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(relative system what nucleus zebrafish cell to the the contrast exploited many in we that, Here in revealing migration organisms. bioprobes, polarization living neutrophil in regulating particularly in understood, positively microtubules well not by is motility migration leukocyte cell control Microtubules Summary work this to equally ` contributed authors *These 1 Yoo Kan Sa and zebrafish polarity live neutrophil in in migration microtubules of role The 5702 o:10.1242/jcs.108324 doi: 5702–5710 125, Science Cell of Journal 2012 August 19 Accepted naHuttenlocher Anna uhrfrcrepnec ( correspondence for Author rga nClua n oeua Biology, Molecular and Cellular in Program 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. nadto otecnetdpnetrl fmcouue in microtubules of role context-dependent the to addition In 4 eateto Zoology, of Department hmtxs irtbl,Nurpi,Zebrafish Neutrophil, Microtubule, , 1, ,Piyn Lam Pui-ying *, [email protected] 5,6, ` 5 eateto Pediatrics, of Department 1, ,Mr .Eichelberg R. 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K. with thank We ANOVA One-way Acknowledgements 5B. Fig. post-test: 7B,C. Tukey Fig. post-test: with Bonferroni were ANOVA values unpaired P One-way Two-tailed values, analyses. paired of following tailed population the overall by of derived distribution Gaussian Assuming Statistics eg . ave .A n utnohr A. Huttenlocher, and A. E. Harvie, Q., A. Deng, Huttenlocher, and M. J. Green, J., P. Cavnar, K., S. Yoo, Q., Deng, R. B. Brinkley, and W. C. Smith, J., B. Hughes, J., L. Wible, C., D. Anderson, References W.M.B., to at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108324/-/DC1 online available GM052932 months. material Association 12 Supplementary after National numbers release Heart for the PMC [grant American in and Deposited A.H.]. Health a S.Y.]; to GM074827 of to by 11PRE4890041 supported Institutes [number was fellowship work critical This EB3-GFP and of discussion Funding gift insightful generous for manuscript. for Starnes the Galjart W. of reading T. N. tol2-lyz-Dendra2-RhoT19N and and of plasmid Dent construction E. for plasmid, Deng Q. plasmid, rne,M,Zne .T n ooh .M. G. Bokoch, and T. F. Zenke, M., Krendel, wn .M n ishe,M W. M. Kirschner, and M. K. Kwan, La memn,T,Bdr .L,Mnly .J,Wrs . Wedlich-So T., Worbs, J., S. Monkley, L., B. Bader, T., ¨mmermann, yli-ieg pcfcgn yoyeCdrn erfs myelopoiesis. zebrafish during C lysozyme gene Dev. specific myeloid-lineage a microtubules. Bioessays 13105. nlmaoydisease. inflammatory spines. dendritic of invasion microtubule dynamic dependent dynamics. function. microtubule of modulator physiological a Sci. not Cell is J. and vivo in C. microtubules J. Bulinski, yolsi rget nteasneo microtubules. of absence the in fragments cytoplasmic control? etohlplrzto n migration. and polarization neutrophil for assay novel a using neutrophils GTPases. active human chemoattractant-stimulated in activation eit etohlatato obceilifcinadtsu injury. tissue and infection bacterial 14 to attraction neutrophil mediate internists. for utrophin. of domain actin-binding the on based Cytoskeleton filaments actin for probes of effects leukocytes: polymorphonuclear colchicine. in and stimulation microtubules chemotactic Cytoplasmic (1982). oe o a2i etohlmtlt n ciertnini erfs hematopoietic zebrafish in retention active and motility neutrophil tissue. in Rac2 for roles E-1mdae rs-akbtenmcouue n h ci cytoskeleton. actin the and microtubules Biol. Cell between cross-talk mediates GEF-H1 ai ekct irto yitgi-needn lwn n squeezing. and flowing -independent by 453 migration Fo leukocyte M., Rapid Keller, K., Hirsch, laevis. Xenopus of extension 4599-4610. convergent during morphology cell 517-528. , 51-55. , 126 e.Cell Dev. 19) F nDrosophila. in GFP (1995). Traffic 314-323. , a.Immunol. Nat. 4 15 294-301. , t 112 19) am-uui:tehbo ellrmcouueassemblies. microtubule cellular of hub the Gamma-tubulin: (1993). ts:Fg E n-a NV ihDnetps-et i.3A. Fig. post-test: Dunnett with ANOVA One-way 7E. Fig. -test: 637-643. , 64 u.J nen Med. Intern. J. Eur. ei.Cl e.Biol. Dev. Cell Semin. 4243-4255. , 822-832. , 5 .Bo.Chem. Biol. J. 21 470-477. , 19) -A-1 esosn soitsdnmclywith dynamically associates (ensconsin) E-MAP-115 (1999). 735-745. , 20) ci n irtblsi elmtlt:wihoei in is one which motility: cell in microtubules and Actin (2004). .Rheumatol. J. 9 960-969. , rtr . rthe,D . Fa R., D. Critchley, R., ¨rster, 20) nesiilluoyemgainadimmune and migration leukocyte Interstitial (2008). 21) euaino elmgainb dynamic by migration cell of Regulation (2011). 274 18) esset ietoa oiiyo el and cells of motility directional Persistent, (1984). 21 20) irtbl-idn h-E controls Rho-GEF microtubule-binding A (2005). 13198-13204. , rnsGenet. Trends 21) ociiei lnclmdcn.Aguide A medicine. clinical in Colchicine (2010). 22 503-508. , 22 o.Bo.Cell Biol. Mol. 1171-1173. , 968-974. , Cell 19) hrceiaino a n and rac of Characterization (1999). 31 20) irtbl smer during asymmetry Microtubule (2002). 21) itntsgaigmechanisms signaling Distinct (2012). 719-729. , 20) rncitoa euainof regulation Transcriptional (2009). 19) entlostmultisystem onset Neonatal (1995). 20) uloieecag factor exchange Nucleotide (2002). 11 324-325. , 13 20) estl fluorescent Versatile (2007). Nature 4470-4483. , t ts:Fg BD Two- 3B–D. Fig. -test: slr .e al. et R. ¨ssler, .Neurosci. J. 310 Development 58-61. , 20) Activity- (2008). el Microbiol. Cell. 21) Dual (2011). lnr R., ¨ldner, elMotil. Cell 28 13094- , (2008). Nature Mech. 132 Nat. , a oc,F . hain .R,Haeusl R., M. Ahmadian, P., F. Horck, van K. Kawakami, and G. Morvan, A., Urasaki, rd,N . agnu .M,Tae,D,Lo,A .adZn .I. L. Zon, and T. A. Look, D., Traver, M., D. Langenau, S., N. Trede, aeoo . esa,S . oca-ttad . ag .adRde,A J. A. Ridley, and R. Garg, B., Wojciak-Stothard, J., S. Heasman, A., Takesono, tae,B,Mria . ilr,T,Eas . un,C . ae,O,Mle,M., Milner, O., Sabet, Y., C. Huang, I., Evans, T., Millard, S., Moreira, B., Stramer, tpnv,T,Semr . ognad .C,Lnbre,G,Drln,B,De B., Dortland, G., Lansbergen, C., C. Hoogenraad, J., Slemmer, T., Stepanova, e,Y,L,R,Zeg .adBsh H. Busch, and Y. Zheng, R., Li, Y., Ren, ml,J . egr . aeia .adBrhdk,A. Bershadsky, and I. Kaverina, B., Geiger, V., J. Small, ed .J,Kly . un . a,M n atn P. Martin, and M. Way, G., Dunn, G., Kelly, J., M. D. Redd, Lichlyter, G. and G. S. Gundersen, W. Sherrod, and S., S. Ratner, A. Alberts, A., T. Cook, F., A. Palazzo, K. Mizuno, and H. Aizawa, M., Nishita, igit .E n o,C Q. C. Doe, and E. S. Siegrist, U. Euteneuer, and B. K. Pryzwansky, M., Schliwa, Sa Sa aiiv .M,Glad .M,Dmia .V,Iaoa .Y,Km,S .and G. S. Komm, Y., O. Ivanova, V., L. Domnina, M., I. Gelfand, M., J. Vasiliev, igi V. Niggli, J. T. Mitchison, and T. A. Look, C., Grabher, P., Niethammer, K. Keren, and A. Mogilner, erdr .M,Aos-err,J . e oo .A,Frhar . Schwartz- H., Furthmayr, A., M. Pozo, del L., J. Alonso-Lebrero, M., J. Serrador, M. W. Bement, P., Forscher, A., C. Mandato, W., A. M., Schaefer, Bista, C., D., O. Neukirchen, Rodriguez, H., J. Yu, K., Kessenbrock, H., A. Crevenna, J., Riedl, aha,J . ern .J,Lu .X,Kni . ok .T n utnohr A. Huttenlocher, and T. A. Look, J., Kanki, X., T. Liu, J., B. Perrin, R., J. Mathias, e,X . ise,W .adShat,M A. M. Schwartz, and B. W. Kiosses, D., X. Ren, a emue,A,Wn,K,Kih,Z . oars . an .M,Soa,K. Shokat, M., K. Hahn, C., Govaerts, A., Z. Knight, K., Wong, A., Keymeulen, Van aeh .L,Ro,R .adGli,J I. J. Gallin, and K. R. Root, L., H. Malech, oua .A,Coe,K . ketjvc,I,Ksnr .L n Huttenlocher, and L. D. Kastner, I., Aksentijevich, M., K. Cooper, A., M. Lokuta, A. Shimizu, and M. Sugai, H., Gonda, T., Hara, T., Katakai, H., J. Lee, nhzMdi,F n erdr .M. J. Serrador, A. and F. M. ´nchez-Madrid, Pozo, del and F. ´nchez-Madrid, rnpsbeeeetietfe h iia i-euneadahgl repetitive highly a O. and transposition. for cis-sequence essential region minimal subterminal the the in sequence identified element transposable immunity. understand to zebrafish of 21) irtblsrglt irtr oaiytruhRoRC inln nT in signaling Rho/ROCK through polarity cells. migratory regulate Microtubules (2010). euae essetmtlt n otc euso nDoohl arpae nvivo. in macrophages Biol. Drosophila in Cell repulsion J. contact W. and motility Wood, persistent and regulates P. Martin, G., Dunn, 20) iulzto fmcouuegot nclue ern i h s fEB3- of N. use Galjart, the protein). and via fluorescent A. neurons 3-green cultured Akhmanova, protein in G., (end-binding growth GFP Cappellen, microtubule van of F., Visualization (2003). Grosveld, I., C. Zeeuw, irtblsgiemgaigcells? migrating guide microtubules arpaeceoai nvv:suiso irtbl ucini erfs wound zebrafish in function microtubule of studies inflammation. vivo: in chemotaxis macrophage microtubules. stable of orientation and Biol. formation Rho-regulated mediates 1 irtbl-soitdgaiencetd xhnefco o a n Rho and Rac for factor exchange nucleotide guanine microtubule-associated a H1, Dev. fibroblasts. V. L. Olshevskaja, rpdadisrl nTcl oaiainadmotility. and polarization cell T in role its and uropod chemotaxis. T-cell for phosphorylation cofilin induces signalling Biol. and Cell. of 1 Mol. kinase sets LIM differential activates for implications neutrophils: human pathways. of migration R771. oteuoo fTlmhctsdrn elpolarization. redistributed cell is during and lymphocytes molecule-3 T of adhesion uropod intercellular the of to region Sa cytoplasmic and the F. Lozano, with J., Calvo, R., Albiez, motility. and activation cell to related 705-717. phenomenon unusual an neutrophils: uropod. the of roles interactions. immune and migration morphogenesis. and movement M. cell C. al. Waterman-Storer, et and Z. Werb, A., F-actin. T. visualize Holak, to D., marker Jenne, F., Bradke, rdeto yrgnprxd eitsrpdwuddtcini zebrafish. in detection wound rapid mediates 459 peroxide hydrogen of gradient in neutrophils of chemotaxis retrograde by inflammation zebrafish. of transgenic Resolution (2006). idn rti h ycl deinadtecytoskeleton. the and adhesion cell by Rho protein binding hAatvt ttebc swl ssgasa h front. the at signals as well as back the at activity RhoA R. H. Bourne, and M. microtubules. with interacts that factor exchange etohlmgain etil,mcouue n irflmn retto and orientation and microtubule, chemotaxis. during Centriole, function migration. neutrophil GTPases. nlmaoydsaeadMcl-el syndrome. Muckle-Wells 95 and disease inflammatory A. fpEMadRoRC inln nlmhct oaiyaduoo formation. uropod and polarity lymphocyte in Biol. signaling Cell J. Rho-ROCK and p-ERM of 394-399. , 996-999. , 20) etohlceoai naptetwt entlostmultisystem neonatal-onset with patient a in chemotaxis Neutrophil (2005). 20) hrceiaino Characterization (2001). 21 3 LSONE PLoS 723-729. , 483-496. , irtblsi erfs etohl 5709 neutrophils zebrafish in Microtubules 20) irtbl-irpinidcdadchemotactic-peptide-induced and Microtubule-disruption-induced (2003). .Bo.Chem. Biol. J. .Cl Sci. Cell J. .Ebyl x.Morphol. Exp. Embryol. J. 189 167 elMtl Cytoskeleton Motil. Cell 22 681-689. , 327-337. , 5 774-783. , e8774. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. .Luo.Biol. Leukoc. J. 17) feto ocmdo h ooooybhvorof behaviour locomotory the on colcemid of Effect (1970). 20) osaiienurpi oaiy I3adCc2augment Cdc42 and PIP3 polarity, neutrophil stabilize To (2006). 116 273 813-822. , 20) h hp fmtl cells. motile of shape The (2009). 20) irtbl-nue otclcl polarity. cell cortical Microtubule-induced (2007). a.Methods Nat. .Cl Biol. Cell J. 34954-34960. , 10hGF hAseii unn nucleotide guanine RhoA-specific a p190RhoGEF, f 20) osre irtbl-ci neatosin interactions microtubule-actin Conserved (2003). a.Cl Biol. Cell Nat. MOJ. EMBO a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 80 Immunity 21) ls-eitdmcouuebundling microtubule Clasp-mediated (2010). 24 r .C,Moear .H n Kranenburg, and H. W. Moolenaar, C., L. er, 19) lnn n hrceiaino GEF- of characterization and Cloning (1998). 63 nhzMdi,F. ´nchez-Madrid, 1281-1288. , 20) toa eldrvdfco 1alpha factor cell-derived Stromal (2002). 75 20) rnigu h er eiigthe defining rear: the up Bringing (2009). 625-640. , 415-422. , 5 605-607. , 20) ucinldseto fteTol2 the of dissection Functional (2006). 666-693. , 19) ekct oaiaini cell in polarization Leukocyte (1999). 19) irtbl ercinit the into retraction Microtubule (1997). 10 .Bo.Chem. Biol. J. 18 17) tutrlaayi fhuman of analysis Structural (1977). 19) euaino h ml GTP- small the of Regulation (1999). 20 353-359. , 501-511. , 367-379. , 5 18) etooesltigin splitting Centrosome (1982). 599-609. , n.AlryAtm Immunol. Asthma Allergy Ann. .Immunol. J. .Cl Biol. Cell J. .Neurosci. J. .Cl Biol. Cell J. 20) iec:aversatile a Lifeact: (2008). MOJ. EMBO 19) osninteracts Moesin (1997). 276 Genetics 3 ur Biol. Curr. 20) tissue-scale A (2009). 957-964. , 4948-4956. , 20) o do How (2002). 20) Imaging (2006). 174 159 138 23 20) h use The (2004). 18 20) mDia (2001). 174 2655-2664. , 20) Roles (2004). 437-445. , 1063-1067. , 1409-1423. , 578-585. , 639-649. , 19 a.Cell Nat. Cell R762- , Nature Genes 31 , Journal of Cell Science itan . ooh .M n aemnSoe,C M. C. Waterman-Storer, and M. G. Bokoch, T., Wittmann, M. C. Waterman-Storer, and T. Wittmann, aemnSoe,C . otyae .A,Lu .P,Brig,K n Salmon, and K. Burridge, P., B. Liu, A., R. Worthylake, M., C. Waterman-Storer, K. Kaibuchi, and J. Noritake, T., Watanabe, o asw . ebuge .J,Mle,A . ie,J .adBmn,W M. W. Bement, and R. J. Sider, L., A. Miller, J., K. Verbrugghe, G., Dassow, von iordv,T,Mle,P .adKvrn,I. Kaverina, and M. P. Miller, T., Vinogradova, 5710 irtbl etblzn ciiyo p8sahi ontemo Rac1. of downstream Op18/stathmin of Chem. activity destabilizing microtubule way? the point microtubules and nfibroblasts. in D. E. migration. cell 20) cina itnedrn cytokinesis. during distance a at Action (2009). smer nmtl el:rl fGlidrvdarray. Golgi-derived of role cells: motile in asymmetry 19) irtbl rwhatvtsRc opooelmlioilprotrusion lamellipodial promote to Rac1 activates growth Microtubule (1999). 279 ora fCl cec 2 (23) 125 Science Cell of Journal 6196-6203. , a.Cl Biol. Cell Nat. rnsCl Biol. Cell Trends 1 45-50. , 15 .Cl Sci. Cell J. 76-83. , 20) elmtlt:cnRoGTPases Rho can motility: Cell (2001). 114 20) euaino irtblsin microtubules of Regulation (2005). .Cl Biol. Cell J. 3795-3803. , 20) irtbl network Microtubule (2009). elCycle Cell 187 20) euainof Regulation (2004). 831-845. , 8 2168-2174. , .Biol. J. o,S . tre,T . eg .adHtelce,A. Huttenlocher, and Q. Deng, W., T. Starnes, K., S. Yoo, o,S . eg . anr .J,W,Y . an .M n utnohr A. Huttenlocher, and M. K. Hahn, I., Y. Wu, J., P. Cavnar, Q., Deng, K., S. Yoo, u . ag . a emue,A,Rne,M n ore .R. H. Bourne, A. Huttenlocher, and and M. K. S. Rentel, Yoo, A., Keymeulen, Van F., Wang, J., Xu, Nash, M., F. Diapouli, D., Caille, B., Colom, M., Beyrau, B., M. Voisin, A., Woodfin, esrta eitsluoyewudatato nvivo. in attraction wound leukocyte mediates that sensor 21) ifrnilrglto fporso n oaiyb IKdrn neutrophil during PI3K by zebrafish. polarity live and in protrusion motility of regulation Differential (2010). rfikn uigifamto nlv zebrafish. live in inflammation during trafficking al. et E. r vivo. G. in Rainger, JAM-C neutrophils M., S. molecule Albelda, adhesion T., Chavakis, B., G. etohlmcouue upesplrt n nac ietoa migration. directional USA enhance Sci. and Acad. polarity Natl. suppress microtubules Neutrophil a.Immunol. Nat. 102 6884-6889. , e.Cell Dev. gltsplrzdtranse polarized egulates 21) ptoeprlpooaeigo neutrophil of photolabeling Spatiotemporal (2011). 12 18 761-769. , 226-236. , .Luo.Biol. Leukoc. J. Nature dteilmgainof migration ndothelial 21) y saredox a is Lyn (2011). 21) h junctional The (2011). 480 89 109-112. , 661-667. , (2005). Proc.