rac1 [[Kasuga 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9148763&query_hl=37&itool=pubmed_docsum]]. Rac1 has a nuclear localization signal [[Williams 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15304504&query_hl=17&itool=pubmed_docsum]]. Rac1 has a 50-fold higher GTP hydrolysis rate than Ras and is less sensitive to the C3 component of Clostridium botulinum toxin compared to RhoA [[Didsbury 1992|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1597193&query_hl=33&itool=pubmed_docsum]]. Activated Cdc42 markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of RacGEF activity. RacGTP binding reduces the RacGEF activity Cerione 2005. Dbl-induced Rac activation can be blocked by dominant negative Rac and dominant negative Trio [[Zheng 2004|http://www.ncbi.nlm.nih.gov/pubmed/14597635]].\n
Mutant of Rac1, which cannot hydrolyze GTP.
96% of the Rac protein in human neutrophils is Rac2 [[Bokoch 1994|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7982999&query_hl=6&itool=pubmed_docsum]]. Rac2 knock-out mice have a defect in chemokinesis [[Glogauer 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15308574&query_hl=13&itool=pubmed_DocSum]]. Rac2 also regulates NADPH oxidase activity [[Bokoch 1991|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1660188&query_hl=25&itool=pubmed_DocSum]], [[Bokoch 1992|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1331090&query_hl=25&itool=pubmed_DocSum]].
See RhoGAPs
Rack1 interacts with Gβγ and is recruited to the leading edge in HL-60 cells. Down-regulation of Rack1 dramatically enhances chemotaxis of cells, whereas overexpression of Rack1 or a fragment of Rack1 that retains Gβγ-binding capacity inhibits cell migration. Rack1 competes specifically with PLC and PI3K for binding to Gβγ [[Hamm 2008|http://www.ncbi.nlm.nih.gov/pubmed/18596232?ordinalpos=20&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Rapamycin brings FRB and FKBP together.
Three highly related mammalian proteins, N-Ras, K-Ras, and H-Ras, form a subfamily of the p21 family of small guanosine triphosphatases (GTPases). These proteins bind GDP in the basal state and become activated by exchange proteins of the son of sevenless ([[SOS]]) or Ras ~GDP-releasing factor (~Ras-GRF) family. The release of GDP and binding of GTP "activates" Ras, allowing it to bind to downstream effectors, including the c-Raf protein ~Ser-Thr kinase, the ~RalGDS exchange factor, and phosphoinositide 3-kinase. Ras is anchored at the plasma membrane by a C-terminal farnesyl group. One function of Ras is to facilitate localization of its cytosolic effectors at the plasma membrane. Mutant forms of Ras that stabilize the ~GTP-bound state contribute to tumor formation [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
adapted from Ben Rhau's protocol\n//be sure to take proper biosafety precautions when handling virus.//\n\n1. approximately 12-18 hours prior to infection, seed 32,000 NIH-3T3 cells per well in a 12-well plate in normal growth media.\n\n2. Prepare a 600 microliter infection cocktail consisting of viral supernatant and Polybrene at a final concentration of 8 micrograms/mL in normal growth media.\n\nThe ratio of viral supernatant to growth media can be very high, such as 500uL supe : 100uL media. Often a range of dilutions is used 500 supe:100 media, 250:350, 125:475 e.g.\n\n3. Remove the media from the NIH-3T3 cells and add the 600 microliter infection cocktail to the cells.\n\n4. Return to the incubator for 4-5 hours.\n\n5. Remove the cocktail and replace with 1.4 mL of normal growth media.\n\n6. Assay/harvest the infected cells at 48 hours post infection.\n\n
from Aynur\n\nDay 1: \n\nPlate 1 million/mL cells per well in a 6 well plate in growth medium (without antibiotic). Add polybrene stock (usually 10 or 4 mg/mL) to 1 mL virus supernatant at 16 μg/mL. Then add the virus to the cells and spin them down for 90 min at 1000 x g at room temperature.\n\nAfter spinning, incubate the cells for 5 h at 37 ºC. Transfer the cell suspension into conical tubes (15 mL) and centrifuge the cells. Resuspend the cell pellet in 2 mL fresh media and transfer the cells in a new 6 well plate. Incubate the cells overnight at 37 ºC.\n\nDay 2:\n\nAdd 2 mL media (without antibiotic).\n\nDay 3:\n\nCheck expression of infected cells, if your construct contains fluorescent proteins. Start with the selection (you might first to do a killing curve with wild type cells to find the right drug concentration).\n
adapted from Orion's original protocol.\n\nGPG-293 cells stably express the genes required for producing packaging proteins for retroviruses. viral vectors we have used with success with this protocol are:\n1. the pMSCV series (Clontech)\n2. the pMXs series\n\n''Recipes''\n\n//Regular growth medium (+ drugs)://\nDMEM (Invitrogen cat.no. 12430)\n+ 10% FBS\n+ additional Glutamine (final 2 mM)\n+ Pen/Strep (final 1x)\n+ tetracycline (final 1 μg/mL)\n+ G418 (final 300 μg/mL)\n+ puromycin (final 2 μg/mL)\nsterilize by filtration.\nwrap bottle in foil.\nstore at 4 degrees.\nmake fresh every two weeks.\n\n//Transfection medium (no drugs)://\nDMEM (GIBCO, product number: 12430)\n+ 10% FBS\n+ additional Glutamine (final 2 mM)\n+ Pen/Strep stock (final 1x)\nsterilize by filtration.\nstore at 4 degrees.\n\n\n''Important notes'' \n1. regular growth media contains light sensitive antibiotics. protect from light with foil. pre-warm media by placing the bottle in the incubator ~ 1 h prior to passaging cells. this helps avoid the annoyance of the water bath.\n2. make up the regular growth media every two weeks (to keep drugs active).\n3. when passaging untransfected cells, supplement the PBS and trypsin with 1 μg/mL tetracycline to suppress VSV-G production.\n4. recommended dilution for passaging: 1:3 to 1:5. these cells grow pretty slowly. the day after passaging, cells will look a bit sick mostly due to adapting to fresh drug from the media. keep them on a routine passaging schedule so that they don't vary in health from too long of an absence from fresh drugs in media.\n5. note that the cells aren't incredibly sticky. pipet carefully, otherwise cells will de-adhere from the plate.\n\n''Protocol''\n\n//Day 0.//\nthis is the day before transfection. plate cells into 10 cm (or 6 cm) tissue culture dishes in regular growth media to achieve 70-80% confluency the next day for transfection.\n\n//Important:// confluency is a critical parameter during the transfection. if the confluency is too low or high, transfection efficiency will drop.\n\n//Day 1.//\n1. pre-warm transfection media. \n2. prepare the transfection mixture. we use the TransIT-293 transfection reagent (MirusBio, cat.no. MIR 2704) according to manufacturer's instructions. form the complexes in plain DMEM or Opti-MEM (Invitrogen cat.no. 31985). wait 15 min for complex formation.\n3. before adding transfection mix to cells: wash cells 1x with PBS minus tetracycline. then add transfection media. finally, add the transfection mixture. \n\n//note:// from here on out, the cells will only be cultured in transfection/drug-free media, which means that viral production and packaging are starting to happen. ''use appropriate personal protective equipment and biosafety measures (bleach, etc) when handling cells.''\n\n//Day 2.//\n~ 24 h post-transfection, change to fresh transfection media.\n\n//Days 3 and 4.//\nchange media once again sometime within these two days. original protocol states "change media on day 3.5. day 4 is also ok." i usually do it as close to day 3.5 to maximize the time of viral production until collection.\n\n//Day 5.//\n1st collection: pipet and keep viral media. replace with fresh transfection media. store viral media at 4 degrees.\n\n//Day 6.//\n2nd collection: collect media and replace cells with fresh transfection media.\n\n//Day 7.//\n3rd collection: collect media and discard cells. \n- filter the viral supernatants through a 0.22 micron filter (Millipore, cat.no. SLFV033RS). \n- viral supes can be stored at 4 deg for up to two weeks.\n \n//notes://\n- typically, virus from 1st collection gives best infection. sometimes i skip the 3rd collection.\n- the viral media can be combined during the collections. if you want to test the infectability of each collection, keep them in separate tubes.\n- viral supes can be frozen and stored at -80 degrees for future use, but transduction efficiency is reduced by ~ 50%. i see a reduction in transduction after ~ 10 days at 4 degrees, so i try to use mine within a week's time from the 1st collection.\n- if virus is not transducing efficiently, you have the option of [[concentrating your virus|http://www.ncbi.nlm.nih.gov/pubmed?term=pmscv%20concentrate]]
Ras homologous. This stub refers to RhoA. Rho activity is required to regulate the myosin-II-mediated contractility and adhesion necessary for normal tail retraction during leukocyte migration [[Bokoch review 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15752980&query_hl=11&itool=pubmed_DocSum]]. Rho injected into Swiss3T3 induce stress fibers. Rho acts on two major effectors, [[ROCK]] and [[mDia1]], among which [[mDia1]] produces straight actin filaments and aligns microtubules [[Narumiya 2006|http://www.ncbi.nlm.nih.gov/pubmed/16943426?ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Activated RhoA is distributed predominantly to the sides and rear of stimulated HL60 cells [[Bourne 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16537448&query_hl=13&itool=pubmed_docsum]]. fMLP induces multiple pseudopods (presumably through deactivation of RhoA) in the presence of dominant-negative variants of Gα12 and Gα13 [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887922&query_hl=19&itool=pubmed_docsum]].
RhoB colocalizes with active [[Src]]. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner [[Frame 2004|http://www.ncbi.nlm.nih.gov/pubmed/15572128]].
RhoG interacts directly with the Armadillo repeats ([[ARM]]) of [[Elmo]] in a ~GTP-dependent manner and forms a ternary complex with [[Dock180]] to induce activation of [[Rac1]]. The [[RhoG]]-[[Elmo]]-[[Dock180]] pathway is required for activation of Rac1 and cell spreading mediated by integrin, as well as for neurite outgrowth induced by nerve growth factor [[Negishi 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12879077&query_hl=14&itool=pubmed_DocSum]]. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP. Importantly, chemotaxis in response to soluble agonists was unaffected by lack of RhoG [[Stephens 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16621998&query_hl=5&itool=pubmed_docsum]]. RhoG depletion does not substantially inhibit cell adhesion, spreading, migration or Rac activation [[Schwartz 2008|http://www.ncbi.nlm.nih.gov/pubmed/18505794?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
See RhoGAPs
Binds cytosolic WAVE complex [[Weiner 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16417406&query_hl=23&itool=pubmed_docsum]].
~GTPase-activating proteins (~GAPs) stimulate the weak intrinsic ~GTP-hydrolysis activity of the GTPases, thereby inactivating them. GAP activity is regulated by several mechanisms, including protein-protein interactions, phospholipid interactions, phosphorylation, subcellular translocation and proteolytic degradation[[Settleman 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15246431&query_hl=5&itool=pubmed_DocSum]].
Guanine nucleotide dissociation inhibitors ([[GDI]]) keep the GTPase in the GDP-bound inactive conformation.
G12 family G protein activates Rho by binding of RhoGEF [[Sternweis 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9641915&query_hl=21&itool=pubmed_DocSum]]. Other members of the RhoGEF family are LARG, PDZ-RhoGEF and GTRAP48. [[Pak1]] associates with the [[DH]][[PH]] domain P115-RhoGEF but not with PDZ-RhoGEF or LARG [[Gutkind 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17224083&query_hl=16&itool=pubmed_docsum]].
Guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP for GTP [[Sondek 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15688002&query_hl=51&itool=pubmed_docsum]], thus activating RhoGTPases. Most RhoGEFs have a catalytic dbl homology ([[DH]]) domain and a pleckstrin homology ([[PH]]) domain which interacts with polyphosphoinositides. However, a newly discovered family does contain DH or PH domains, but still activate RhoGTPases [[Meller review 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16254241&query_hl=36&itool=pubmed_docsum]]
RhoGTPases are molecular switches that cycle between two conformational states: a ~GTP-bound “active” state and a ~GDP-bound “inactive” state involved in cytoskeleton rearrangements. They have a [[prenyl]] group, which localizes them to the membrane. They activate two types of actin nucleators, [[WASP]]/[[WAVE]] proteins and Diaphanous-related formins (DRFs), which induce different types of actin organization [[Ridley 2006 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16949823&query_hl=32&itool=pubmed_DocSum]]. RhoGTPases control [[superoxide generation]] and cell motility [[Bokoch 2005 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15752980&query_hl=17&itool=pubmed_docsum]].\n\nFor review of all major RhoGTPases see [[Der 2004 review|http://www.ncbi.nlm.nih.gov/pubmed/15020670]].
Showed that activated [[Cdc42]] pulls down [[IQGAP]] [[Cerione 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9305904&query_hl=39&itool=pubmed_DocSum]]. \n\nShowed the minimal binding surface for Cdc42 on [[Pak]] [[Cerione 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9760238&query_hl=39&itool=pubmed_DocSum]].\n\nShowed that [[alpha-Pix]] exists as a dimer, which allows its DH and PH domains to function in trans as a RacGEF. It can function as a GEF for either [[Cdc42]] or [[Rac]] as a monomer [[Cerione 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15306850&query_hl=39&itool=pubmed_DocSum]].\n\nShowed that activated Cdc42 can bind to [[alpha-Pix]] enhancing its GEF activity for [[Rac]]. Conversely, activated Rac inhibits the GEF activity of [[alpha-Pix]] [[Cerione 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15649357&query_hl=39&itool=pubmed_DocSum]].\n\nShowed that [[DOCK11]] is a GEF for Cdc42 that enhances its GEF activity upon activated Cdc42 binding [[Cerione 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16968698&query_hl=39&itool=pubmed_DocSum]].\n\nShowed that show that knocking-down beta-Pix or overexpressing a beta-Pix mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. The phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells [[Cerione 2010|http://www.ncbi.nlm.nih.gov/pubmed/20375009]].
Showed that CA Rac disrupts chemotaxis in Dicty [[Firtel 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10805781&query_hl=57&itool=pubmed_DocSum]].\n\nShowed that PI3K is necessary (blocked by LY294002) for chemotaxis in Dicty [[Firtel 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11389841&query_hl=57&itool=pubmed_DocSum]].\n\nShowed that PI3K localizes to the leading edge and [[PTEN]] localizes to the back in Dicty [[Firtel 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12062104&query_hl=57&itool=pubmed_DocSum]].\n\nShowed that PIP3 accumulates transiently after addition of chemoattractant in Dicty [[Firtel 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12802064&query_hl=57&itool=pubmed_DocSum]].\n\nShowed which of the six PI3K proteins in Dicty were responsible for chemotaxis [[Firtel 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17331950&query_hl=57&itool=pubmed_DocSum]].\n\nShowed that [[Ras]] activation does not occur in [[Gβγ]] null cells and basal [[Ras]] activity is elevated in [[LY294002]] treated and [[PTEN]] KO cells. Showed that RBD recruitment precedes CRAC under cAMP stimulation in Dicty. The experiments presented are consistent with a Ras PI3K positive feedback loop, but not conclusive [[Firtel 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17635933&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
All the morphological effects of Rnd proteins that have been described so far are related to an inhibition of ~RhoA-mediated contraction, indicating that Rnd proteins inhibit Rho. Studies have recently shown that Rnd1 and Rnd3 interact with [[p190]] RhoGAP, and recruit this protein at sites where Rho should be inhibited [[Hansen 2003|http://www.ncbi.nlm.nih.gov/pubmed/12842009?dopt=Abstract&holding=npg]]. Rnd3 might also increase the intrinsic GAP activity of [[p190]] RhoGAP and overexpression of Rnd3 decreases the fraction of RhoA that is bound to GTP [[Hansen 2003|http://www.ncbi.nlm.nih.gov/pubmed/12842009?dopt=Abstract&holding=npg]]. In addition, the effects of Rnd1 or Rnd3 protein expression are much less pronounced in [[p190]] ~RhoGAP-knockout cells. This set of data provides convincing evidence that Rnd1 and Rnd3 effects are mainly mediated by [[p190]] RhoGAP [[Chardin 2006 review|http://www.ncbi.nlm.nih.gov/pubmed/16493413?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Showed that cAR1 and cAR3 are partially redundant cAMP receptors in Dicty [[Insall 1994|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7949426&query_hl=1&itool=pubmed_DocSum]].\n\nShowed that both [[WASP]] and [[Scar1]] interact with the p21 subunit of the [[Arp2/3]] complex through their carboxyl termini [[Insall 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9889097&query_hl=1&itool=pubmed_DocSum]].\n\nShowed that calcium signaling is not required for chemotaxis in Dicty [[Insall 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10970875&query_hl=1&itool=pubmed_DocSum]].\n\nShowed that the [[Pir121]] mutants continually extends new pseudopods by widening and splitting existing leading edges rather than by initiating new pseudopods, consistent with an overactivation of SCAR phenotype. The double mutant resembles a [[SCAR]] mutant [[Insall 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12956949&query_hl=1&itool=pubmed_DocSum]].\n\nShowed that disruption of the gene that encodes [[Nap1]] causes loss of [[SCAR]] function. Cells lacking [[Nap1]] are small, rounded, with diminished actin polymerization, small pseudopods, and defects in adhesion. Despite these defects, [[Nap1]] mutant cells move and chemotax surprisingly effectively [[Insall 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16581519&query_hl=1&itool=pubmed_DocSum]].\n\nShowed that 1) pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo, 2) pseudopods are made at the same rate whether cells are moving up or down gradients, 3) directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones, and 4) [[LY294002]] affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI3K regulates the underlying mechanism of cell movement, rather than control of direction [[Insall 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17220879&query_hl=1&itool=pubmed_DocSum]].\n\nShowed that Dicty have WAVE complex "waves" and that SCAR/WAVE is essential for mitosis [[Insall 2010|http://www.ncbi.nlm.nih.gov/pubmed/20530573]].
Showed that several genes were required for actin-based lamella in S2 cells with a high-through put genomic approach [[Vale 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12975351&query_hl=29&itool=pubmed_docsum]].
Showed that a stable polarity axis in yeast bud formation could be generated through a positive feedback loop in which a stochastic increase in the local concentration of activated Cdc42 on the plasma membrane enhanced the probability of actin polymerization and increased the probability of further Cdc42 accumulation to that site [[Li 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12560471&query_hl=2&itool=pubmed_DocSum]].\n\nShowed that yeast bud polarity is coupled to both actin and signaling pathways [[Li 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15353546&query_hl=2&itool=pubmed_DocSum]].\n\n
SCAR for suppressor of cAMP receptor ([[cAR1]]) is another name for [[WAVE]] and was first identified in Dictyostelium as a [[WASP]]-related protein [[Saxe 1998|http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=9732292&dopt=Abstract]]. ~Scar1-GFP is recruited only to protruding lamellipodia and was absent from filopodia in B16F1 melanoma cells [[Small 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11257497&query_hl=43&itool=pubmed_DocSum]]. Both [[WASP]] and Scar1 interact with the p21 subunit of the [[Arp2/3]] complex through their carboxyl termini [[Insall 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=9889097]].
SH2 domains bind phosphorylated tyrosine residues.
Protein domain, which binds proline rich sequences. For an exhaustive review on different proline-rich binding domains, see [[Li 2005 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16134966&query_hl=148&itool=pubmed_DocSum]].
The Src-homology 2 (SH2)-containing phosphatases (SHIP1 and SHIP2) dephosphorylate the 5 position of the inositol ring to produce PI(3,4)P2. Loss of SHIP2 causes a dramatic increase in insulin sensitivity, suggesting that this phosphatase critically regulates PI3K signaling downstream of insulin [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]]. Genetic inactivation of SHIP1 leads to severe defects in neutrophil polarization and motility. By directing where PIP3 accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis [[Sasaki 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17173042&query_hl=3&itool=pubmed_docsum]].
SHIP! null neutrophils appear to be overadhered and resemble Rac1 null neutrophils [[Sasaki 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17173042&query_hl=3&itool=pubmed_docsum]].
Streptolysin-O (SLO) is a thiol-activated, membrane-damaging protein toxin of Mr 69,000 that is produced by most strains of beta-hemolytic group A streptococci. Native, primarily water-soluble toxin molecules bind to cholesterol-containing target membranes to generate large transmembrane pores of up to 30-nm diameter [[Sziegoleit 1985|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=3880730]].
Son of sevenless (SOS) is an exchange factor that stimulates GDP release from [[Ras]] to allow GTP binding and formation of the active state. Proline-rich regions of SOS bind to the Src-homology 3 (SH3) domains of Grb2. Phosphorylation of receptor or adaptor proteins on certain Tyr residues results in binding to the [[SH2]] domain of [[Grb2]], thereby recruiting the [[Grb2]]-[[SOS]] complex to the membrane where Ras resides. SOS also has a pleckstrin homology ([[PH]]) domain and dbl homology domain. The dbl homology domain can stimulate GDP/GTP exchange on [[Rac]], and the [[PH]] domain regulates the [[Rac]] exchange activity and mediates binding to the membrane [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]]. SOS binds PACSINs through the proline-rich region of SOS to coordinate actin polymerization by contributing to the local regulation of Rac activity [[McPherson 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11352907&query_hl=133&itool=pubmed_docsum]].
SST2 encodes a GTPase activating protein that stimulates GTP hydrolysis on the G protein alpha subunit Gpa1p. SST2 was original identified as a mutant that was supersensitive to alpha factor [[Otte 1992|http://www.ncbi.nlm.nih.gov/pubmed/7050665?dopt=Abstract]]. If you knockout the yeast RGS protein SST2, which GAPs the alpha subunit for the pheromone receptor, the cells are hypersensitive to pheromone [[Drubing 2003|http://www.ncbi.nlm.nih.gov/pubmed/12598904?dopt=Abstract]].
Showed that neutrophils take their first step upgradient [[Zigmond 1974|http://www.nature.com/nature/journal/v249/n5456/pdf/249450a0.pdf]].\n\nShowed that neutrophils can respond to a 1% gradient across their surface [[Zigmond 1977|http://www.jcb.org/cgi/reprint/75/2/606]]. \n\nShowed that neutrophils form ruffles over their cell body except the tail when fMLP is increased from 10 nM to 100 nM. Showed that cells migrating in 1 nM fMLP exposed to 100 nM fMLP persisted in the direction of migration while those allowed to become apolar were randomized relative to the old direction of migration. Examined development of polarity in neutrophils and showed morphological asymmetry toward the gradient within 1 min of gradient application. Observed that generation of new pseudopods occurred at the front. [[Zigmond 1981|http://www.ncbi.nlm.nih.gov/pubmed/7251666?ordinalpos=52&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that actin assembly in neutrophil lysate (LSS) could be induced by GTPγS-loaded in Cdc42 and GTPγS, but only by GTPγS-loaded in Cdc42 in HSS [[Zigmond 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9230078&query_hl=33&itool=pubmed_docsum]].\n\nShowed that [[Latrunculin]] inhibits f-actin accumulation at a concentration where [[Latrunculin]] should be sequestered inside a neutrophil. This suggests an accessory species, which facilitates [[Latrunculin]] binding to G-actin [[Zigmond 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12112154&query_hl=9&itool=pubmed_docsum]].
is the queen of the WeinerLab
See [[SCAR]] or [[WAVE]].
See [[SCAR]].
Showed that deficiency of [[WASP]] in both human and murine neutrophils results in profound defects in clustering of beta2 integrins, leading to defective adhesion and transendothelial migration under conditions of physiologic shear flow [[Simon 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16901726&query_hl=97&itool=pubmed_DocSum]].\n\nDeveloped a microfluidic device to image neutrophil rolling, adhesion, and transendothelial migration [[Simon 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17389960&query_hl=97&itool=pubmed_DocSum]].
Showed that GTPγS and [[Aluminum Fluoride]] stimulated actin assembly in electropermeabilized cells but had only marginal effects on intact cells and that stimulated could be blocked by GDPβS [[Grinstein 1989|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=2510721&query_hl=90&itool=pubmed_DocSum]].\n\nShowed that although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Showed that inhibition of PIP2 hydrolysis or increased PIP2 generation prevented the actin disassembly necessary for the completion of phagocytosis [[Grinstein 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15809313&query_hl=90&itool=pubmed_DocSum]].\n\nShowed that [[coronin-1]] is required for an early step in phagosome formation, consistent with a role in actin polymerization [[Grinstein 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15829569&query_hl=90&itool=pubmed_DocSum]].\n\nShowed that in primary human neutrophils, coronins-1-4 and -7 are expressed. Coronin-1 accumulates at the leading edge of migrating neutrophils and at the nascent phagosome. Inhibition of coronin function by transduction of a dominant-negative form of the protein leads to inhibition of chemotaxis and a reduction in neutrophil spreading and adhesion [[Grinstein 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17442961&query_hl=90&itool=pubmed_DocSum]].\n\nShowed that cell migration decreases with decreasing pH in neutrophils and confirmed the role of NHE1 in this process [[Grinstein 2008|http://www.ncbi.nlm.nih.gov/pubmed/18094149?ordinalpos=12&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Reconstituted NADPH oxidase activity in response to GTPgS in permeabilized neutrophils. 1) They used inhibitors in their cell permeabilization to eliminate types of proteins in their active fraction. For instance, wormannin has no effect on superoxide production, so PI3K is not likely to be involved in superoxide generation. 2) The determined the time course for important components leaking out of the cell. For instance, two of the phox proteins leaking our almost immediately, while it takes more than 30 minutes for Rac to leak out. 3) They also tried phox affinity columns, but without success [[Cockcroft 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10548486&query_hl=4&itool=pubmed_DocSum]]. He also reconstituted PLD activation by Arf in permeabilized HL60s [[Cockcroft 1994|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8290961&dopt=Abstract]].
Involved in post-synaptic density. Binds cortactin. Homer family proteins, which contain EVH1 domains bind Shank [[Kiim 2000 review|http://www.ncbi.nlm.nih.gov/pubmed/10806096]]. IRSp53 links postsynaptic shank1 to the small G-protein cdc42 [[Kreienkamp 2002|http://www.ncbi.nlm.nih.gov/pubmed/10806096]]. NMDA receptors are linked to the cytoskeleton by the PSD-95 protein complex. Shank forms a ternary complex with PSD-95 by binding via its PDZ domain to the C terminus of ~PSD-95-associated protein GKAP [[Sheng 1999|http://www.ncbi.nlm.nih.gov/pubmed/10433268?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_SingleItemSupl.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&logdbfrom=pubmed]]. In brain, the PDZ domain of Shank forms a complex with the C-terminus of [[beta-Pix]] and [[beta-Pix]] -associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42 [[Kim 2003|http://www.ncbi.nlm.nih.gov/pubmed/12626503]]. See also [[Gundelfinger 2002 revew|http://www.ncbi.nlm.nih.gov/pubmed/12065602]].
Showed that N-WASP-WIP complex-mediated actin polymerization is activated by phosphatidylserine-containing membranes depending on membrane curvature in the presence of Toca-1 or FBP17 and in the absence of Cdc42 and PIP2 [[Suetsugu 2008|http://www.ncbi.nlm.nih.gov/pubmed/18923421]].\n\nA nice review of the proposed functions of membrane curvatures mediated by the BAR domain superfamily proteins [[Suetsugu 2010|http://www.ncbi.nlm.nih.gov/pubmed/20435640]].
Paper to reference:\n\nShowed that single molecule actin molecules undergo retrograde flow and measured lifetime and distribution [[Watanabe 2002|http://www.ncbi.nlm.nih.gov/pubmed/11834838]].\n\nShowed that actin flows back at different speeds depending on location within cell [[Danuser 2004|http://www.ncbi.nlm.nih.gov/pubmed/15375270]].\n\nShowed that agonist increased cAR mobility at leading edge of Dicty [[Schmidt 2008|http://www.ncbi.nlm.nih.gov/pubmed?term=18469015]].\n\nShowed that Crac-GFP is highly dynamic in Dicty [[Ueda 2006|http://www.ncbi.nlm.nih.gov/pubmed?term=16507590]].\n\nModeled how noise affects directional sensing in Dicty [[Ueda 2008|http://www.ncbi.nlm.nih.gov/pubmed?term=17416630]].\n\nShowed that cAR and Gβγ are coupled. Possible tracking programs [[Tian Jin 2010|http://www.ncbi.nlm.nih.gov/pubmed/20876874]].
Proteins, People and Protocols
The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration [[Mizuno 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16230460&query_hl=5&itool=pubmed_docsum]]. In mammalian cells, human SSH homologs (hSSHs) suppressed LIMK1-induced actin reorganization. Furthermore, SSH and the hSSHs dephosphorylated P cofilin in cultured cells and in cell-free assays [[Mizuno 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11832213&query_hl=5&itool=pubmed_DocSum]].\n\nSlingshot-1L, a cofilin-activating phosphatase, localizes to focal adhesions and interacts with coronin 2A [[Bear 2009|http://www.ncbi.nlm.nih.gov/pubmed/19654210?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]..
See [[SOS]].
The use of microfluidics and the Chavrier system to dissect components of polarization.
Arabidopsis [[Dock180]] homolog, which binds the [[SCAR]]/[[WAVE]] complex by yeast-2-hybrid and Rop GTPases [[Hulskamp 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17267444&query_hl=11&itool=pubmed_docsum]].
A new class of actin nucleator, which relies on [[WH2]] domains to 'stich' together actin filaments [[Mullins 2005|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=15674283&ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and biochemically interacts with a [[Formin]] [[Cappuccino]] to enhance its nucleation [[Mullins 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17923532&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
See [[Spire]].
Tissue culture protocol\n\nThawing cells:\n• Because cells are frozen in media that contains 10% DMSO, thaw the cells quickly using the 37C water bath. Transfer cells from cryotube to 10mL of fresh media. Spin cells at 400g for 2min at room temperature. Decant the media. Resuspend in fresh media. Aliquot into flack containing media of choice. \n\nSplitting Adherant Cells:\n• When cells reach a confluence of 70-95% remove media from the flask. Wash cells with 10mL of pre-warmed PBS (lacking Ca+2/Mg+2) 2-times. Aspirate of buffer after each wash. Don’t cut corners, by washing the cells only once. The trypsin will be less affective (trust me, your only making your job more difficult). Add 2mL of trypin solution to the flask containing no media. Rock the media. If you wash the cells well with PBS prior to adding the PBS, you should be able to trypsinize the cells in about 2 minutes. Quench the reaction by adding 10mL of warm media. Transfer media and cells to 15mL conical an spin cells at 400g for 2 minutes. Decant the media and resuspend the cell pellet in fresh warm media. Triturate cells really well. You can even vortex the cells to break apart the clumps. Aliqout some of these cells into a flask containing fresh media to split the cells. \n\n3T3 cells + FLIP-IN construct (zeocinR) (from Lim lab)\n• Media_Dibecco Modified Eagles (D-MEM) High glucose, 10% FBS, 5mL of antibiotics/antimycotics. \n• After thawing cells, plate in 30mL of media using a T75 flask. After 24hrs at 37C, add [zeucin]f = 100μg/mL (stock is 100mg/mL). Zeocin is light sensitive. \n• Split cells every 2-3 days\n\nB16F1 (mouse melanoma cells)\n• Media_MEM Eagles with Earle’s, 10% FBS, 5mL of antibiotics/antimycotics, 5mL of 100x stock non-essential amino acids. \n• After thawing cells, plate in 30mL of media using a T75 flask.\n• Split cells every 2-3 days\n\nB16F10 (mouse melanoma cells)\n• Media_ Dibecco Modified Eagles (D-MEM) High glucose, 10% FBS, 5mL of antibiotics/antimycotics.\n• After thawing cells, plate in 30mL of media using a T75 flask.\n• Split cells every 2-3 days\n\nSpanish PAE (porcin aortic epithelia cells)\n• Media_F12 Ham’s, 10% FBS, 5mL of antibiotics/antimycotics.\n• After thawing cells, plate in 30mL of media using a T75 flask.\n• Split cells every 2-3 days\n\nRacV12 PAE (porcin aortic epithelia cells)\n• Media_F12 Ham’s, 10% FBS, 5mL of antibiotics/antimycotics.\n• After thawing cells, plate in 30mL of media using a T75 flask. After 24hrs at 37C, add [hygromycin]f = 60μg/mL (stock is 60mg/mL) and [puromycin]f = 0.3μg/mL \n• The hygromycin and puromycin can be added to a bottle of media and stored in the deli fridge. \n• Split cells every 2-3 days\n\nFreezing Cells:\n• Assume you have 30mL in a T75 flask that contains the strain of interest at 100% confluency. \n• Trpysinize cells as if you were going to split the cells. Quench the reaction with 10 mL of media. \n• Spin down cells. Remove media\n• Bring them up in freezing media.\n• Resuspend cells in 4.5mL of pre-chilled growth media + 10% sterile DMSO (cold). This will be enough for 3 aliqouts/cryotubes. If the cells contain a cassette with a selectable marker, don’t use media with the antibiotic. It is OK to use media that contain the normal antitbiotic/antimycotic mixture. \n• Prelabel tubes with EtOH resistance ink. Date? Cells type?\n• Slow freeze in “Mr. Frosty” using 100% isopropanol in the container. Alternatively, use styrofoam rack to slow freeze the cells in the -80C freezer.
Thawing cells:\nbecause cells are frozen in media that contains 10% DMSO, thaw the cells quickly using the 37C water bath. transfer cells from cryotube to 10 mL of fresh pre-warmed media. spin cells at 400 x g for 2 min at room temperature. aspirate media and resuspend in fresh growth media in a new dish.\n\nPassaging Adherent Cells:\nwhen cells reach a confluence of 70-95%:\n- aspirate media.\n- wash cells with 10 mL of pre-warmed D-PBS (lacking Ca+2/Mg+2)\n- add 0.5 - 1.0 mL of trypsin. ensure the bottom of dish is covered. place in incubator for 2 min (or, for 293T cells, leave in the hood for 1 min).\n- quench the reaction by adding pre-warmed growth media to 10 mL total volume. \n- (optional. this step depends on your cell line): transfer cell solution to 15 mL conical an spin cells at 400g for 2 minutes. aspirate media and resuspend cells in fresh growth media.\n- dilute your cells to the appropriate density in new dishes.\n\nNIH-3T3 cells (mouse fibroblast):\n• D-MEM High glucose (Invitrogen cat no 11995), 10% bovine calf serum, plus Pen/Strep/Glutamine. \n• passage cells at 1:10 dilution every 2-3 days. DO NOT LET THEM OVERGROW.\n\nLx-293T cells (human embryonic kidney, for viral production):\n• D-MEM High glucose (DME-H21) from CCF, 10% fetal bovine serum, plus Pen/Strep/Glutamine.\n• passage cells at 1:5 dilution every 2-3 days.\n\nHT-1080 cells (human fibrosarcoma):\n• D-MEM High glucose (Invitrogen cat no 11995), 10% fetal bovine serum, plus Pen/Strep/Glutamine. \n• passage cell at 1:10 every 2-3 days.\n\n3T3 cells + FLIP-IN construct (zeocinR) (from Lim lab)\n• Media_Dibecco Modified Eagles (D-MEM) High glucose, 10% FBS, 5mL of antibiotics/antimycotics. \n• After thawing cells, plate in 30mL of media using a T75 flask. After 24hrs at 37C, add [zeucin]f = 100μg/mL (stock is 100mg/mL). Zeocin is light sensitive. \n• Split cells every 2-3 days\n\nB16F1 cells (mouse melanoma):\n• Media_MEM Eagles with Earle’s, 10% FBS, 5mL of antibiotics/antimycotics, 5mL of 100x stock non-essential amino acids. \n• After thawing cells, plate in 30mL of media using a T75 flask.\n• Split cells every 2-3 days\n\nB16F10 (mouse melanoma cells)\n• Media_ Dibecco Modified Eagles (D-MEM) High glucose, 10% FBS, 5mL of antibiotics/antimycotics.\n• After thawing cells, plate in 30mL of media using a T75 flask.\n• Split cells every 2-3 days\n\nSpanish PAE (porcin aortic epithelia cells)\n• Media_F12 Ham’s, 10% FBS, 5mL of antibiotics/antimycotics.\n• After thawing cells, plate in 30mL of media using a T75 flask.\n• Split cells every 2-3 days\n\nRacV12 PAE (porcin aortic epithelia cells)\n• Media_F12 Ham’s, 10% FBS, 5mL of antibiotics/antimycotics.\n• After thawing cells, plate in 30mL of media using a T75 flask. After 24hrs at 37C, add [hygromycin]f = 60μg/mL (stock is 60mg/mL) and [puromycin]f = 0.3μg/mL \n• The hygromycin and puromycin can be added to a bottle of media and stored in the deli fridge. \n• Split cells every 2-3 days\n\nFreezing Cells:\n• freezing media is typically 2X serum concentration + 10% DMSO.\n• trypsinize cells, quench reaction with growth media to 10 mL total volume. \n• centrifuge cells and remove media. while spinning, pre-label cryovials with ETOH resistant ink. date, cell type, your initials, # of cells, etc.\n• for each vial, resuspend in 1 mL of freezing media.\n• if cells have been cultured with a selectable marker, do not add antibiotic to the freezing media.\n• aliquot to cryovials and slow freeze in “Mr. Frosty” using 100% isopropanol in the container. alternatively, use styrofoam rack to slow freeze the cells in the -80 C freezer.\n
Splitting HL-60 cells—Weiner Lab Sept 2006 \n\nBackground: Neutrophils are a great system for chemotaxis, but mature neutrophils live for only a short time and are difficult to transfect. For many of our studies, we use HL-60 cells originally derived from a human leukemia cell line. These cells can be propagated indefinitely in culture and can be differentiated to cells that behave very much like human neutrophils by adding 1.5% DMSO. They are easily infected with retroviruses and lentiviruses and can also be transiently transfected via electroporation.\n\nCulture Media: 1 Liter RPMI/Hepes\n+ 200mls Heat Inactivated Fetal Bovine Serum \n+ 12 mls antibiotic/antimycotic\n\nCell Culture: HL-60 cells are happiest when kept at a density between 0.1 and 2 million cells/ml. Count cells on hemacytometer (Add 10 µl to hemacytometer. The total # of cells in 16 squares divided by 100 = # million cells/ml), counting only round, non-clumped cells. I usually split cells on Tues and Friday, splitting to 0.15 million/ml on Tuesday and 0.1 million/ml on Friday. \n\nCell Differentiation: I also differentiate cells Tues and Friday by splitting to 0.15 million per ml in Culture Media + cells + 1.5% DMSO final. So if cells were at 1 million/ml, would add 31.4 mls media, 600 microliters DMSO, mix well, then add 8 mls cells. Note—add DMSO and mix well prior to adding cells. DMSO is somewhat viscous and denser than culture media. Use only hybrimax DMSO for differentiation. Cells will not need to be split following addition of DMSO. Cells stop proliferating upon differentiation and typically achieve a density of 1-2 million/ml at 7 days post differentiation. Cells are best at 6 days after adding DMSO but can be used 5-8 days after adding DMSO.\n\nFreezing cells—spin down cells at 400g for 5min, resuspend in ice cold freezing media (Culture media + 10% DMSO) at 20 million cells/ml. Aliquot 1.8mls each into cryotubes, sandwich between 2x 15ml falcon Styrofoam, put at -80C for 2 days, then transfer to liquid nitrogen.\n\nThawing cells: 1. quickly thaw cells at 37C just until last bit of ice has melted. \n2. Resuspend in 20mls ice cold cell culture media, \n3. spin 400g for 5min (as little as 2 min), \n4. aspirate supernatant.\n5. Resuspend pellet in 20mls prewarmed complete media and put into T75.\n6. Grow until at 1-2 million/ml (usually takes 5 days or so) before splitting.\nBest to wait until next split before differentiating.\nGood idea to amplify cells and freeze more if stock is limiting.\n\n[img[culture|http://img508.imageshack.us/img508/9466/figure1rgbjk7.gif]]\n\n''Fig. 1. Passaging and differentiating HL-60 cells.'' When cells reach a density between 1-2 million cells/ml, split to 0.15 million cells/ml in a total volume of 10 ml prewarmed culture media. Differentiate cells in culture media plus 1.5% DMSO; cells take ~5 days to become migratory.
Specifically Rac associated. See [[Sra1]].
Specifically [[Rac1]]-associated protein 1 [[Klambt 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15269173&query_hl=58&itool=pubmed_docsum]] interacts with WAVE2 and [[Abi1]] and translocates to the tips of membrane protrusions after injection with constitutively active [[Rac]] [[Stradal 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14765121&query_hl=60&itool=pubmed_docsum]]. EM of Sra1 colocalizes with Rac at actin sites [[Kaibuchi 2008|http://www.ncbi.nlm.nih.gov/pubmed/18497446?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
A tyrosine kinase, which phosphorylates [[WASP]] and a host of other proteins. See wikipedia article [[Src|http://en.wikipedia.org/wiki/Src_(gene)]]. Src activity is suppressed by Latrunculin or cytocholasin and its translocation to the membrane disrupted by Scar1 VCA domain [[Frame 2004|http://www.ncbi.nlm.nih.gov/pubmed/15572128]].
Stathmin/Op18 binds αβ tubulin dimers, inhibits tubulin polymerization, and promotes microtubule catastrophe. Phosphorylation of Op18 at several sites, particularly Ser16, inhibits the binding of Op18 to microtubules and prevents its destabilizing activity. Phosphorylation of Op18 at Ser16 is stimulated by the epidermal growth factor receptor in a [[Rac]]/[[Cdc42]]-dependent manner, and this effect was blocked by expression of the [[Pak1]] autoinhibitory domain. [[Bokoch 2003 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12676796]]
Showed that cell polarity can arise solely through positive feedback given limiting components [[Altshuler 2008|http://www.nature.com/nature/journal/v454/n7206/full/nature07119.html]].
Strategy for purification of MBP-Streptolysin O from E. coli\n\nAndrew Houk and Arthur Millius 8/8/2006\n\nPreviously\n\nWe used BL21 strain of E. coli (this strain is optimized for protein expression). The MBP-SLO was under an IPTG-inducible promoter (Lac repressor). We used chemically competent cells w/37C 30 second heat shock in presence of the plasmid, let them recover in absence of antibiotic, then streaked individual cells on plates w/ampicillin. Let them grow over night at 37C. Pick individual colony (use well spaced colonies, watch out for satellite colonies). Place colony in 50mL starter culture (50mL of media containing amp) shake overnight at 37C. Dilute bt. 1:50 to 1:200 in 3L (using wide-bottom 1L Erlenmeyer flasks). Grow until OD600 is 0.5-0.8 then induce with final concentration of 0.4mM IPTG. Incubate 90 minutes. Add them to 1L containers spin them down. Resuspend in PBS 30mL or so. Spin them down again. Remove a small amount of bacteria out and bring them back up in sample buffer. Snap froze the rest in liquid nitrogen then stored at -80C.\n\n\nOverview\n\nWe will use affinity purification of the Maltose Binding Protein tag on the streptolysin O. This tag does not need to be cleaved off as the fusion protein is as active as cleaved SLO [Weller, Muller, Messner et al Eur. J. Biochem. 236, 34-39 (1996)]\n\nBuffers\nLysis Buffer = 10mM NaPO4, 0.5M NaCl, 10mM beta-mercaptoethanol, 10mM EDTA, pH=7, 1 tablet protease inhibitor per 50 ml, 0.5 mg/mL lysozyme (add immediately before use)\n\n \n\nWash Column buffer = 100mM NaCl, 20mM HEPES, 10mM beta-mercaptoethanol, 1mM EDTA, 1mM PMSF\n\n \n\nElution Column buffer = 100mM NaCl, 20mM HEPES, 10mM beta-mercaptoethanol, 1mM EDTA, 10mM maltose, 1mM PMSF, 1 protease inhibitor tablet per 50 ml\n\nProcedure\n\nThis protocol begins with a pellet of E. coli overexpressing MBP-SLO.\n\nResuspend cells in 40mL lysis buffer (without lysozyme) and freeze them down.\n\nThaw the cells and add lysozyme to 0.5mg/mL, incubate 15min at 4C\n\nCombine aliquots, dilute 1:2 in wash column buffer, and sonicate 30 secs on, 30 secs off for 10 minutes or until viscosity noticeable diminshes\n\nKeep 200 µl of the lysate for later analysis with SDS-PAGE\n\nCentrifuge the sonicated cells at 40,000g for 1 hour at 4C (to clear the lysate and prevent it from clogging up the column). [For FPLC we would filter prior to adding to column in addition to the spin]\n\nDuring the spin prepare the two columns:\n\nAdd the amylose resin to the column (5cm x 2.5cm). Prepare 5 ml of amylose resin (NEB) by adding ~7.5 ml to empty column. Let settle. Add 15 ml to column and at the same time add the frit. Press frit to bottom of column flesh with top of beads. Wash with 10 ml more of column buffer. Cap to prevent column drying.\n\nKeep 200 µl of supernatant from centrifuged lysate for later analysis with SDS-PAGE\n\nAfter the centrifugation dilute the lysate 1:2 with wash column buffer and apply the diluted lysate to the first column.\n\nCollect the first flowthrough and add to the second column.\n\nCollect the second flowthrough in 50 ml falcon tubes and store on ice.\n\nWash both columns with 10 total volumes (10 times the amount of resin in the column) of the wash column buffer (-PMSF). In this case, add 30 ml and check with Bradford assay. Add 10 ml, 5 ml, and then 5 ml checking for cleared protein after each.\n\nNote: For Bradford assay, prepare a 96 well plate with 180uL of Bradford reagent (diluted 1:4 in ddH2O) and add 20uL of each fraction to the appropriate well.\n\nKeep 200 µl of last fraction for later analysis with SDS-PAGE.\n\nElute with the elution column buffer\n\nCollect 2mL fractions and check for eluted protein with Bradford assay.\n\nCombine the peak fractions and dialyze overnight (10,000 MWCO from Pierce) into the wash column buffer (-PMSF). \n\nDay two:\n\nRemove buffer, replace with fresh wash column buffer, and dialyze again for at least two hours (ideally four hours).\n\nRun a gradient SDS-PAGE of 10 µl from all of the samples. Dilute the final sample and BSA standard to attain relative concentration.
See [[SLO]]
fMLP also stimulates superoxide production in neutrophils. This activity was fully reconstitued with four proteins [[Abo 1994|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1512217&query_hl=28&itool=pubmed_DocSum]]. The relevant GEF [[P-Rex1]] was purified on the basis of Rac activation with with PIP3 and Gbetagamma as inputs [[Stephens 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11955434&query_hl=5&itool=pubmed_DocSum]]. Superoxide production can also be stimulated by GTPgS [[Cockcroft 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10548486&query_hl=4&itool=pubmed_DocSum]], downregulates Rho activation (via Rhotekin pull-down) and is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading [[Bar-Sagi 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12598902&query_hl=8&itool=pubmed_docsum]]. Superoxide generation kills neutrophils through activation of proteases by potassium influx [[Segal 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11907569&query_hl=20&itool=pubmed_docsum]].
Recombining protein modules to understand protein circuits. The state of synthetic biology in 2010 [[Lim 2010 review|http://www.ncbi.nlm.nih.gov/pubmed/20485291]].
Systems biology [[Kitano 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11872829&query_hl=11&itool=pubmed_docsum]] focuses on a process carried out by a set of components that would not occur given any individual component. A great introduction is in the article: Can a biologist fix a radio? [[Lazebnik 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12242150&query_hl=7&itool=pubmed_docsum]]. Bistability [[Ferrell 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11891111&query_hl=9&itool=pubmed_docsum]] was shown elegantly in oocyte maturation [[Ferrell 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9572732&query_hl=1&itool=pubmed_docsum]] and in the cell cycle [[Ferrel 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12629549&query_hl=9&itool=pubmed_docsum]].
T cells belong to group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and NK cells by the presence of a special receptor on their cell surface that is called the T cell receptor (TCR). The abbreviation "T", in T cell, stands for thymus since it is the principal organ for their development.
Total Internal Reflection Fluoresence occurs when light reflects a refractive index interface at the critical angle to generate an evanescent wave (based in quantum tunneling).
See TorC2
Identified [[WAVE]] through [[VCA]] homology to [[WASP]]. Showed that ectopically expressed [[WAVE]] induces the formation of actin filament clusters and that CA Rac induces the translocation of endogenous [[WAVE]] from the cytosol to membrane ruffling areas [[Takenawa 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9843499&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that WAVE1 and WAVE3 have a similar expression profile, with WAVE3 being expressed in the brain. Showed that WAVE2 is enriched in leukocytes [[Takenawa 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10381382&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that [[WAVE]] is phosphorylated by MAP kinases in Swiss 3T3 cells and that this phosphorylation is required for PDGF-induced membrane ruffling [[Takenawa 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10488099&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that a [[WAVE]] mutant lacking the acidic domain still has an effect on actin [[Takenawa 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10833423&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that activated [[Rac]] binds to the amino terminus of IRSp53, and the carboxy-terminal SH3 domain of IRSp53 binds to [[WAVE]] [[Takenawa 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11130076&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that WASP-interacting protein ([[WIP]]) interacts directly with N-WASP and actin and that microinjection of [[WIP]] into NIH3T3 fibroblasts induces filopodia [[Takenawa 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11331876&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that full-length IRSp53 binds [[Rac]] much less efficiently than the N-terminal fragment (IP), but that full-length IRSp53 associates with [[Rac]] in the presence of WAVE2 [[Takenawa 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12054568&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that WAVE2 and IRSp53 colocalized in the tips of protruding lamellipodia and filopodia in B16 cells and in the absence of Ena/VASP [[Takenawa 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12734400&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that WAVE2 null mice have defects in angiogenesis and formation of lamellipodia in epithelial cells [[Takenawa 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12879075&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that WAVE1 and WAVE2 have different roles in fibroblasts [[Takenawa 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14536061&query_hl=95&itool=pubmed_docsum]].\n\nShowed that [[N-WASP]] and [[Grb2]] are involved in [[podosome]] formation [[Takenawa 2008|http://www.ncbi.nlm.nih.gov/pubmed/18606851?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\nShowed that the proline-rich sequence of [[Eps8]] interacts with the SH3 domain of IRSp53. Showed that CA Rac can induce further Rac activation and that this activity is inhibited by a truncated version of [[Eps8]] [[Takenawa 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15289329&query_hl=79&itool=pubmed_DocSum]].\n\nShowed that the basic region of WAVE binds PIP3 and PIP2. The amino-terminal region of WAVE including the basic region is localized to the leading edge upon PDGF stimulation or CA Rac. This recruitment could occur in the presence of DN Rac, but is completely block by PI3K inhibitors [[Takenawa 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15107862&query_hl=95&itool=pubmed_docsum]].\n\nShowed that disruption of WAVE2 by RNAi prevent Rac-induced membrane ruffling in B16F10 cells [[Takenawa 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15608687&query_hl=95&itool=pubmed_docsum]].\n\nShowed that WAVE localization is retained in IRSp53 knock-down cells. Showed that purified WAVE2 complex is activated by IRSp53 in a Rac-dependent manner with PIP3-containing liposomes [[Takenawa 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16702231&query_hl=95&itool=pubmed_docsum]].\n\nShowed that the Rac-binding domain (RBD) domain of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. The Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain [[Takenawa 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17003044&query_hl=95&itool=pubmed_docsum]].\n\nShowed that Rac-induced development of cadherin-dependent adhesions required WAVE2-dependent actin reorganization [[Takenawa 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17164293&query_hl=95&itool=pubmed_docsum]].\n\nShowed that in vitro WAVE2 is directly phosphorylated by a MAP kinase, i.e. extracellular signal-regulated kinase ERK2. The proline-rich region and the [[VCA]] region of WAVE2 were phosphorylated. Phosphorylation of the [[VCA]] enhances its affinity for [[Arp2/3]] and this reduces Arp2/3-mediated actin polymerization in vitro [[Takenawa 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17202194&query_hl=95&itool=pubmed_docsum]].\n\nShowed that [[N-WASP]] and [[Grb2]] are involved in [[podosome]] formation [[Takenawa 2008|http://www.ncbi.nlm.nih.gov/pubmed/18606851?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nReported a casein kinase2 [[CK2]] binding protein [[CKIP-1]] that contains a [[PH]] domain and inactivates [[Akt]]. Stable CKIP-1 expression caused [[Akt]] inactivation and cell growth inhibition [[Takenawa 2007|http://www.ncbi.nlm.nih.gov/pubmed/17942896?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Talin binds [[Vinculin]] and [[Actin]] and activates [[Integrins]] in the formation of focal adhesion complexes. Talin may be cleaved by [[Calpain]].
Taxol binds tightly to polymers and inhibits MT depolymerization. Taxol also prevents colchicine-induced activation of ROCK [[Niggli 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=12571279]].
Tetraspanins are small membrane proteins involved in a multitude of biological processes, such as fertilization, parasite and viral infection, synaptic contacts at neuromuscular junctions, platelet aggregation, maintenance of skin integrity, immune response induction, metastasis suppression and tumour progression. Two tetraspanins CD82 and CD9 suppress metastasis while two tetraspanins CD151 and CD8 promote tumor progression [[Zoeller review 2009|http://www.ncbi.nlm.nih.gov/pubmed/19078974?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
this was a protocol (ca. 2003) used by the members of Henry Bourne's lab for lentiviral infection of HL-60 cells (top).\nwe have since updated/optimized the protocol for generating Weiner lab HL-60 cell lines (bottom).\n\nLast modified by DQ, sept 2012\n---------------------------------------\nfrom the Bourne Lab:\n\nReagents:\n~ polybrene, 8 mg/mL. dilute this 1:50 in media. Final [4 ug/mL].\n~ Cells at 2 million/mL.\n~ 25-30X concentrated lentivirus*.\n\nProtocol:\n1. in a 96-well, mix together:\n\n40 uL cells\n31 uL virus*\n2.5 uL diluted polybrene\n26.5 uL medium\ntotal = 100 uL for 80,000 cells.\n\nfor no-virus-control, add 57.5 uL media.\n\n2. In 96-well plate, fill up unused wells with water (prevents evaporation).\n\n3. After 5.5 h, dilute 1:2 (add 100 uL media) and leave O/N.\n\n4. Next morning: transfer cells to eppy, spin (500 x g, 5 min), wash 2x DPBS, resuspend in 400 uL media in 24-well plate.\n\n5. 2 days post-inf: 200 uL FACS, add 200 uL fresh media.\n\n6. 5 days post-inf: FACS.\n\n.* conc virus using centricon column (Centricon Plus-80 unit, Millipore) or via ultracentrifugation. Viral volume determined by optimal titer (3-4 x 107 particles/mL) and MOI of 50.\n\n------------------------------------------------\nWeiner lab modifications:\n\nReagents:\n~ polybrene, 5 mg/mL. dilute this 1:50 in media. Final [4 ug/mL]. (current stock is 4 mg/mL; dilute this 1:40)\n~ Cells at 2 million/mL.\n~ 20-25X concentrated lentivirus**.\n\nProtocol:\n1. in a 48- or 24-well, mix together:\n\n80 uL cells\n62 uL virus**\n8 uL diluted polybrene\n50 uL medium\ntotal = 200 uL for 160,000 cells.\n\nfor no-virus control, add 112 uL media.\n\n2. In a 48- or 24-well plate, fill up unused wells with water (prevents evaporation).\n\n3. After 5.5 h, dilute 1:2 (add 200 uL media) in the 48- or 24-well plate and leave O/N (should be a final cell density at 0.4 mil/mL).\n\n4. Next morning: take a look at your cells-- hopefully they are round and happy! transfer cells to eppy, spin (500 x g, 5 min), wash 2x DPBS (0.75 - 1.0 mL), resuspend in 400-800 uL media in 24-well plate (the goal is to keep the density at or around 0.4 mil/mL).\n\n5. keep monitoring cell growth/health. 2 days post-inf: assess via FACS or microscopy. add drug for selection if desired.\n\n6. if cells look good, expand the population for frozen storage.\n\n.** conc virus using Lenti-X Concentrator. I did not calculate viral titer.\n
Showed that that Arp2/3 complex is incorporated into the network exclusively at the lamellipodium tip, like actin, at sites coincident with WAVE complex accumulation. Capping protein likewise showed a turnover similar to actin and Arp2/3 complex, but was confined to the tip [[Stradal 2008|http://www.ncbi.nlm.nih.gov/pubmed/18309290?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Tiam-1, a [[GEF]] for the small GTPase Rac and implicated in tumor invasion and metastasis, is expressed in the developing CNS. Tiam-1 contributes to cytoskeletal reorganization required during cell migration and neurite extension in defined neuronal population [[Salinas 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9204476&query_hl=7&itool=pubmed_DocSum]]. Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo [[Chen 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=10893266]]. Tiam1 interacts with the p21-Arc [Arp (actin-related protein) complex] subunit of the [[Arp2/3]] complex and Tiam1 co-localizes with the [[Arp2/3]] complex at sites of actin polymerization. Blocking [[Arp2/3]] activation with a [[WASP]] (~Wiskott-Aldrich syndrome protein) inhibitor leads to subcellular relocalization of Tiam1 and decreased [[Rac]] activation [[Collard 2006|http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=16599904&dopt=Abstract]]. Tiam1 is translocated to the membrane of [[Myosin IIA]]-null cells, colocalizing with [[Rac]] at membrane edges in ES cells. Suppression of Tiam1 by siRNA knockdown looks like a [[Myosin IIA]]-null morphological phenotype [[Yamada 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17310241&query_hl=3&itool=pubmed_docsum]]. Tiam1 catalyzes exchange when [[Rac]] is bound to liposomes [[Antonny 2003|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=12471028&ordinalpos=14&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. Tiam1 co-IPs with [[Par3]] and [[PKC]], co-localizes with them, and Tiam1 KO has defects in persistent polarity in keratinocytes [[Collard 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17825562&ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
See [[Tiam-1]]
Showed that cAMP gradient induces a stable accumulation of PIP3 at the front in a latrunculin treated cell, but when the gradient is withdrawn and reapplied, PIP3 accumulation occurs at the back of the cell [[Jin 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17606871&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Showed that microtubules in vitro coexist in growing and shrinking populations which interconvert rather infrequently [[Mitchison 1984|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=6504138&query_hl=69&itool=pubmed_DocSum]].\n\nShowed that monomeric actin is ATP bound [[Mitchison 1995|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=7787248&query_hl=15&itool=pubmed_DocSum]].\n\nPurified the [[Arp2/3]] complex from Xenopus and platelet extracts and showed that it was capable of nucleating actin assembly [[Mitchison 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9000076&query_hl=15&itool=pubmed_DocSum]].\n\nShowed that depletion of cofilin from Xenopus extracts results in an increased length in Listeria comet tails. This indicates that cofilin is required for actin turnover [[Mitchison 1997|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9087446&query_hl=15&itool=pubmed_DocSum]].\n\nShowed that new actin assembly occurs at the tip of a filopodia in a growth cone at a rate of 1 µm/min and disassembly at a rate of 0.6 µm/min with photoactivation and photobleaching [[Mitchison 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10477762&query_hl=23&itool=pubmed_docsum]].\n\nShowed that Xenopus fibroblasts turnover actin filaments in lamellipodia with speckle analysis [[Mitchison 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11834838&query_hl=15&itool=pubmed_DocSum]].\n\nShowed that hydrostatic pressure can drive localized protrusion in cells by blebbing [[Mitchison 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15902261&query_hl=15&itool=pubmed_DocSum]].
Showed that expression of over 100 different constitutively active GTPases falls into 9 categories based on morphology in NIH3T3 cells [[Meyer 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12732140&query_hl=12&itool=pubmed_DocSum]].\n\nShowed that you could couple GTPase activation to its recruitment in an inducible fashion with a modified Chavrier system [[Meyer 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15908919&query_hl=12&itool=pubmed_DocSum]].\n\nShowed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system [[Meyer 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16239477&query_hl=12&itool=pubmed_DocSum]].\n\nShowed that GTPases with polybasic clusters are targeting to the membrane by PIP2 and PIP3, by inducibly depleting PIP2 and PIP3 from the membrane [[Meyer 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17095657&query_hl=12&itool=pubmed_DocSum]].\n\nDeveloped a 96-well electroporation chamber to transfect HL-60 cells in a high-throughput manner [[Meyer 2008|http://www.ncbi.nlm.nih.gov/pubmed/18408727?ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n
Transducer of [[Cdc42]] activity. Toca-1 is a member of the [[PCH]] family of proteins [[Stanley 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17296299&query_hl=119&itool=pubmed_DocSum]] and binds both N-WASP and [[Cdc42]]. N-WASP-WIP complex and Toca-1, are required for [[Cdc42]]-induced actin assembly [[Kirschner 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=15260990]]. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. Toca-1 binds both N-WASP through the SH3 domain and [[Cdc42]] through the HR1 domain [[Negishi 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16885158&query_hl=136&itool=pubmed_docsum]].
Mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase [[Firtel 2005|http://www.ncbi.nlm.nih.gov/pubmed/16079174?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Dictyostelium deficient in TorC2 or [[PKB]] (the Dicty name for Akt) activity show reduced phosphorylation of the endogenous substrates (Talin, PI4P 5-kinase, two Ras GEFs, and a RhoGap) and are impaired in chemotaxis. This occurs at the leading edge even in the absence of PIP(3) [[Devereotes 2008|http://www.ncbi.nlm.nih.gov/pubmed/18635356?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Type the text for 'Torstein Wittman'
Trio is a unique RhoGEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG, [[Rac1]] and RhoA, respectively. Dbl-homology ([[DH]]) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology ([[PH]]) domains localize RhoGEFs near their downstream targets. Trio GEFD1 interacts through its [[PH]] domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells [[Debant 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11146652&query_hl=14&itool=pubmed_docsum]].
Prevents [[Cofilin]] localization.
Tuba is a novel scaffold protein that functions to bring together [[Dynamin]] with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Direct binding partners include N-WASP and [[Ena]]/[[VASP]] proteins. A Dbl homology domain present in the middle of Tuba upstream of a Bin/amphiphysin/Rvs [[BAR]] domain activates [[Cdc42]], but not [[Rac]] and [[Rho]] [[De Camilli 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14506234&query_hl=43&itool=pubmed_docsum]].
Ultrasensitivity is enhanced sensitivity beyond Michaelis-Menton kinetics and can be acheived through "cooperative ultrasensitivity", which could occur for an any enzyme with a Hill coefficient greater than one, through "multistep ultrasensitivity", which an enzyme acts or binds at more than one step or place, or through "zero-order ultrasensivity", where converter enzymes operating under saturating conditions amplify the response to the signal [[Koshland 1981|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=6947258&query_hl=59&itool=pubmed_docsum]].
| !date | !user | !location | !storeUrl | !uploadDir | !toFilename | !backupdir | !origin |\n| 6/12/2006 18:14:10 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 18:16:6 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 18:16:29 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 18:28:32 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 18:56:27 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 19:4:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 19:10:40 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 19:14:4 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 6/12/2006 19:53:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Proteins]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 22:43:31 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Proteins]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/12/2006 23:59:48 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Proteins]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 7/12/2006 0:1:0 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Proteins]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 7/12/2006 10:26:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 7/12/2006 18:31:55 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/12/2006 9:59:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/12/2006 12:44:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 8/12/2006 17:34:58 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/12/2006 11:47:54 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 12:42:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 17:40:5 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 18:16:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 18:51:3 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 18:53:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/12/2006 19:22:31 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Macrophage%20B16F10%20B16F1%203T3%20%5B%5BNatural%20killer%20cells%5D%5D%20%5B%5BB%20cell%5D%5D%20%5B%5BT%20cell%5D%5D%20Lymphocyte%20Fibroblast%20Neutrophil%20Monocytes%20%5B%5BWelcome%20to%20your%20tiddlyspot.com%20site!%5D%5D%20GettingStarted]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 19:27:34 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Macrophage%20B16F10%20B16F1%203T3%20%5B%5BNatural%20killer%20cells%5D%5D%20%5B%5BB%20cell%5D%5D%20%5B%5BT%20cell%5D%5D%20Lymphocyte%20Fibroblast%20Neutrophil%20Monocytes%20%5B%5BWelcome%20to%20your%20tiddlyspot.com%20site!%5D%5D%20GettingStarted]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/12/2006 19:57:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Macrophage%20B16F10%20B16F1%203T3%20%5B%5BNatural%20killer%20cells%5D%5D%20%5B%5BB%20cell%5D%5D%20%5B%5BT%20cell%5D%5D%20Lymphocyte%20Fibroblast%20Neutrophil%20Monocytes%20%5B%5BWelcome%20to%20your%20tiddlyspot.com%20site!%5D%5D%20GettingStarted]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/12/2006 20:53:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/12/2006 1:19:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/12/2006 11:53:57 | Iowa | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 11/12/2006 11:54:2 | Iowa | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 11/12/2006 14:5:45 | Iowa | [[/|http://chemotaxis.tiddlyspot.com/#%5B%5BCell%20Lines%5D%5D]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/12/2006 15:59:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/12/2006 20:37:40 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 12/12/2006 11:7:43 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/12/2006 14:56:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/12/2006 15:7:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 6/1/2007 17:0:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/1/2007 16:26:40 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 22/1/2007 11:10:0 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 22/1/2007 11:39:40 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 22/1/2007 23:44:36 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 23/1/2007 19:52:37 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 26/1/2007 11:54:3 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 27/1/2007 13:50:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/2/2007 11:44:11 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/2/2007 12:46:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/2/2007 13:15:36 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/2/2007 13:24:9 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 12/2/2007 23:14:59 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/2/2007 13:58:24 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 17/2/2007 13:59:9 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 23/2/2007 19:30:1 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 7/3/2007 21:57:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/3/2007 17:45:20 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 19/3/2007 17:48:4 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 19/3/2007 17:53:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 19/3/2007 17:59:34 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/3/2007 11:55:31 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 21/3/2007 13:49:24 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 22/3/2007 13:3:0 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 22/3/2007 13:33:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 22/3/2007 17:17:9 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/3/2007 20:37:45 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 26/3/2007 17:32:14 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 30/3/2007 10:29:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 30/3/2007 10:30:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 31/3/2007 10:4:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 31/3/2007 14:42:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 1/4/2007 19:14:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/4/2007 21:3:38 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/4/2007 22:46:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/4/2007 23:3:44 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 2/4/2007 17:8:48 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/4/2007 12:40:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/4/2007 13:41:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/4/2007 19:44:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 5/4/2007 15:36:18 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/4/2007 16:1:55 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/4/2007 16:25:24 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/4/2007 16:52:32 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/4/2007 17:19:5 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/4/2007 19:22:35 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 6/4/2007 16:8:35 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/4/2007 16:19:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/4/2007 12:3:48 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/4/2007 20:53:22 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/4/2007 14:58:0 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2007 11:4:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Arno]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2007 11:24:31 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#Arno]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/4/2007 15:16:34 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2007 16:8:14 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2007 22:52:20 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2007 23:25:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2007 10:55:11 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2007 17:18:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2007 20:9:32 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2007 20:42:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2007 23:3:29 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/4/2007 15:9:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/4/2007 17:42:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/4/2007 14:7:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/4/2007 14:11:40 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 16/4/2007 16:30:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 16/4/2007 20:28:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/4/2007 21:56:3 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/4/2007 22:15:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/4/2007 13:22:24 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/4/2007 17:36:6 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 17/4/2007 17:37:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/4/2007 23:22:54 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 18/4/2007 19:17:50 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 18/4/2007 22:54:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/4/2007 12:48:20 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/4/2007 13:3:11 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/4/2007 15:52:15 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/4/2007 19:22:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/4/2007 20:36:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/4/2007 20:56:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/4/2007 10:33:0 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/4/2007 10:51:29 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 25/4/2007 23:33:50 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 27/4/2007 12:18:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/4/2007 22:58:13 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 30/4/2007 21:38:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 1/5/2007 17:43:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 2/5/2007 14:0:22 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/5/2007 19:23:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/5/2007 13:20:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/5/2007 10:14:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/5/2007 13:9:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 23/5/2007 13:47:6 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 30/5/2007 15:23:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 31/5/2007 20:29:22 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/6/2007 10:27:3 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/6/2007 13:55:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 21/6/2007 15:47:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/7/2007 14:9:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 31/7/2007 18:30:43 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/8/2007 14:3:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/8/2007 14:3:34 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 3/8/2007 11:11:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/8/2007 11:22:44 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/8/2007 18:22:9 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/8/2007 18:24:35 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 24/8/2007 9:22:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/9/2007 7:42:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 27/9/2007 16:33:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 1/10/2007 19:20:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/10/2007 19:25:59 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/10/2007 22:28:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/10/2007 13:13:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 22/10/2007 19:8:41 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/11/2007 22:53:29 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/11/2007 20:14:4 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 10/11/2007 15:4:38 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/11/2007 20:0:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/11/2007 20:2:11 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/11/2007 15:14:11 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 27/11/2007 12:16:41 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/11/2007 11:10:27 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/11/2007 12:2:40 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/11/2007 14:5:20 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/11/2007 12:31:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/11/2007 15:56:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#People%20%5B%5BRon%20Vale%5D%5D%20Proteins%20Nck%20n-Chimaerin%20Nap1%20%5B%5BMyosin%20IIB%5D%5D%20%5B%5BMyosin%20IIA%5D%5D%20Tiam1%20%5B%5BMyosin%20phosphatase%5D%5D%20%5B%5BMyosin%20II%5D%5D%20MLCK%20MLC%20Microtubules%20%5B%5BPertussis%20toxin%5D%5D%20Pak1%20Merlin%20Mena%20LIMK%20Kette]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/11/2007 18:16:36 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/#People%20%5B%5BRon%20Vale%5D%5D%20Proteins%20Nck%20n-Chimaerin%20Nap1%20%5B%5BMyosin%20IIB%5D%5D%20%5B%5BMyosin%20IIA%5D%5D%20Tiam1%20%5B%5BMyosin%20phosphatase%5D%5D%20%5B%5BMyosin%20II%5D%5D%20MLCK%20MLC%20Microtubules%20%5B%5BPertussis%20toxin%5D%5D%20Pak1%20Merlin%20Mena%20LIMK%20Kette]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/11/2007 18:27:49 | Arthur | [[chemotaxis.html|file:///Volumes/arthurmillius/Arthur/Wiki/December%202007/chemotaxis.html]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/11/2007 19:47:13 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/11/2007 19:48:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/11/2007 19:55:49 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/11/2007 20:1:53 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/11/2007 20:2:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 2/12/2007 15:10:11 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/12/2007 21:11:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 5/12/2007 11:54:45 | Iowa | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/12/2007 11:56:25 | Iowa | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 5/12/2007 12:3:38 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/12/2007 13:45:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/1/2008 14:3:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/2/2008 2:31:31 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 2/7/2008 19:17:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 3/7/2008 17:57:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 23/7/2008 11:51:58 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 31/7/2008 13:51:51 | Sarah | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 31/7/2008 14:2:58 | Sarah | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 31/7/2008 14:29:48 | Sarah | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 31/7/2008 14:31:9 | Sarah | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/8/2008 11:38:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 18/8/2008 21:15:44 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 18/8/2008 21:15:55 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/8/2008 21:26:36 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/8/2008 16:6:32 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 26/8/2008 11:7:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 28/8/2008 9:2:52 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/8/2008 9:3:5 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 30/8/2008 11:50:21 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/9/2008 13:20:14 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 25/9/2008 15:17:22 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 26/9/2008 12:55:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 8/10/2008 17:45:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/10/2008 17:36:8 | Iowa | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/11/2008 17:1:36 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 12/11/2008 22:27:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/12/2008 11:5:57 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/12/2008 16:28:19 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/12/2008 16:52:37 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 5/1/2009 11:25:0 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 7/1/2009 22:56:51 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 8/1/2009 12:17:32 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/1/2009 17:56:42 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 15/1/2009 12:59:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 15/1/2009 14:3:54 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 15/1/2009 14:54:1 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 30/1/2009 14:47:54 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/2/2009 15:57:41 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/2/2009 19:27:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 28/2/2009 13:32:9 | Delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 12/3/2009 10:48:54 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/3/2009 22:9:27 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/3/2009 11:21:25 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/4/2009 14:27:44 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/4/2009 11:32:54 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2009 12:9:13 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2009 12:27:38 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/4/2009 11:23:11 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/4/2009 18:48:47 | Sarah | [[/|http://chemotaxis.tiddlyspot.com/#%5B%5BSplitting%20HL60%20cells%5D%5D]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/4/2009 21:32:27 | bryant | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/4/2009 21:35:20 | Bryant | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 2/5/2009 16:24:34 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 9/5/2009 18:57:44 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/5/2009 22:26:49 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 12/5/2009 23:59:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/5/2009 12:31:55 | Bryant | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/5/2009 12:32:36 | Bryant | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/5/2009 12:32:57 | Bryant | [[index.html|http://chemotaxis.tiddlyspot.com/index.html]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/5/2009 15:58:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/5/2009 17:50:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/5/2009 12:51:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 29/5/2009 13:14:38 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 3/6/2009 10:59:29 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 3/6/2009 11:5:41 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 3/6/2009 15:58:1 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/6/2009 12:2:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 4/6/2009 12:15:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/6/2009 16:5:51 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 16/6/2009 16:27:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 18/6/2009 18:10:56 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 21/6/2009 21:48:11 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 30/6/2009 12:51:4 | Delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/7/2009 16:42:19 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/7/2009 16:43:9 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/7/2009 17:16:36 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/7/2009 17:47:58 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/7/2009 17:49:43 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/7/2009 18:15:51 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/7/2009 18:36:39 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/7/2009 18:37:5 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/7/2009 13:21:13 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/7/2009 16:40:45 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/7/2009 18:18:59 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/7/2009 11:32:40 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 16/7/2009 11:35:26 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/7/2009 11:41:3 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 21/7/2009 14:18:50 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/7/2009 16:47:40 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/7/2009 16:47:59 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/7/2009 16:53:6 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 25/7/2009 16:53:35 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 7/8/2009 14:46:54 | AnnaPayne-Tobin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/8/2009 12:37:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 11/8/2009 12:39:38 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 1/9/2009 12:29:18 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 2/9/2009 15:0:16 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/9/2009 11:48:43 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 15/9/2009 14:37:9 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 18/9/2009 11:35:21 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 21/9/2009 17:53:20 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 21/9/2009 17:55:53 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 24/9/2009 21:5:53 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/9/2009 17:36:20 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/9/2009 17:37:14 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/9/2009 17:39:0 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 12/10/2009 16:0:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 12/10/2009 16:0:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/10/2009 13:2:39 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/10/2009 15:11:49 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 12/11/2009 13:11:17 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/11/2009 18:24:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 17/11/2009 18:27:31 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/12/2009 12:34:15 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 27/1/2010 16:46:9 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 5/2/2010 11:27:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/2/2010 12:10:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 18/2/2010 13:13:7 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 18/2/2010 13:24:6 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 18/2/2010 18:8:35 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 18/2/2010 18:13:44 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 1/3/2010 20:7:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/3/2010 20:8:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/3/2010 15:58:50 | Sheel | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/3/2010 22:33:49 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 10/3/2010 22:42:58 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 10/3/2010 22:49:48 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 12/3/2010 12:36:15 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 20/4/2010 14:31:51 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/4/2010 14:34:27 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 28/5/2010 21:59:7 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 24/6/2010 17:13:59 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 24/6/2010 17:49:30 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 24/6/2010 18:10:58 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 24/6/2010 18:11:32 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 24/6/2010 18:20:29 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 24/6/2010 18:46:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 24/6/2010 18:53:36 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 28/6/2010 12:52:4 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/6/2010 13:22:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/6/2010 17:11:41 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/6/2010 18:14:12 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/6/2010 21:25:5 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 28/6/2010 21:50:37 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 29/6/2010 22:35:44 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 1/7/2010 17:34:51 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 6/7/2010 17:56:59 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 6/7/2010 18:2:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/7/2010 12:10:57 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 8/7/2010 12:12:33 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/7/2010 12:27:14 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 15/7/2010 13:11:14 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 26/7/2010 22:46:22 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 30/7/2010 15:47:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/8/2010 9:48:57 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 27/8/2010 18:19:29 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 16/9/2010 20:5:25 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/11/2010 11:51:55 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 19/11/2010 12:33:54 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 28/12/2010 11:3:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/1/2011 10:38:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/1/2011 15:55:28 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 19/1/2011 18:38:23 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 19/1/2011 19:33:48 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/1/2011 15:19:52 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/1/2011 16:43:26 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 20/1/2011 19:1:46 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 26/1/2011 14:41:2 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 9/2/2011 22:51:15 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 4/3/2011 19:41:8 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 4/3/2011 20:13:22 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/3/2011 14:35:23 | Julie | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 10/3/2011 14:56:21 | Julie | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/4/2011 18:15:28 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2011 18:28:57 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 13/4/2011 23:13:28 | YourName | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 13/4/2011 23:18:32 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2011 11:2:5 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2011 11:2:48 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2011 11:3:25 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2011 12:9:42 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2011 13:20:2 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2011 13:32:21 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2011 14:18:22 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2011 14:25:49 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 14/4/2011 14:56:31 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2011 14:57:21 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 14/4/2011 14:59:32 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 31/8/2011 13:46:34 | Arthur | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 26/9/2011 14:28:33 | YourName | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/11/2011 14:50:7 | Andrew | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 17/8/2012 14:13:47 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 17/8/2012 14:17:15 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 5/9/2012 15:33:56 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 5/9/2012 15:36:52 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 10/9/2012 14:38:1 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . | Ok |\n| 10/9/2012 14:49:36 | delquin | [[/|http://chemotaxis.tiddlyspot.com/#%5B%5BThe%20Bourne%20Infection!%5D%5D]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/9/2012 11:34:45 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/9/2012 11:34:52 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/9/2012 11:35:8 | | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 11/9/2012 11:35:27 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 18/9/2012 10:25:54 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 18/9/2012 10:28:37 | delquin | [[index.html|http://chemotaxis.tiddlyspot.com/index.html]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/11/2012 12:10:31 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/11/2012 12:11:51 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 19/11/2012 12:12:48 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |\n| 3/12/2012 12:22:12 | delquin | [[/|http://chemotaxis.tiddlyspot.com/]] | [[store.cgi|http://chemotaxis.tiddlyspot.com/store.cgi]] | . | index.html | . |
/***\n|''Name:''|UploadPlugin|\n|''Description:''|Save to web a TiddlyWiki|\n|''Version:''|3.4.4|\n|''Date:''|Sep 30, 2006|\n|''Source:''|http://tiddlywiki.bidix.info/#UploadPlugin|\n|''Documentation:''|http://tiddlywiki.bidix.info/#UploadDoc|\n|''Author:''|BidiX (BidiX (at) bidix (dot) info)|\n|''License:''|[[BSD open source license|http://tiddlywiki.bidix.info/#%5B%5BBSD%20open%20source%20license%5D%5D ]]|\n|''~CoreVersion:''|2.0.0|\n|''Browser:''|Firefox 1.5; InternetExplorer 6.0; Safari|\n|''Include:''|config.lib.file; config.lib.log; config.lib.options; PasswordTweak|\n|''Require:''|[[UploadService|http://tiddlywiki.bidix.info/#UploadService]]|\n***/\n//{{{\nversion.extensions.UploadPlugin = {\n major: 3, minor: 4, revision: 4, \n date: new Date(2006,8,30),\n source: 'http://tiddlywiki.bidix.info/#UploadPlugin',\n documentation: 'http://tiddlywiki.bidix.info/#UploadDoc',\n author: 'BidiX (BidiX (at) bidix (dot) info',\n license: '[[BSD open source license|http://tiddlywiki.bidix.info/#%5B%5BBSD%20open%20source%20license%5D%5D]]',\n coreVersion: '2.0.0',\n browser: 'Firefox 1.5; InternetExplorer 6.0; Safari'\n};\n//}}}\n\n////+++!![config.lib.file]\n\n//{{{\nif (!config.lib) config.lib = {};\nif (!config.lib.file) config.lib.file= {\n author: 'BidiX',\n version: {major: 0, minor: 1, revision: 0}, \n date: new Date(2006,3,9)\n};\nconfig.lib.file.dirname = function (filePath) {\n var lastpos;\n if ((lastpos = filePath.lastIndexOf("/")) != -1) {\n return filePath.substring(0, lastpos);\n } else {\n return filePath.substring(0, filePath.lastIndexOf("\s\s"));\n }\n};\nconfig.lib.file.basename = function (filePath) {\n var lastpos;\n if ((lastpos = filePath.lastIndexOf("#")) != -1) \n filePath = filePath.substring(0, lastpos);\n if ((lastpos = filePath.lastIndexOf("/")) != -1) {\n return filePath.substring(lastpos + 1);\n } else\n return filePath.substring(filePath.lastIndexOf("\s\s")+1);\n};\nwindow.basename = function() {return "@@deprecated@@";};\n//}}}\n////===\n\n////+++!![config.lib.log]\n\n//{{{\nif (!config.lib) config.lib = {};\nif (!config.lib.log) config.lib.log= {\n author: 'BidiX',\n version: {major: 0, minor: 1, revision: 1}, \n date: new Date(2006,8,19)\n};\nconfig.lib.Log = function(tiddlerTitle, logHeader) {\n if (version.major < 2)\n this.tiddler = store.tiddlers[tiddlerTitle];\n else\n this.tiddler = store.getTiddler(tiddlerTitle);\n if (!this.tiddler) {\n this.tiddler = new Tiddler();\n this.tiddler.title = tiddlerTitle;\n this.tiddler.text = "| !date | !user | !location |" + logHeader;\n this.tiddler.created = new Date();\n this.tiddler.modifier = config.options.txtUserName;\n this.tiddler.modified = new Date();\n if (version.major < 2)\n store.tiddlers[tiddlerTitle] = this.tiddler;\n else\n store.addTiddler(this.tiddler);\n }\n return this;\n};\n\nconfig.lib.Log.prototype.newLine = function (line) {\n var now = new Date();\n var newText = "| ";\n newText += now.getDate()+"/"+(now.getMonth()+1)+"/"+now.getFullYear() + " ";\n newText += now.getHours()+":"+now.getMinutes()+":"+now.getSeconds()+" | ";\n newText += config.options.txtUserName + " | ";\n var location = document.location.toString();\n var filename = config.lib.file.basename(location);\n if (!filename) filename = '/';\n newText += "[["+filename+"|"+location + "]] |";\n this.tiddler.text = this.tiddler.text + "\sn" + newText;\n this.addToLine(line);\n};\n\nconfig.lib.Log.prototype.addToLine = function (text) {\n this.tiddler.text = this.tiddler.text + text;\n this.tiddler.modifier = config.options.txtUserName;\n this.tiddler.modified = new Date();\n if (version.major < 2)\n store.tiddlers[this.tiddler.tittle] = this.tiddler;\n else {\n store.addTiddler(this.tiddler);\n story.refreshTiddler(this.tiddler.title);\n store.notify(this.tiddler.title, true);\n }\n if (version.major < 2)\n store.notifyAll(); \n};\n//}}}\n////===\n\n////+++!![config.lib.options]\n\n//{{{\nif (!config.lib) config.lib = {};\nif (!config.lib.options) config.lib.options = {\n author: 'BidiX',\n version: {major: 0, minor: 1, revision: 0}, \n date: new Date(2006,3,9)\n};\n\nconfig.lib.options.init = function (name, defaultValue) {\n if (!config.options[name]) {\n config.options[name] = defaultValue;\n saveOptionCookie(name);\n }\n};\n//}}}\n////===\n\n////+++!![PasswordTweak]\n\n//{{{\nversion.extensions.PasswordTweak = {\n major: 1, minor: 0, revision: 3, date: new Date(2006,8,30),\n type: 'tweak',\n source: 'http://tiddlywiki.bidix.info/#PasswordTweak'\n};\n//}}}\n/***\n!!config.macros.option\n***/\n//{{{\nconfig.macros.option.passwordCheckboxLabel = "Save this password on this computer";\nconfig.macros.option.passwordType = "password"; // password | text\n\nconfig.macros.option.onChangeOption = function(e)\n{\n var opt = this.getAttribute("option");\n var elementType,valueField;\n if(opt) {\n switch(opt.substr(0,3)) {\n case "txt":\n elementType = "input";\n valueField = "value";\n break;\n case "pas":\n elementType = "input";\n valueField = "value";\n break;\n case "chk":\n elementType = "input";\n valueField = "checked";\n break;\n }\n config.options[opt] = this[valueField];\n saveOptionCookie(opt);\n var nodes = document.getElementsByTagName(elementType);\n for(var t=0; t<nodes.length; t++) \n {\n var optNode = nodes[t].getAttribute("option");\n if (opt == optNode) \n nodes[t][valueField] = this[valueField];\n }\n }\n return(true);\n};\n\nconfig.macros.option.handler = function(place,macroName,params)\n{\n var opt = params[0];\n if(config.options[opt] === undefined) {\n return;}\n var c;\n switch(opt.substr(0,3)) {\n case "txt":\n c = document.createElement("input");\n c.onkeyup = this.onChangeOption;\n c.setAttribute ("option",opt);\n c.className = "txtOptionInput "+opt;\n place.appendChild(c);\n c.value = config.options[opt];\n break;\n case "pas":\n // input password\n c = document.createElement ("input");\n c.setAttribute("type",config.macros.option.passwordType);\n c.onkeyup = this.onChangeOption;\n c.setAttribute("option",opt);\n c.className = "pasOptionInput "+opt;\n place.appendChild(c);\n c.value = config.options[opt];\n // checkbox link with this password "save this password on this computer"\n c = document.createElement("input");\n c.setAttribute("type","checkbox");\n c.onclick = this.onChangeOption;\n c.setAttribute("option","chk"+opt);\n c.className = "chkOptionInput "+opt;\n place.appendChild(c);\n c.checked = config.options["chk"+opt];\n // text savePasswordCheckboxLabel\n place.appendChild(document.createTextNode(config.macros.option.passwordCheckboxLabel));\n break;\n case "chk":\n c = document.createElement("input");\n c.setAttribute("type","checkbox");\n c.onclick = this.onChangeOption;\n c.setAttribute("option",opt);\n c.className = "chkOptionInput "+opt;\n place.appendChild(c);\n c.checked = config.options[opt];\n break;\n }\n};\n//}}}\n/***\n!! Option cookie stuff\n***/\n//{{{\nwindow.loadOptionsCookie_orig_PasswordTweak = window.loadOptionsCookie;\nwindow.loadOptionsCookie = function()\n{\n var cookies = document.cookie.split(";");\n for(var c=0; c<cookies.length; c++) {\n var p = cookies[c].indexOf("=");\n if(p != -1) {\n var name = cookies[c].substr(0,p).trim();\n var value = cookies[c].substr(p+1).trim();\n switch(name.substr(0,3)) {\n case "txt":\n config.options[name] = unescape(value);\n break;\n case "pas":\n config.options[name] = unescape(value);\n break;\n case "chk":\n config.options[name] = value == "true";\n break;\n }\n }\n }\n};\n\nwindow.saveOptionCookie_orig_PasswordTweak = window.saveOptionCookie;\nwindow.saveOptionCookie = function(name)\n{\n var c = name + "=";\n switch(name.substr(0,3)) {\n case "txt":\n c += escape(config.options[name].toString());\n break;\n case "chk":\n c += config.options[name] ? "true" : "false";\n // is there an option link with this chk ?\n if (config.options[name.substr(3)]) {\n saveOptionCookie(name.substr(3));\n }\n break;\n case "pas":\n if (config.options["chk"+name]) {\n c += escape(config.options[name].toString());\n } else {\n c += "";\n }\n break;\n }\n c += "; expires=Fri, 1 Jan 2038 12:00:00 UTC; path=/";\n document.cookie = c;\n};\n//}}}\n/***\n!! Initializations\n***/\n//{{{\n// define config.options.pasPassword\nif (!config.options.pasPassword) {\n config.options.pasPassword = 'defaultPassword';\n window.saveOptionCookie('pasPassword');\n}\n// since loadCookies is first called befor password definition\n// we need to reload cookies\nwindow.loadOptionsCookie();\n//}}}\n////===\n\n////+++!![config.macros.upload]\n\n//{{{\nconfig.macros.upload = {\n accessKey: "U",\n formName: "UploadPlugin",\n contentType: "text/html;charset=UTF-8",\n defaultStoreScript: "store.php"\n};\n\n// only this two configs need to be translated\nconfig.macros.upload.messages = {\n aboutToUpload: "About to upload TiddlyWiki to %0",\n backupFileStored: "Previous file backuped in %0",\n crossDomain: "Certainly a cross-domain isue: access to an other site isn't allowed",\n errorDownloading: "Error downloading",\n errorUploadingContent: "Error uploading content",\n fileLocked: "Files is locked: You are not allowed to Upload",\n fileNotFound: "file to upload not found",\n fileNotUploaded: "File %0 NOT uploaded",\n mainFileUploaded: "Main TiddlyWiki file uploaded to %0",\n passwordEmpty: "Unable to upload, your password is empty",\n urlParamMissing: "url param missing",\n rssFileNotUploaded: "RssFile %0 NOT uploaded",\n rssFileUploaded: "Rss File uploaded to %0"\n};\n\nconfig.macros.upload.label = {\n promptOption: "Save and Upload this TiddlyWiki with UploadOptions",\n promptParamMacro: "Save and Upload this TiddlyWiki in %0",\n saveLabel: "save to web", \n saveToDisk: "save to disk",\n uploadLabel: "upload" \n};\n\nconfig.macros.upload.handler = function(place,macroName,params){\n // parameters initialization\n var storeUrl = params[0];\n var toFilename = params[1];\n var backupDir = params[2];\n var uploadDir = params[3];\n var username = params[4];\n var password; // for security reason no password as macro parameter\n var label;\n if (document.location.toString().substr(0,4) == "http")\n label = this.label.saveLabel;\n else\n label = this.label.uploadLabel;\n var prompt;\n if (storeUrl) {\n prompt = this.label.promptParamMacro.toString().format([this.toDirUrl(storeUrl, uploadDir, username)]);\n }\n else {\n prompt = this.label.promptOption;\n }\n createTiddlyButton(place, label, prompt, \n function () {\n config.macros.upload.upload(storeUrl, toFilename, uploadDir, backupDir, username, password); \n return false;}, \n null, null, this.accessKey);\n};\nconfig.macros.upload.UploadLog = function() {\n return new config.lib.Log('UploadLog', " !storeUrl | !uploadDir | !toFilename | !backupdir | !origin |" );\n};\nconfig.macros.upload.UploadLog.prototype = config.lib.Log.prototype;\nconfig.macros.upload.UploadLog.prototype.startUpload = function(storeUrl, toFilename, uploadDir, backupDir) {\n var line = " [[" + config.lib.file.basename(storeUrl) + "|" + storeUrl + "]] | ";\n line += uploadDir + " | " + toFilename + " | " + backupDir + " |";\n this.newLine(line);\n};\nconfig.macros.upload.UploadLog.prototype.endUpload = function() {\n this.addToLine(" Ok |");\n};\nconfig.macros.upload.basename = config.lib.file.basename;\nconfig.macros.upload.dirname = config.lib.file.dirname;\nconfig.macros.upload.toRootUrl = function (storeUrl, username)\n{\n return root = (this.dirname(storeUrl)?this.dirname(storeUrl):this.dirname(document.location.toString()));\n}\nconfig.macros.upload.toDirUrl = function (storeUrl, uploadDir, username)\n{\n var root = this.toRootUrl(storeUrl, username);\n if (uploadDir && uploadDir != '.')\n root = root + '/' + uploadDir;\n return root;\n}\nconfig.macros.upload.toFileUrl = function (storeUrl, toFilename, uploadDir, username)\n{\n return this.toDirUrl(storeUrl, uploadDir, username) + '/' + toFilename;\n}\nconfig.macros.upload.upload = function(storeUrl, toFilename, uploadDir, backupDir, username, password)\n{\n // parameters initialization\n storeUrl = (storeUrl ? storeUrl : config.options.txtUploadStoreUrl);\n toFilename = (toFilename ? toFilename : config.options.txtUploadFilename);\n backupDir = (backupDir ? backupDir : config.options.txtUploadBackupDir);\n uploadDir = (uploadDir ? uploadDir : config.options.txtUploadDir);\n username = (username ? username : config.options.txtUploadUserName);\n password = config.options.pasUploadPassword; // for security reason no password as macro parameter\n if (!password || password === '') {\n alert(config.macros.upload.messages.passwordEmpty);\n return;\n }\n if (storeUrl === '') {\n storeUrl = config.macros.upload.defaultStoreScript;\n }\n if (config.lib.file.dirname(storeUrl) === '') {\n storeUrl = config.lib.file.dirname(document.location.toString())+'/'+storeUrl;\n }\n if (toFilename === '') {\n toFilename = config.lib.file.basename(document.location.toString());\n }\n\n clearMessage();\n // only for forcing the message to display\n if (version.major < 2)\n store.notifyAll();\n if (!storeUrl) {\n alert(config.macros.upload.messages.urlParamMissing);\n return;\n }\n // Check that file is not locked\n if (window.BidiX && BidiX.GroupAuthoring && BidiX.GroupAuthoring.lock) {\n if (BidiX.GroupAuthoring.lock.isLocked() && !BidiX.GroupAuthoring.lock.isMyLock()) {\n alert(config.macros.upload.messages.fileLocked);\n return;\n }\n }\n \n var log = new this.UploadLog();\n log.startUpload(storeUrl, toFilename, uploadDir, backupDir);\n if (document.location.toString().substr(0,5) == "file:") {\n saveChanges();\n }\n var toDir = config.macros.upload.toDirUrl(storeUrl, toFilename, uploadDir, username);\n displayMessage(config.macros.upload.messages.aboutToUpload.format([toDir]), toDir);\n this.uploadChanges(storeUrl, toFilename, uploadDir, backupDir, username, password);\n if(config.options.chkGenerateAnRssFeed) {\n //var rssContent = convertUnicodeToUTF8(generateRss());\n var rssContent = generateRss();\n var rssPath = toFilename.substr(0,toFilename.lastIndexOf(".")) + ".xml";\n this.uploadContent(rssContent, storeUrl, rssPath, uploadDir, '', username, password, \n function (responseText) {\n if (responseText.substring(0,1) != '0') {\n displayMessage(config.macros.upload.messages.rssFileNotUploaded.format([rssPath]));\n }\n else {\n var toFileUrl = config.macros.upload.toFileUrl(storeUrl, rssPath, uploadDir, username);\n displayMessage(config.macros.upload.messages.rssFileUploaded.format(\n [toFileUrl]), toFileUrl);\n }\n // for debugging store.php uncomment last line\n //DEBUG alert(responseText);\n });\n }\n return;\n};\n\nconfig.macros.upload.uploadChanges = function(storeUrl, toFilename, uploadDir, backupDir, \n username, password) {\n var original;\n if (document.location.toString().substr(0,4) == "http") {\n original = this.download(storeUrl, toFilename, uploadDir, backupDir, username, password);\n return;\n }\n else {\n // standard way : Local file\n \n original = loadFile(getLocalPath(document.location.toString()));\n if(window.Components) {\n // it's a mozilla browser\n try {\n netscape.security.PrivilegeManager.enablePrivilege("UniversalXPConnect");\n var converter = Components.classes["@mozilla.org/intl/scriptableunicodeconverter"]\n .createInstance(Components.interfaces.nsIScriptableUnicodeConverter);\n converter.charset = "UTF-8";\n original = converter.ConvertToUnicode(original);\n }\n catch(e) {\n }\n }\n }\n //DEBUG alert(original);\n this.uploadChangesFrom(original, storeUrl, toFilename, uploadDir, backupDir, \n username, password);\n};\n\nconfig.macros.upload.uploadChangesFrom = function(original, storeUrl, toFilename, uploadDir, backupDir, \n username, password) {\n var startSaveArea = '<div id="' + 'storeArea">'; // Split up into two so that indexOf() of this source doesn't find it\n var endSaveArea = '</d' + 'iv>';\n // Locate the storeArea div's\n var posOpeningDiv = original.indexOf(startSaveArea);\n var posClosingDiv = original.lastIndexOf(endSaveArea);\n if((posOpeningDiv == -1) || (posClosingDiv == -1))\n {\n alert(config.messages.invalidFileError.format([document.location.toString()]));\n return;\n }\n var revised = original.substr(0,posOpeningDiv + startSaveArea.length) + \n allTiddlersAsHtml() + "\sn\st\st" +\n original.substr(posClosingDiv);\n var newSiteTitle;\n if(version.major < 2){\n newSiteTitle = (getElementText("siteTitle") + " - " + getElementText("siteSubtitle")).htmlEncode();\n } else {\n newSiteTitle = (wikifyPlain ("SiteTitle") + " - " + wikifyPlain ("SiteSubtitle")).htmlEncode();\n }\n\n revised = revised.replaceChunk("<title"+">","</title"+">"," " + newSiteTitle + " ");\n revised = revised.replaceChunk("<!--PRE-HEAD-START--"+">","<!--PRE-HEAD-END--"+">","\sn" + store.getTiddlerText("MarkupPreHead","") + "\sn");\n revised = revised.replaceChunk("<!--POST-HEAD-START--"+">","<!--POST-HEAD-END--"+">","\sn" + store.getTiddlerText("MarkupPostHead","") + "\sn");\n revised = revised.replaceChunk("<!--PRE-BODY-START--"+">","<!--PRE-BODY-END--"+">","\sn" + store.getTiddlerText("MarkupPreBody","") + "\sn");\n revised = revised.replaceChunk("<!--POST-BODY-START--"+">","<!--POST-BODY-END--"+">","\sn" + store.getTiddlerText("MarkupPostBody","") + "\sn");\n\n var response = this.uploadContent(revised, storeUrl, toFilename, uploadDir, backupDir, \n username, password, function (responseText) {\n if (responseText.substring(0,1) != '0') {\n alert(responseText);\n displayMessage(config.macros.upload.messages.fileNotUploaded.format([getLocalPath(document.location.toString())]));\n }\n else {\n if (uploadDir !== '') {\n toFilename = uploadDir + "/" + config.macros.upload.basename(toFilename);\n } else {\n toFilename = config.macros.upload.basename(toFilename);\n }\n var toFileUrl = config.macros.upload.toFileUrl(storeUrl, toFilename, uploadDir, username);\n if (responseText.indexOf("destfile:") > 0) {\n var destfile = responseText.substring(responseText.indexOf("destfile:")+9, \n responseText.indexOf("\sn", responseText.indexOf("destfile:")));\n toFileUrl = config.macros.upload.toRootUrl(storeUrl, username) + '/' + destfile;\n }\n else {\n toFileUrl = config.macros.upload.toFileUrl(storeUrl, toFilename, uploadDir, username);\n }\n displayMessage(config.macros.upload.messages.mainFileUploaded.format(\n [toFileUrl]), toFileUrl);\n if (backupDir && responseText.indexOf("backupfile:") > 0) {\n var backupFile = responseText.substring(responseText.indexOf("backupfile:")+11, \n responseText.indexOf("\sn", responseText.indexOf("backupfile:")));\n toBackupUrl = config.macros.upload.toRootUrl(storeUrl, username) + '/' + backupFile;\n displayMessage(config.macros.upload.messages.backupFileStored.format(\n [toBackupUrl]), toBackupUrl);\n }\n var log = new config.macros.upload.UploadLog();\n log.endUpload();\n store.setDirty(false);\n // erase local lock\n if (window.BidiX && BidiX.GroupAuthoring && BidiX.GroupAuthoring.lock) {\n BidiX.GroupAuthoring.lock.eraseLock();\n // change mtime with new mtime after upload\n var mtime = responseText.substr(responseText.indexOf("mtime:")+6);\n BidiX.GroupAuthoring.lock.mtime = mtime;\n }\n \n \n }\n // for debugging store.php uncomment last line\n //DEBUG alert(responseText);\n }\n );\n};\n\nconfig.macros.upload.uploadContent = function(content, storeUrl, toFilename, uploadDir, backupDir, \n username, password, callbackFn) {\n var boundary = "---------------------------"+"AaB03x"; \n var request;\n try {\n request = new XMLHttpRequest();\n } \n catch (e) { \n request = new ActiveXObject("Msxml2.XMLHTTP"); \n }\n if (window.netscape){\n try {\n if (document.location.toString().substr(0,4) != "http") {\n netscape.security.PrivilegeManager.enablePrivilege('UniversalBrowserRead');}\n }\n catch (e) {}\n } \n //DEBUG alert("user["+config.options.txtUploadUserName+"] password[" + config.options.pasUploadPassword + "]");\n // compose headers data\n var sheader = "";\n sheader += "--" + boundary + "\sr\snContent-disposition: form-data; name=\s"";\n sheader += config.macros.upload.formName +"\s"\sr\sn\sr\sn";\n sheader += "backupDir="+backupDir\n +";user=" + username \n +";password=" + password\n +";uploaddir=" + uploadDir;\n // add lock attributes to sheader\n if (window.BidiX && BidiX.GroupAuthoring && BidiX.GroupAuthoring.lock) {\n var l = BidiX.GroupAuthoring.lock.myLock;\n sheader += ";lockuser=" + l.user\n + ";mtime=" + l.mtime\n + ";locktime=" + l.locktime;\n }\n sheader += ";;\sr\sn"; \n sheader += "\sr\sn" + "--" + boundary + "\sr\sn";\n sheader += "Content-disposition: form-data; name=\s"userfile\s"; filename=\s""+toFilename+"\s"\sr\sn";\n sheader += "Content-Type: " + config.macros.upload.contentType + "\sr\sn";\n sheader += "Content-Length: " + content.length + "\sr\sn\sr\sn";\n // compose trailer data\n var strailer = new String();\n strailer = "\sr\sn--" + boundary + "--\sr\sn";\n //strailer = "--" + boundary + "--\sr\sn";\n var data;\n data = sheader + content + strailer;\n //request.open("POST", storeUrl, true, username, password);\n try {\n request.open("POST", storeUrl, true); \n }\n catch(e) {\n alert(config.macros.upload.messages.crossDomain + "\snError:" +e);\n exit;\n }\n request.onreadystatechange = function () {\n if (request.readyState == 4) {\n if (request.status == 200)\n callbackFn(request.responseText);\n else\n alert(config.macros.upload.messages.errorUploadingContent + "\snStatus: "+request.status.statusText);\n }\n };\n request.setRequestHeader("Content-Length",data.length);\n request.setRequestHeader("Content-Type","multipart/form-data; boundary="+boundary);\n request.send(data); \n};\n\n\nconfig.macros.upload.download = function(uploadUrl, uploadToFilename, uploadDir, uploadBackupDir, \n username, password) {\n var request;\n try {\n request = new XMLHttpRequest();\n } \n catch (e) { \n request = new ActiveXObject("Msxml2.XMLHTTP"); \n }\n try {\n if (uploadUrl.substr(0,4) == "http") {\n netscape.security.PrivilegeManager.enablePrivilege("UniversalBrowserRead");\n }\n else {\n netscape.security.PrivilegeManager.enablePrivilege("UniversalXPConnect");\n }\n } catch (e) { }\n //request.open("GET", document.location.toString(), true, username, password);\n try {\n request.open("GET", document.location.toString(), true);\n }\n catch(e) {\n alert(config.macros.upload.messages.crossDomain + "\snError:" +e);\n exit;\n }\n \n request.onreadystatechange = function () {\n if (request.readyState == 4) {\n if(request.status == 200) {\n config.macros.upload.uploadChangesFrom(request.responseText, uploadUrl, \n uploadToFilename, uploadDir, uploadBackupDir, username, password);\n }\n else\n alert(config.macros.upload.messages.errorDownloading.format(\n [document.location.toString()]) + "\snStatus: "+request.status.statusText);\n }\n };\n request.send(null);\n};\n\n//}}}\n////===\n\n////+++!![Initializations]\n\n//{{{\nconfig.lib.options.init('txtUploadStoreUrl','store.php');\nconfig.lib.options.init('txtUploadFilename','');\nconfig.lib.options.init('txtUploadDir','');\nconfig.lib.options.init('txtUploadBackupDir','');\nconfig.lib.options.init('txtUploadUserName',config.options.txtUserName);\nconfig.lib.options.init('pasUploadPassword','');\nsetStylesheet(\n ".pasOptionInput {width: 11em;}\sn"+\n ".txtOptionInput.txtUploadStoreUrl {width: 25em;}\sn"+\n ".txtOptionInput.txtUploadFilename {width: 25em;}\sn"+\n ".txtOptionInput.txtUploadDir {width: 25em;}\sn"+\n ".txtOptionInput.txtUploadBackupDir {width: 25em;}\sn"+\n "",\n "UploadOptionsStyles");\nconfig.shadowTiddlers.UploadDoc = "[[Full Documentation|http://tiddlywiki.bidix.info/l#UploadDoc ]]\sn"; \nconfig.options.chkAutoSave = false; saveOptionCookie('chkAutoSave');\n\n//}}}\n////===\n\n////+++!![Core Hijacking]\n\n//{{{\nconfig.macros.saveChanges.label_orig_UploadPlugin = config.macros.saveChanges.label;\nconfig.macros.saveChanges.label = config.macros.upload.label.saveToDisk;\n\nconfig.macros.saveChanges.handler_orig_UploadPlugin = config.macros.saveChanges.handler;\n\nconfig.macros.saveChanges.handler = function(place)\n{\n if ((!readOnly) && (document.location.toString().substr(0,4) != "http"))\n createTiddlyButton(place,this.label,this.prompt,this.onClick,null,null,this.accessKey);\n};\n\n//}}}\n////===\n
Type the text for 'Uri Alon'
Vertebrates have three [[Ena]]-related genes: [[Mena]], [[EVL]] and VASP. VASP was identified as a major protein kinase A [[PKA]] substrate in platelets (Halbrugge et al., 1990) and subsequently found to localize to focal adhesions, actin stress fibers, the lamellipodial leading edge and to filopodial tips as do [[Mena]] and [[EVL]] [[Gerler 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12432060&query_hl=16&itool=pubmed_docsum]]. VASP promotes processive filament elongation. Dicty VASP null cells have defects in persistence suggesting a role in chemotaxis [[Firtel 2002|http://www.ncbi.nlm.nih.gov/pubmed/12388544?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and VASP binds directly to the IL8 receptor [[Richmond 2009|http://www.ncbi.nlm.nih.gov/pubmed/19435808?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. \n\nThe protein has three parts: an N-terminal EVH1 domain that links VASP to key ligands, a central proline-rich region (PRR) that contains at least three separate binding sites for profi- lin–actin and actin monomers, and a C-terminal EVH2 domain that interacts with F-actin possibly to cap the barbed end [[Goode 2009 review|http://www.ncbi.nlm.nih.gov/pubmed/19168341]]. Clustering of VASP molecules may be required for their ability to protect and accelerate barbed end elongation [[Small 2008|http://www.ncbi.nlm.nih.gov/pubmed/18923426]].
[[Verprolin]], [[Cofilin]], Acidic. The V (also called the WH2 domain) portion binds actin [[Takenawa 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9473482&query_hl=10&itool=pubmed_DocSum]], while the CA portion binds [[Arp2/3]] [[Insall 1998|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=9889097&query_hl=4&itool=pubmed_DocSum]]. The C portion can also bind actin [[Mullins 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16403731&query_hl=102&itool=pubmed_DocSum]].
Vav proteins are guanine nucleotide exchange factors (GEFs) for the [[Rho]]/[[Rac]] family of GTPases. In mammals, there are three family members; Vav1 is specifically expressed in the hematopoietic system, whereas Vav2 and Vav3 are more ubiquitously expressed. Vav proteins have been implicated in cytoskeletal reorganization, including formation of the immunological synapse, phagocytosis, platelet aggregation, spreading, and transformation. All Vav proteins contain several characteristic structural motifs that enable their function as signal transducer proteins. These include a [[DH]], which exhibits [[GEF]] activity towards the RhoGTPases; [[PH]] domain, SH2 and two SH3 domains, an acidic-rich (Ac) region and a ‘calponin-homology’ (CH) region, which functions as an actin-binding domain in other proteins. Vav proteins are the only known RhoGEFs that combine in the same protein the [[DH]]/[[PH]] motifs and the structural hallmark of signal transducer proteins, the SH2 and SH3 domains [[Katzav 2004 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=14607270]].
Showed systematically that HL-60 cells are similar to human neutrophils [[Niggli 2002|http://www.ncbi.nlm.nih.gov/pubmed/11950599?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=13]]
The yeast homolog of [[WIP]].
Showed that the lamellipodia has actin branch angles from 15 to 90 degrees instead of just 70 degrees [[Small 2008|http://www.ncbi.nlm.nih.gov/pubmed/18278037]].\n\nAnalyzed dynamics of Arp2/3 and WAVE complex in B16F10 cells by FRAP [[Small 2008|http://www.ncbi.nlm.nih.gov/pubmed/18309290]].\n\nShowed that there are no branches in the lamellipodia [[Small 2010|http://www.ncbi.nlm.nih.gov/pubmed/20418872]].
Vinculin bind [[Talin]] and is activated by PIP2 [[Burridge 1996|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=8632828&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
[[Adaptation]]\n\n[[Biochemistry]]\n[[Bead assay]]\n\n[[Calcium Signaling]]\n[[C. Elegans Polarity]]\n[[Chaotropic]]\n[[Chemokinesis]]\n[[Chemotaxis]]\n[[Clock]]\n[[Complement Cascade]] \n[[Cool Papers]]\n[[Cytoplast]]\n\n[[Diffusion]]\n[[Dictyostelium]]\n[[Directional Sensing]]\n\n[[EM]]\n[[Epithelial polarity]]\n[[Feed Forward Loop]]\n[[Filopodia]]\n[[Fugetaxis]]\n\n[[Haptotaxis]]\n\n[[Lamella]]\n[[Lamellipodia]]\n[[Leading Edge]]\n[[Local Activation Global Inhibition]]\n[[Local Stimulation - Microfluidics]]\n\n[[Microscopy]]\n\n[[Permeabilized Cells]]\n[[Polarization]]\n[[Postive Feedback]]\n\n[[Single Molecule]]\n[[Spatial Signaling]]\n[[Superoxide Production]]\n[[Synthetic Biology]]\n[[Systems Biology]]\n\n[[TIRF]]\n\n[[Ultrasensitivity]]\n[[Yeast Polarity]]\n
Yeast [[PI3K]].
Another name for [[N-WASP]] expressed in hematopoeitic cells. Neutrophils from WASP knockout mice have defects in chemotaxis and when combined with mDia knockout mice have severe defects in chemotaxis [[Alberts 2009|http://www.ncbi.nlm.nih.gov/pubmed/19265163?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. They also have defects in actin polarization.
WASP (Wiskott–Aldrich syndrome protein)-family verprolin homology protein. WAVE contains a WAVE homology domain (WHD), a basic region, a pro-rich region, and a [[VCA]] domain. The basic region is necessary and sufficient to bind PIP2 and PIP3 [[Takenawa 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15107862&query_hl=1&itool=pubmed_docsum]].
Consists of [[Pir121]], [[Hem-1]], WAVE2, [[Abi1]], and [[Hspc300]]. These proteins are only present in the complexed form, with the exception of [[Hspc300]], which displays a free pool. The complex is organized around a core of [[Nap]] and [[Abi]]. [[Sra]] is a peripheral subunit recruited on the [[Nap]] side, whereas the [[WAVE]] and [[Hspc300]] subunits are recruited on the [[Abi]] side of the core [[Kirschner 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15070726&query_hl=7&itool=pubmed_docsum]]. In Drosophila [[S2]] cells, a [[SCAR]] null-like phenotype results when either [[Abi2]], [[Pir121]] or [[Nap1]] is removed and leads to [[SCAR]] degradation [[Vale 2003|http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=12975351&dopt=Abstract]]. Three papers obfuscate WAVE complex biology: [[Greengard 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16862120&query_hl=3&itool=pubmed_docsum]], [[Kirschner 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12181570&query_hl=5&itool=pubmed_DocSum]], and [[Scita 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15048123&query_hl=20&itool=pubmed_docsum]].
WAVE1 is expressed in neurons and fibroblasts, but not in neutrophils. [[Takenawa 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10381382&query_hl=4&itool=pubmed_docsum]]. WAVE1 may have a role independent of [[Arp2/3]] activation in actin reorganization [[Takenawa 2000|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10833423&query_hl=14&itool=pubmed_DocSum]].
WAVE2 is expressed ubiquitously. WAVE2 deficient mice show defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization [[Kirschner 2003|http://www.ncbi.nlm.nih.gov/pubmed/12853475?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. In the presence of a ~RacDN, a truncated version of WAVE2 (containing a WHD and basic domain) still localizes to the membrane. This localization is abolished the presence of [[Wortmannin]] [[Takenawa 2004|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15107862&query_hl=1&itool=pubmed_docsum]]. The VCA domain of WAVE2 is thought to be phosphorylated by Erk2. This phosphorylation increases WAVE2 affinity for Arp3, but paradoxically decreases WAVE2 VCA activity in an actin pyrene polymerization assay. A WAVE2 mutant with all its phosphorylation sites replaced with alanine still rescued a WAVE2-null MEF [[Takenawa 2007|http://www.ncbi.nlm.nih.gov/pubmed/17202194?dopt=AbstractPlus]]. Fibroblasts containing a phospho-defective WAVE2 exhibit an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus [[Cory 2007|http://www.ncbi.nlm.nih.gov/pubmed/18032787?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
The WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP not through a proline-rich sequence, but through a consensus sequence (ESRFYFHPISD) that is conserved in WIP, CR16, WICH, and yeast verprolin [[Way 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12372256&query_hl=30&itool=pubmed_docsum]]. The WH1 domain fold is unexpectedly similar to that of the pleckstrin homology domain and PTB domains [[Lim 1999|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10338211&query_hl=30&itool=pubmed_docsum]].
WH2 domains (also called the V or Verprolin domains) transiently attach to actin filaments and allow N-WASP attachment to actin comet tails on lipid coated beads [[Taunton 2007|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17350575&query_hl=24&itool=pubmed_docsum]].
[[WASP]] interacting protein. WIP retards N-WASP/[[Cdc42]]-activated actin polymerization mediated by the [[Arp2/3]] complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia [[Takenawa 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?itool=abstractplus&db=pubmed&cmd=Retrieve&dopt=abstractplus&list_uids=11331876]]. WIP also interacts with [[Profilin]], globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments [[Jones 2006 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16546573&query_hl=139&itool=pubmed_DocSum]]. See also WICH [[Takenawa 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11829459&query_hl=74&itool=pubmed_docsum]].
WRC labeling\n\nArthur Millius 7/24/07\n\nOverview\n\nTo label recombinant WRC with Alexa 594 and purify away from dye.\n\nBuffers\n\nKMEI + 20% glycerol = 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole, 20% glycerol\nTCEP = 1 M TCEP\n\nProcedure\n\nAll steps done at 4 C.\n\nQuickly thaw 100 µl recombinant WRC at 335 nM, which is ~18 µg. Add 1.1 µl 1 M TCEP. Rotate for >2 hours. Note: I incubated for 5.5 hours.\n\nBuffer exchange with micro bio-spin 30 column\n\n-Invert column to resuspend settled gel\n-Tap column to remove air bubbles\n-Snap off tip place in 2 ml centrifuge tube\n-Allow buffer to drain by gravity (2 min)\n-Centrifuge 2 min at 1000 x g\n-Apply 500 µl of KMEI + 20% glycerol and centrifuge 1000 x g for 1 min\n-Discard and repeat wash twice\n-Place column in a clean 1.5 ml microcentrifuge tube. Apply sample on (10 – 75 µl) directly onto the top center of the gel. Centrifuge the column for 4 min at 1000 x g.\n\nNote: I recovered 125 µl of recombinant WRC.\n\nTake a 25 µl aliquot and add 1 mM DTT (1 µl of 25 mM).\n\nAdd 3 µM alexa 594, 11 µl of 30 µM alexa 594 maleimide (1.5 µl 2 mM alexa 594 in 98.5 µl KMEI + 20% glycerol) to 100 ml WRC in KMEI + 20% glycerol.\n\nAfter 5 minutes, 15, 30, and 60 minutes take a ~25 µl aliquot and add 1 mM DTT to quench the reaction.\n\nRemove 5 µl for a gel from each aliquot.\n\nTake remaining 20 µl and do buffer exchange in KMEI + 20% glycerol with micro bio-spin 30 column.\n\nNote: I recovered ~45 µl from the buffer exchange. I took 10 µl for a gel.
WRP, a RacGAP that binds directly to WAVE1 through its SH3 domain and specifically inhibits [[Rac]] function in vivo [[Scott 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12447388&query_hl=21&itool=pubmed_docsum]]. WRP also contains an FCH domain (the first half of a [[BAR]] domain).
''rules'' the //chemotaxis// field [[Weiner 2007|http://www.ncbi.nlm.nih.gov/pubmed/17696648?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]
This document is a ~TiddlyWiki from tiddlyspot.com. A ~TiddlyWiki is an electronic notebook that is great for managing todo lists, personal information, and all sorts of things.\n\n@@font-weight:bold;font-size:1.3em;color:#444; //What now?// &nbsp;&nbsp;@@ Before you can save any changes, you need to enter your password in the form below. Then configure privacy and other site settings at your [[control panel|http://chemotaxis.tiddlyspot.com/controlpanel]] (your control panel username is //chemotaxis//).\n<<tiddler tiddlyspotControls>>\n@@font-weight:bold;font-size:1.3em;color:#444; //Working online// &nbsp;&nbsp;@@ You can edit this ~TiddlyWiki right now, and save your changes using the "save to web" button in the column on the right.\n\n@@font-weight:bold;font-size:1.3em;color:#444; //Working offline// &nbsp;&nbsp;@@ A fully functioning copy of this ~TiddlyWiki can be saved onto your hard drive or USB stick. You can make changes and save them locally without being connected to the Internet. When you're ready to sync up again, just click "upload" and your ~TiddlyWiki will be saved back to tiddlyspot.com.\n\n@@font-weight:bold;font-size:1.3em;color:#444; //Help!// &nbsp;&nbsp;@@ Find out more about ~TiddlyWiki at [[TiddlyWiki.com|http://tiddlywiki.com]]. Also visit [[TiddlyWiki Guides|http://tiddlywikiguides.org]] for documentation on learning and using ~TiddlyWiki. New users are especially welcome on the [[TiddlyWiki mailing list|http://groups.google.com/group/TiddlyWiki]], which is an excellent place to ask questions and get help. If you have a tiddlyspot related problem email [[tiddlyspot support|mailto:support@tiddlyspot.com]].\n\n@@font-weight:bold;font-size:1.3em;color:#444; //Enjoy :)// &nbsp;&nbsp;@@ We hope you like using your tiddlyspot.com site. Please email [[feedback@tiddlyspot.com|mailto:feedback@tiddlyspot.com]] with any comments or suggestions.
Showed that both the basic domain and GBD domain are required for repression and regulation of N-WASP [[Lim 2000|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=11052943&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].\n\nShowed that PIP2 binds the polybasic region of N-WASP to potentiate actin assembly [[Lim 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15664188&query_hl=14&itool=pubmed_docsum]].
Western Blot \nArthur Millius 1/9/06\n\nOverview:\n\nTo run a Western blot on our cytosol and plasma membrane fractions of pig leukocyte preparation on 12/20/06. We will probe with antibodies to Hem-1, GDI, and transferring receptor to 1) determine the linear range of sensitivity for the antibodies given a series of dilutions from cytosol and membrane and to 2) determine whether membrane or cytosol wave complex degraded during the preparation.\n\nWestern blot protocol:\n\nGeneral Idea:\n\nRun a protein SDS-PAGE gel. Transfer it onto membranes. Block the membranes then probe with primary antibody. Wash the membrane and probe with the secondary antibody. Use the Odyssey infrared imaging system (LI-COR Biosciences) to detect the antibody.\n\nSample Preparation\n \nControl\n1) Cytosol from wild-type sucrose gradient (HS3 Leuk)\n2) Plasma membrane before high speed spin post-supernatant (CT PM)\n3) Plasma membrane after HS3 (CT PMC)\n\n\nSamples were previously frozen without loading buffer in liquid nitrogen on 12/20/06. Samples were thawed and 6x loading buffer (LB) was added to them to a final loading buffer concentration of 1x. For HS3 Leuk, 100 µl was taken from a 1 ml aliquot. 20 µl was added to 100 µl HS3 Leuk and 100 µl CT PM. 10 µl was added to the 50 µl aliquot of CT PMC.\n\nSamples were boiled for ~5 minutes at ~90C.\n\nSamples were diluted (µl) in 1x loading buffer as follows:\n\nControl HS3 Leuk\n1/5 = 20 in 80\n1/10 = 10 in 90\n1/20 = 5 in 95\n1/100 = 1 in 99\n1/300 = 1 in 299\n\nControl CT PM and CT PMC\n1/20 = 5 in 95\n1/100 = 1 in 99\n1/300 = 1 in 299\n1/600 = 50 of 1/300 in 50 \n1/1200 = 25 of 1/300 in 75\n\nSamples were boiled again right before loading for 5 minutes at ~90C.\n\nRunning the gels\n\nUse 4-12% BT NuPAGE gradient gels.\nAfter removing the combs we first rinsed out the wells with distilled water.\nLoad the gels into gel chambers, and add 200 ml running buffer (1x MOPS) + 500 µl antioxidant to the inner chamber. Check for leakage between the outer and inner chambers in either gel box. Note: The antioxidant is necessary to prevent DTT in the loading buffer from running away from the protein during the run.\n\nFill the outer chamber with running buffer.\nWash out all of the wells with a pipetman with running buffer.\nThe loading order for each gel is shown below:\n\nGel 1 (blotted with rabbit anti-hem1):\n1 = MW\n2 = HS3 Leuk 1/5\n3 = HS3 Leuk 1/10\n4 = HS3 Leuk 1/20\n5 = HS3 Leuk 1/50\n6 = HS3 Leuk 1/100\n7 = HS3 Leuk 1/300\n8 = CT PMC 1/20\n9 = CT PMC 1/100\n10 = CT PMC 1/300\n11 = CT PMC 1/600\n12 = CT PMC 1/1200\n13 = CT PM 1/20\n14 = CT PM 1/100\n15 = CT PM 1/300\n16 = CT PM 1/600\n17 = CT PM 1/1200\n\nGel 2a (blotted with rabbit anti-GDI)\n1 = MW\n2 = HS3 Leuk neat\n3 = HS3 Leuk 1/5\n4 = HS3 Leuk 1/10\n5 = HS3 Leuk 1/20\n6 = CT PMC neat\n7 = CT PM neat\n\nGel 2b (blotted with goat anti-transferrin receptor)\n8= MW\n9 = HS3 Leuk neat\n10 = CT PMC 1/100\n11 = CT PMC 1/300\n12 = CT PMC 1/600\n13 = CT PMC 1/1200\n14 = CT PM 1/100\n15 = CT PM 1/300\n16 = CT PM 1/600\n17 = CT PM 1/1200\n\n\nTransferring the proteins to a nitrocellulose membrane\n\nCarefully crack open the gel case with the spatula and use the spatula to cut off the wells and cut off the bottom edge of the gel where it is attached to the plastic case.\n\nThe semi-dry transfer apparatus is set up as shown:\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nThe gels and nitrocellulose membranes were equilibrated with transfer buffer for ~5 minutes prior to assembly of the transfer apparatus. Just before assembly, equilibrate Whatman paper with transfer buffer. Note: It is important to use a broken pipet to roll the bubbles out of the Whatman paper prior to assembly of the assembly. When handling the gel, always do so at the bottom where the SDS concentration is highest and the gel is strongest. Never touch the membrane without gloves, try to use forcepts as much as possible and touch it at the corner. Label each membrane in the top left on the side that will encounter protein.\n\nAssemble the apparatus as shown above.\nUse a broken pipet to roll the bubbles out of the apparatus prior to placing the upper plate on the apparatus.\nRun the transfer in constant current mode at 120mA per membrane for 30 minutes. Note: When the transfer is complete the MW markers should be visible on the membrane but no longer visible in the gel or in the Whatman paper below the membrane (its presence in the Whatman below the membrane would indicate that the proteins were transferred through the membrane onto the Whatman paper).\n\n\nDevelopment of the Membrane\n\nBlock the membrane in a mixture of 50% TBS, 50% Odyssey block (10 ml per membrane, I used 30 ml total) Note: It is critical that Tween is not present in the blocking solution as it will stick to the membrane, which would be detrimental since Tween is fluorescent.\n\nAt this point I cut the membrane for gel 2 in half (at the middle of the MW marker in lane 8).\n\nPour back 30 ml into a 50 ml falcon and add to 0.2% tween.\n\nPlace ~9 ml into square containers, and place one membrane in each container.\n\nProbe gel 2a for GDI (Rabbit 1:1000), and the other half (gel 2b) for transferrin receptor (Goat 1:500). The membrane for gel 1 was probed for Hem-1 (Rabbit 1:2000). The antibodies were diluted in a 50/50 solution of Odyssey Block and TBS with Tween present at a final concentration of 0.2% Note: Tween will not stick to membranes if they have already been blocked.\n\nSpecifically, I poured the 9 ml from the square container with the membrane into a 15 ml falcon, added appropriate amount of antibody depending on volume of liquid (some were less than 9 ml), and added it back to square container.\n\nThe membranes were incubated with primary antibodies overnight at 4C.\n\nThe antibodies were saved in a 15 ml Falcon tube with 0.2% azide.\n\nThe membranes were given 3 quick washes then 3x10 minute washes in TBS-T.\n\nWe incubated the membranes in secondary antibody for one hour at room temperature, 15 ml per dish, Alexa-680 conjugated antibody against Rabbit and Goat. The antibody was diluted 1:2000 in 50/50 Odyssey Block/TBS with Tween present at 0.2%. I added 5 µl for the anti-goat for 10 ml and 10 µl for the anti-rabbit for 20 ml Note: Cover the dishes with aluminum foil to prevent the fluorophores from being photobleached.\n\nThe membranes were again given (3) quick washes then 3x10 minute washes in TBS-T. The membranes were given (2) final 10 minute washes in TBS Note: The purpose of this was to wash out all of the Tween, thus reducing background fluorescence\n\nVisualize the membranes using the Odyssey System in the Lim lab (username = limlab and password = theking). Flatten membrane with roller at bottom edge of glass. Invert membrane so that molecular weight marker (upper-left hand corner of membrane) is on the bottom left portion of the glass. Select “membrane” for the setting, draw box around coordinates that membrane occupies, scan at 700, and use intensity so that bands do not bleach.
Wiskostatin induces folding of the isolated, unstructured GBD into its autoinhibited conformation, suggesting that wiskostatin functions by stabilizing N-WASP in its autoinhibited state [[Kirshner 2004|http://www.ncbi.nlm.nih.gov/pubmed/15235593?ordinalpos=13&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
Wortmannin, a metabolite of the fungus Penicillium funiculosum, is a specific inhibitor of phosphoinositide 3-kinases (PI3Ks). It can also inhibit other PI3K-related enzymes such as mTOR, DNA-PK, some phosphatidylinositol 4-kinases, myosin light chain kinase (MLCK) and mitogen-activated protein kinase (MAPK) at high concentrations. It is an ATP analog, which irreversibly attaches to PI3K preventing ATP hydrolysis. Wortmannin confusing results:\n\n[[Bourne 2002|http://www.nature.com/ncb/journal/v4/n7/full/ncb810.html]]\n[[Condeelis 2006|http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRT-4MCW996-P&_user=4430&_coverDate=11%2F21%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059594&_version=1&_urlVersion=0&_userid=4430&md5=d7f93e52153201554885542e0448b6af]]\n[[Bourne 2006|http://www.jcb.org/cgi/content/full/174/3/437]]
Use special gold-bottomed 96-well plate for XCelligence Assays (all equipment located in Pathway to Medicine Area). Careful not to touch bottom of plate, can damage wires or make reading gold surface difficult. Always handle by top or by edges. Half-columns (sets of 4 wells) all share a ground wire -- have to run experiment on all 4 wells, or at least make sure there is liquid in all 4 wells. Otherwise will get an error b/c wells not grounded properly.\n\n\n\n1. Make fresh mHBSS + Ca + 1% BSA (low-endotoxin, A9306 from sigma), as the buffer to be used for all experiments. Serum will stimulate cells and make readings less clear.\n2. Plate 50 ul of 100 ug/ml fibronectin in wells that you are using. Tap edges of plate to make sure that fibronectin settles to bottom of well. Allow to sit covered for 1 h at room temp.\n3. While fibronectin is plating, count cells to be used. With HL-60s, find that 250,000 cells/well is a good density. Spin down appropriate amount of cells 400 g for 5 min and resuspend in buffer. Will be adding 80 ul of cells to each well, so make sure that this 80 ul contains 250,000 cells. Do this step close to the end of hour, don't want cells in mHBSS too long.\n4. Start up software (RTCA software).\nLogin for program (RTCA Software): \nusername: Administrator \npassword: administrator\n\nSelect plate that isn't being used, go to Plate->Release to close experiment if one is open. Make sure that the plate reader you want to use is unlocked.\n5. Aspirate fibronectin from all wells, try to minimize touching the bottom of the well (easiest to use multichannel Rainin pipette). Look at wells to see whether all liquid removed.\n6. Add 50 ul buffer to all wells. Tap sides to make sure no bubbles at bottom of well. Replace plastic cover of plate with metal spacer (allows the wells to be accessible during the experiment). Insert into one of the plate reader positions (notch side away from you). Make sure plate slides smoothly onto pins before securing it -- there should be no resistance. Then lock plate in software. Let plate sit for 15 minutes.\n7. Go to Experimental Notes Tab, select folder to save work to, and create a name for the file.\n8. Go to Layout Tab. Turn on appropriate wells, and label them with what the contents of the well will be (click Apply after entering info for each set of wells). Should run all experiments in triplicate (for each experiment, have a row of 3 wells that is all the same condition).\nGo to Schedule Tab, Right click and Add Step to take a background measurement. Take a few sweeps (maybe 5 or so), every 30 sec. Start this step.\n9. At end of sweeps, unlock plate in software, and then remove from plate reader. Add 80 ul cells to each well. I like to shake tube every 3 wells so that cells don't settle significantly and give diff. #'s of cells in the wells.\n10. Return plate to reader, secure and lock. Add a step to Schedule Tab, take 100 measurements, and measure every 3 min. Start this step. If you look at the results in the Plot Tab, cell index should rise as cells settle and stick to the bottom, and plateau after about 30 min. or so. Wait until you see the cell index plateau before adding drug/agonist.\n11. While the cells are settling, make drug/agonist that you wish to add to cells in a regular (not XCelligence) 96 well plate. You will be adding 70 ul to each well, so calculate your final concentrations appropriately (70 ul to a final volume of 200 ul). \n12. When cell index plateaus, click to abort the current step (the fast forward button). Add a new step, where you sample every 30 s, and take 100 sweeps. Start this step.\n13. After a few measurements have been made for each well, add 70 ul of each drug/agonist to appropriate wells. Use multichannel Rainin pipette to get better synchronous addition to the different wells.\n14. Allow experiment to run until interesting results no longer seen in Plot tab. Can abort experiment at that point. Can copy results from Plot tab to Excel spreadsheet to get actual data values, as well as graphs, well layout info. \n\nCleanup: if not all wells used in plate, aspirate liquid from used wells, and return plate (covered with plastic lid now) to drawer for future use. \n\nGeneral limitations: can't take readings much faster than every 20-30s if all wells turned on. By looking at fewer wells, can go faster. Also, there is some way to take less accurate readings that can make it go faster (ask rep from Roche)\nAverage option in Plot tab is useful, can use it to see what average of each condition is, or look at individual wells to discard if one looks bad.\nNormalized Cell Index in Plot tab can give you rough idea of what fold-increase you see upon agonist addition, drag black arrow to select time point to normalize to.\n \n\n\n
Inhibitor of ROCK. Y-27632 causes mutiple leading edge formation. PhAkt accumulates near the cell periphery in each of these leading edges. Additionally, cell is more likely to reverse direction instead of performing a U-turn when treated with Y-27632. [[Bourne 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887922&query_hl=11&itool=pubmed_docsum]].
Yeast Polarity\n\nThe new players:\nRsr1 aka Bud1\nBud2\nBud5\nCdc24\nBem1\nSte5,7,11\nFus3\nFar1\n\nThe usual suspects:\nHeterotrimeric G-proteins\nF-actin\nFormins\nCdc42\n\nNew plot twist:\n\nCdc42 has a very different lifestyle in yeast than it does in mammalian cells. In mammalian cells, most Rho-family GTPases spend most of their time in the cytosol where they exist in complex with GDI. They don’t spend much time at the membrane in mammalian cells unless they are GTP-bound. In yeast, there is a GDI; however, it is completely inconsequential to polarity and there is a lot of Cdc42 (bound to GDP or GTP) at the plasma membrane as well as internal vesicles.\n\nGeneral notes about yeast morphology:\n\nActin plays a very different role in yeast morphology as opposed to mammalian cells. In mammalian cells, the shape of the F-actin network defines the shape of the cell. In yeast, the cell wall defines the shape of the cell. Actin regulates yeast morphology by directing the transport and exocytosis of vesicles containing cell wall biosynthetic machinery. The primary actin structures involved in this polarized secretion process are long cables of F-actin. These cables are tracks along which vesicles are carried by myosin. Generally, if a yeast cell wants to bud or shmoo at a particular site, it builds a bunch of F-actin cables pointing towards that site. Vesicles are then transported along the cables until they arrive at the site and get exocytosed. The cell wall is then reorganized by the newly secreted enzymes to build the new bud/shmoo.\n\nGeneral notes about polarity:\n\nWhen an organism polarizes, two general (and obvious) steps occur. The cell needs to decide the direction in which it will polarize (orientation) and the cell needs to segregate the activity of its signals into distinct regions (polarization). Yeast cells polarize in two situations: budding (cell division) and shmooing (mating). The polarization system is the same during both processes, but the proteins used to orient the axis of polarity are different. \n\nWhat is the orientation program and how does it work?\n\nS. cerevisiae cells proliferate when a “daughter” cell buds off the side of a “mother” cell. Thus over time a yeast cell will give birth to numerous daughter cells. Each birth leaves a scar on the surface of the mother cell – remnants of the extra cell wall that is deposited at the bud site during budding. A while back, someone stained haploid yeast cells with calcofluor (a cell wall stain) and found that all of the scars were clustered around each other. This led to the realization new buds tend to form at the bud scars in haploid cells. Similar studies revealed that in diploid cells, the new bud tends to form at the exact opposite end of the cell from the previous bud. The proteins responsible for choosing the new bud site (in both haploids and diploids) have been identified genetically. These proteins constitute the orientation system for budding. They consist of the Ras-related GTPase Rsr1/Bud1 as well as a whole family of other Bud-proteins. These were identified in a screen for mutants whose bud-site selection was randomized. Bud5 is a GEF for Rsr1 while Bud2 is a GAP for Rsr1. These proteins are required for orienting the bud site in both haploid and diploid yeast. There are also several proteins who act specifically to promote axial or bipolar budding patterns. Bud6-9 are required for the bipolar budding pattern in diploids. Bud3, Bud4, Bud10 and Axl1 are required for the axial budding pattern in haploids. Interestingly the deletion of any of these proteins causes haploid yeast to display a bipolar budding pattern, implying that the bipolar pattern is the default pattern and it is repressed in haploid yeast. Rsr1 is localized uniformly over the yeast plasma membrane; however, Bud2 and Bud5 are both localized to the presumptive bud site before bud formation. I don’t know how Bud2/5 get localized, which is the key determinant of bud site selection. Importantly, and I can’t emphasize this enough, none of these proteins are required for the budding process itself. You could delete any of them, and the cells would still form normal buds – they would just do so in an altered pattern. \n\nDuring mating, gradients of pheromones direct the growth of “shmoos” between cells of opposite mating type. Shmoos are phallic projections by which yeast cells conjugate (connect) with one another during mating. The orientation system in this case consists of: the pheromone receptors (which are GPCRs), the heterotrimeric G-proteins, a MAPK cascade (consisting of Ste7, Ste11 and Fus3 – all scaffolded by Ste5) and Far1. Far1 is a scaffold that interacts with both heterotrimeric G-proteins and the polarization system to orient shmoo growth. Importantly, the orientation system is not required for cells to form shmoos. If uniform pheromone is present or if Far1 is eliminated, yeast cells will still form shmoos – at a random orientation with respect to the gradient. In this case, the shmoos form at the bud scars, due to input from Bud1/Rsr1.\n\nThus, the orientation system is not required for polarity in yeast – i.e. the yeast will bud/shmoo in a randomly chosen direction if the bud site selection program is eliminated or mating factor is applied uniformly.\n\nHow does the polarization system work?\n\nCdc42 is the key signal that must be polarized for budding and shmooing to occur. If Cdc42 activity is uniform then yeast will simply expand uniformly instead of budding or shmooing. GTP-bound Cdc42 activates the formin Bni1, which generates F-actin cables – along which cell wall remodelers are transported to the bud/shmoo site. There is now strong evidence, though not quite ironclad, that Cdc42 activity is amplified by positive feedback. The primary mechanism for positive feedback is active Cdc42 binding and recruiting the scaffolding protein Bem1. Bem1, in turn, binds Cdc24 which is a GEF for Cdc42. The cycle is thus: Cdc42 – Bem1 – Cdc24 – Cdc42 repeat until Cdc42 activity is high. There is now data that Bem1 and Cdc24 form a tripartite complex with a PAK (either Cla4 or Ste20\n\nThere is a secondary, actin dependent positive feedback that works by active transport of vesicular Cdc42 along the actin cables to the bud site (remember there is a lot of vesicular Cdc42 in budding yeast). The evidence for this feedback is that constitutively active Cdc42 can still polarize in yeast – and this polarity is actin dependent. In addition to the cable-mediated positive feedback there are also actin patches which disperse membrane-bound Cdc42 via endocytosis. Thus, there appear to be actin-dependent positive and negative feedback loops in yeast. Importantly, the polarization of endogenous Cdc42 is not actin dependent, but it is dependent on Bem1 and Cdc24. Therefore, it appears as though the Bem1/Cdc24 feedback is more important than either of the actin-dependent feedback loops. Obviously, the cell does not polarize without actin - because the directed transport of vesicles containing cell wall machinery does not occur. However, the primary intracellular polarity signal Cdc42 does polarize without actin.\n\nThere have been no experiments aimed at determining what limits the positive feedback. Obviously, if the positive feedback exists then something must limit it – or else Cdc42 would become uniformly active within the cell. A few ideas are the presence of a global inhibitor, negative crosstalk between signals at the bud and elsewhere (analogous to Rac and Rho crosstalk in neutrophils) or the depletion of limiting components from the cytosol. There have been lots of genetic screens done by now and nobody has identified either a global inhibitor or mutual negative crosstalk. If either of those mechanisms exist they must be highly redundant, or they would have been identified genetically by now. It seems to me that depletion of limiting components (for example: Cdc42, Cdc24 or Bem1) is what prevents Cdc42 activity from spreading throughout the cell. IMO, the recent study by Altschuler and Wu supports the notion of a simple limiting component model operating in yeast.\n\nHow does the orientation system orient the polarization system?\n\nDuring mating, the scaffold Far1 directly interacts with both heterotrimers and Bem1 and Cdc24. Thus, Far1 probably links receptor signaling with Cdc42 activation. There is likely some sort of adaptation mechanism to sharpen up the internal pattern of heterotrimer activation relative to the external pheromone gradient. Rsr1 directly interacts with both Cdc24 and Bem1 so the spot of Rsr1 defines where Cdc42 will become active. Interestingly, Rsr1-GDP binds Bem1 while Rsr1-GTP binds Cdc24. I have no idea how Rsr1 itself gets localized to an axial or bipolar pattern. If you do, please elaborate.\n\n
Showed that DOCK2 null T and B lymphocytes have defects in migration, chemokine-induced Rac activation, and actin polymerization [[Fukui 2001|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11518968&query_hl=31&itool=pubmed_docsum]].\n\nShowed that DOCK2 associates with [[Elmo]] through the SH3 domain on DOCK2. Showed that this interaction is required for [[Rac]] activation (Pak pulldown) and actin assembly [[Fukui 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12829596&query_hl=26&itool=pubmed_docsum]].\n\nShowed that DOCK2 null neutrophils have a defect in [[Rac1]] and [[Rac2]] activation (Pak pulldown), superoxide production (luminol), actin assembly (phalloidin), PIP3 accumulation (PH-Akt). DOCK2 associates with PIP3 and translocates to the leading edge in a PI3K dependent manner ([[LY294002]] inhibition) [[Fukui 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16943182&query_hl=31&itool=pubmed_docsum]].\n\nShowed that a Shigella effector IpgB1 harnesses the host protein Elmo1 by binding to the RhoG portion of Elmo1 to induce membrane ruffling and phagocytosis. By mimicking RhoG activity, IpgB1 allows Shigella to be engulfed [[Fukui 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17173036&ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
Atypical PKC. Same as [[PKCz]].
Pak-interactive exchange factor (alpha-Pix) was identified through its ability to bind the Cdc42/Rac target p21-activated kinase ([[Pak]]). However, unlike many GEFs, PIXα binds to activated forms of [[Cdc42]] and [[Rac]]. Activated [[Cdc42]] markedly enhances its ability to associate with GDP bound [[Rac1]], resulting in a significant activation of ~RacGEF activity. RacGTP binding reduces the ~RacGEF activity [[Cerione 2005|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15649357&query_hl=11&itool=pubmed_docsum]]. [[Gβγ]]-mediated activation of [[Pak1]] requires PIXα (in transfected Cos7 cells), and PIXα null mice have defects in [[Cdc42]] activation, but not [[Rac]] activation [[Wu 2003|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=12887923&query_hl=35&itool=pubmed_docsum]].
Includes a family of actin binding and bundling proteins [[ABPs]], which correlate most closely with retrograde actin flow [[Waterman-Storer 2007|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17204653&ordinalpos=7&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
beta-Pix is necessary and sufficient for [[Rac1]] recruitment to membrane ruffles and to focal adhesions. The ~Rac1-beta-Pix interaction is required for [[Rac1]] activation by beta-Pix as well as for [[Rac1]]-mediated spreading. [[Pak1]] regulates the ~Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced [[Rac1]] activation [[Hordijk 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16492808&query_hl=41&itool=pubmed_docsum]].\n\nbeta-Pix forms a homotrimer and together with [[GIT1]] becomes a heteropentameric complex [[Rittinger 2009| http://www.ncbi.nlm.nih.gov/pubmed/19136011?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=11]]. Stat3 interacts with beta-Pix to regulate Rac1 activity in MEFs [[Cao 2009|http://www.ncbi.nlm.nih.gov/pubmed/19861492?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1]]. \n\nbpix may directly bind Abi-1 [[Rudel 2006|http://www.ncbi.nlm.nih.gov/pubmed/16940963]]
cyclic AMP or adenosine 3',5'-monophosphate
cyclic AMP receptor. See [[Devreotes 1991|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=1989903&query_hl=4&itool=pubmed_DocSum]].
cyclic guanine monophosphate
See [[Coronin]].
Formylated methionine-leucine-phenylalanine. Formyl peptides are di-, tri-, tetrapeptides of bacterial origin. They are released from bacteria in vivo or after decomposition of the cell and trigger activation of formyl peptide receptors in neutrophils.
See [[mDia1]] or [[mDia2]].
mDia1 is of particular potential relevance to neutrophil chemotaxis because it is activated by RhoA and has been shown to play key roles in migration of several cell types, including T lymphocytes. mDia1-deficient neutrophils have impaired capacity to polymerize actin, polarize, and migrate with directionality in response to chemoattractants, potentially through RhoA-ROCK signaling [[Alberts 2009|http://www.ncbi.nlm.nih.gov/pubmed/19265163?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
mDia2 binds the [[WAVE complex]] [[Weiner 2006|http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16417406&ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]] and is recruited to the leading edge in an [[Abi1]]-dependent manner [[Svitkina 2007|http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371%2Fjournal.pbio.0050317]]. mDia2-dependent filopodia correlates with its disengagement from WAVE [[Innocenti 2008|http://www.ncbi.nlm.nih.gov/pubmed/18516090?ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. mDia2 contains a functional nuclear localization signal in the N terminus and at least one functional nuclear export signal in the C terminus, which is sufficient for nuclear import in vitro [[Miki 2009|http://www.ncbi.nlm.nih.gov/pubmed/19117945?ordinalpos=3&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
n-Chimaerin is a ~GTPase-activating protein (GAP) mainly for [[Rac1]] and less so for [[Cdc42]] in vitro. The GAP activity of n-chimaerin is regulated by phospholipids and phorbol esters. Microinjection of [[Rac1]] and [[Cdc42]] into mammalian cells induces formation of the actin-based structures lamellipodia and filopodia, respectively, with the former being prevented by coinjection of the chimaerin GAP domain. Strikingly, microinjection of the full-length n-chimaerin into fibroblasts and neuroblastoma cells induces the simultaneous formation of lamellipodia and filopodia [[Lim 1996|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=8756665&query_hl=2&itool=pubmed_DocSum]].
The p110 catalytic subunits of class Ia phosphoinositide 3-kinases (PI3Ks) form stable heterodimeric complexes with [[p85]] family regulatory subunits. In mammals at least three distinct genes encode class Ia catalytic subunits: p110alpha, p110beta, and p110delta. These proteins all have N-terminal regions that mediate binding to the regulatory subunit, followed by a [[Ras]] binding domain, a C2 domain, an alpha-helical phosphoinositide kinase domain, and a catalytic domain. These enzymes can phosphorylate the D-3 position of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI4P), and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in vitro [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
A [[Rnd]] or [[Rac]] mediated RhoGAP. See [[p190A]] or [[p190B]].
Intriguingly, [[p190A]] RhoGAP is a GSK3-beta substrate required for polarized cell migration [[Settleman 2008|http://www.ncbi.nlm.nih.gov/pubmed/18502760?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. Src promotes Rho inactivation and cytoskeletal reorganization via activation of p190A [[Frame 1999|http://www.ncbi.nlm.nih.gov/pubmed/10036244?dopt=Abstract]].\n\nSee [[Settleman 2005 review|http://www.ncbi.nlm.nih.gov/pubmed/16019436?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] for review of p190A and [[p190B]].
Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP). p190B colocalizes with constitutively active Rac1 in membrane ruffles and activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in ~GTP-bound RhoA [[Hansen 2008|http://www.ncbi.nlm.nih.gov/pubmed/18948007?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. p190B can GAP both Rho and Rac [[Hall 1995|http://www.ncbi.nlm.nih.gov/pubmed/8537347?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]] and this preference is modulated by PS and PIP2 [[Settleman 2004|http://www.ncbi.nlm.nih.gov/pubmed/14699145?ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]]. p190B can be directly phosphorylated by the insulin and ~IGF-1 receptors, and this phosphorylation is required to cause the insulin/~IGF-1-induced translocation of p190B from a largely cytoplasmic distribution to lipid rafts within the plasma membrane [[Settleman 2003|http://www.ncbi.nlm.nih.gov/pubmed/12705864?dopt=Abstract]]. Knockdown of p190B reduces expression of matrix metalloproteases and leaders to the abrogation of tube formation of HUVEC cells in matrigel [[Moreau 2008|http://www.ncbi.nlm.nih.gov/pubmed/18505793?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].\n\nIntriguingly, [[p190A]] RhoGAP is a GSK3-beta substrate required for polarized cell migration [[Settleman 2008|http://www.ncbi.nlm.nih.gov/pubmed/18502760?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]].
The p85 family of regulatory subunits of phosphoinositide 3-kinase (PI3K) form a tight heterodimeric complex with the [[p110]] catalytic subunit. The regulatory subunit provides thermal stability to the [[p110]] subunit but also suppresses its activity. Binding of Src homology 2 (SH2) domains in the regulatory subunit to phosphoTyr-X-X-Met motifs in activated receptors or adaptors recruits PI3K to the membrane and relieves the inhibition. In mammals, at least three different genes for class Ia regulatory subunits exist, p85alpha, p85beta and p55gamma (also called p55PIK). In mammals, p85a is the most ubiquitous and abundant regulatory subunit. Deletion of all isoforms of p85a in the mouse results in perinatal lethality [[Cantley 2002 review|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12040186&dopt=Abstract]].
Cytosolic IRES-GFP
Podosomes, or invadiapodia, are actin rich structures involved in trans-endothelial migration. Podosome formation involves [[N-WASP]] and matrix-metalloproteases (to degrade the ECM) [[Jones 2006 review|http://www.ncbi.nlm.nih.gov/pubmed/16546557?ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]]. Trans-endothelial migration can occur through endothelial cells (transcellular diapedesis) or between endothelial cells (paracellular diapedesis). In lymphocytes, these structures are dependent on [[Src]] kinase and the actin regulatory protein [[WASP]] [[Carman 2006|http://www.ncbi.nlm.nih.gov/pubmed/17570692?ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum]].
geranyl = 10, farnesyl = 15. For a so-so review see [[Waldmann 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16983387&query_hl=15&itool=pubmed_DocSum]].\n\nRac can be prenylated enzymatically [[Pick 2006|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16702219&query_hl=25&itool=pubmed_docsum]] and [[Pick 2002|http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11896062&query_hl=27&itool=pubmed_docsum]].
See [[Superoxide Production]]
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