oadTyo iktsLbrtr,AgneNtoa aoaoy eot IL Lemont, Laboratory, National USA. Argonne 60439, Laboratory, Ricketts Taylor Howard hcg,Ciao L667 USA. 60637, IL Chicago, Chicago, fai pigmeiaeadfraino ceramide-enriched of formation and compartments. sphingomyelinase endocytic acid of ai eii enne,Mtha orte aioC iul,CrsiaTam Christina Miguel*, C. Danilo Corrotte, Matthias Fernandes, Cecilia Maria of repair wounds the membrane and invasion plasma trypanosome mechanical for required is exocyst The REPORT SHORT eevd2 aur 04 cetd2 coe 2014 October 22 Accepted 2014; January 24 Received " of Brazil. University SP, Biology, Campinas, of Institute 13083-862 Biology, Campinas-UNICAMP, Animal Maryland, of Department of address: USA. University *Present 20742-5815, Genetics, MD Molecular Park, and College Biology Cell of contact Department of sites with associated be extracellular to of observed were Sec8 and Exo70 invasion efficient containing vacuoles to internalized recruited recently is complex exocyst The DISCUSSION AND RESULTS Ca in involved proteins includes machinery This al., et (Fernandes 2011). invasion cell host for machinery repair membrane parasite protozoan The INTRODUCTION Resealing Infection, Trypanosome, WORDS: KEY Both repair. by and Ca injury require invasion membrane processes plasma cell with host elements mechanistic of process The ABSTRACT n ehncllso earsaeauiu eurmn o the for requirement unique delivery. membrane a targeted on share dependence of a with repair repair consistent exocyst, the lesion affect mechanical Thus, not toxins. and pore-forming does by caused but lesions wounds, smaller mechanical of resealing u,20;Msaek ta. 03 i n u,2012.). Guo, and Liu 2003; and al., He et 2004; Moskalenko al., 2009; et Guo, spatially (Boyd membrane which plasma 1995), 2012; the Novick, to Munson, vesicles and targets TerBush and 1996; Heider al., 2000; et theTerBush as al., known et (also of Exo84 (Guo and involvement EXOC1–EXOC8) Exo70 Sec15, the Sec10, Sec8, investigated Sec6, Sec3, exocytosisSec5, of composed we requires complex protein octameric 2001), that conserved a process exocyst, al., a et through periphery, (Reddy cell the at Because 2010). a al., et 2008; Tam al., and et Idone 2011; 1992) acid al., lysosomal et of (Fernandes release al., (ASM) sphingomyelinase the by triggered et endocytosis of (Tardieux form rapid exocytosis lysosomal dependent rSc elto inhibits depletion in accumulate Sec8 complex, or EXOC4, exocyst and the EXOC7 nascent Here, as of known traffic. (also components membrane Sec8 respectively), and of Exo70 regulation that spatial show we for need a suggesting uhrfrcrepnec ([email protected]) correspondence for Author 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,2–2doi:10.1242/jcs.150573 27–32 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. .cruzi T. § rsn drs:Dprmn fMcoilg,Uiest of University Microbiology, of Department address: Present .cruzi T. aulsada ie fmcaia onig Exo70 wounding. mechanical of sites at and vacuoles 2+ tigrdeoyoi flssms exocytosis lysosomes, of exocytosis -triggered ihtehs lsammrn,a identified as membrane, plasma host the with .cruzi T. rpnsm cruzi Trypanosoma .cruzi T. .cruzi T. .cruzi T. n srqie for required is and nesclsi oaie fashion polarized a in cells enters nainadCa and invasion nae tprpea sites, peripheral at invades rpnsm cruzi Trypanosoma ` tlzsteplasma the utilizes rsn address: Present .cruzi T. 2+ -dependent invasion shares 2+ - ngnrlzdlssmleoyoi rgee yitaellrCa intracellular by involved triggered exocytosis was lysosomal exocyst generalized facilitating might the in site, whether exocyst invasion determine the To the invasion. that at parasite suggested lysosomes results of exocytosis our promote Thus, 1992). al., et invasion in involved the process at lysosomes a site, host of fusion and recruitment promotes rti hog h og otepam ebae el were Cells membrane. plasma the to Golgi the through protein netdwith infected odtos x7-eiin el hwdcmaal rhigher or comparable of showed levels cells Exo70-deficient conditions, aaie iacsil oanti- to internalized Recently (inaccessible microscopy. confocal parasites by imaged and fixed nlxb emaiiainwt h oefrigtxnSLO with treatment toxin or 2B) Ca pore-forming (Fig. substrate the the the from scraping with 2A), (Fig. permeabilization by enzyme influx lysosomal the of exocytosis measured we transients, Ca cytosolic by driven events endocytic by and utilized process invasion unique The Ca Generalized Wosye l,20;Fradse l,21) Ca 2011). al., et cells Fernandes host 2003; with al., contact et initiate (Woolsey trypomastigotes when triggered hfigclst 37 to reticulum. cells endoplasmic Shifting the in retained and misfolded is ts045–YFP niie nclsdpee nteeoytcmoetExo70 component exocyst the in depleted cells in inhibited eoeifcin ttersrcietmeaue(39 temperature restrictive VSVG-ts045–YFP a the the with At temperature-sensitive transduced of infection. before were formation encoding post- cells the whether construct HeLa investigated to we vacuole. Hsu so 2007; contribute parasitophorous 2007), al., al., et vesicles He et 2000; Golgi Liu al., 2004; et al., Guo 1999; et al., et (Guo vesicles 1A,B). (Fig. regions parasite specific internalized antibodies around with portions parasite to exposed of staining the by iN Fg DE.Cletvl,teerslssgetthat specific with at parasitophorous associated addition suggest sites nascent membrane peripheral localized to results regulate complex might these exocyst vacuoles control the with Collectively, of treated recruitment cells 1D,E). of (Fig. intracellular that of siRNA with number compared the in when decrease parasites significant a caused Sec8 2003). al., et Woolsey al., 1992; et al., (Fernandes et is compartment Tardieux lysosomal 2011; that or endosomal membrane the from intracellular form that to recruited suggesting studies complements to finding earlier directed new not This 1C), vacuoles. is (Fig. parasitophorous traffic nascent time-point membrane Golgi-derived any that at indicating VSVG-ts045–YFP with associated eoaiiaei el eltdo x7 n xoe oCa to exposed and Exo70 of depleted cells in hexosaminidase h xcs ope euae h xctsso post-Golgi of exocytosis the regulates complex exocyst The N nefrne(Ni-eitddpeino x7 or Exo70 of depletion (RNAi)-mediated interference RNA .cruzi T. 2+ b erimn fEo0adSc a akdystronger markedly was Sec8 and Exo70 of Recruitment . hxsmnds erto nrsos oCa to response in secretion -hexosaminidase oohr ooyi ntson.Udralthree all Under shown). (not ionomycin ionophore .cruzi T. 2+ .cruzi T. dpneteoyoi flssmsi not is lysosomes of exocytosis -dependent ˚ loscretfligadtafcigo the of trafficking and folding correct allows C ticesn eid fe hfigt 37 to shifting after periods increasing at ` ,§ cnann aulsi agl derived largely is vacuoles -containing .cruzi T. n om .Andrews W. Norma and .cruzi T. .cruzi T. naino otcls(Tardieux cells host of invasion .cruzi T. entry. 2+ niois eenot were antibodies) rnins hc are which transients, eursexocytic requires 2+ ˚ 2+ )VSVG- C) nlx as influx, signaling .cruzi T. " ˚ 27 C, b 2+ 2+ -

Journal of Cell Science nainsts(adexe l,19) h nraei generalized localized in at increase highly The observed 1992). the Ca al., events et for (Tardieux fusion sites required invasion and is possibility recruitment complex the lysosome out exocyst rule the not do that results these However, exocytosis. HR REPORT SHORT 28 also a had we components 2012), exocyst Munson, of and depletion vesicles the (Heider targeting whether spatially membrane investigated in plasma role the a plays to exocyst the the that with Given injury after SLO resealing toxin wounds membrane mechanical pore-forming plasma of for repair the not for but required is exocyst The Ca in defect intrinsic an of of consequence inhibition the cruzi that suggests T. result This controls. with compared ciao fAp/,pooigatnflmn ulainand nucleation kinetic filament a 2012). actin al., as cytoskeleton, et promoting acts (Liu actin branching Arp2/3, Exo70 cortical that of the demonstration activator in the changes with to consistent related be might 2+ dpnetlssmleoyoi nEo0dpee cells Exo70-depleted in exocytosis lysosomal -dependent naini el eltdi x7 n e8i o a not is Sec8 and Exo70 in depleted cells in invasion 2+ tigrdlysosomal -triggered .cruzi T. xoue el eeicbtdudrcniin htwere that conditions under SLO incubated (Ca After and were wounds. permissive (mechanical) cells SLO) polarized of toxin exposure, repair (pore-forming the non-polarized on effect differential y rpdu oie el rae ihcnrlo Exo70- plasma Ca or of their presence resealing the control in of permeabilization with capable SLO after equally treated membrane-impermeant membrane were Cells the siRNA iodide. to specific propidium exposed and dye repair, membrane one ysrpn rmtesbtaewr o bet block to able not were substrate the from scraping by plasma wounded their with which repair pores SLO, of transmembrane to diameter stable toxin cells external forms an the and the cholesterol with 2E) of to (Fig. binds permeabilization ability Sec8 after or the 2D) membrane affect cells (Fig. not Exo70 Flow propidium-iodide-positive of did panels). depletion left of that 2C, confirmed (Fig. quantification influx cytometric iodide propidium allowing niae yteboki pae(i.2,rgtpnl) As panels). right 2C, (Fig. uptake Ca in without block expected, the by indicated ncnrs,Eo0dpee el htwr mechanically were that cells Exo70-depleted contrast, In ora fCl cec 21)18 73 doi:10.1242/jcs.150573 27–32 128, (2015) Science Cell of Journal 2+ rnnprisv n Ca (no non-permissive or ) gen n hfe rm39 from shifted VSVG-ts045–YFP and expressing (green) Confocal cells (C) HeLa anti- DAPI. of only and images show staining panel Sec8 each or of Exo70 right the on 10 images bars: Scale permeabilization. before civdfrec protein. each for achieved that indicating blots western or Exo70- Student’s and siRNA; control Sec8-specific with treated cells (between aaiergosacsil oanti- to accessible regions extracellular parasite indicate arrows yellow internalized trypomastigotes; (blue). fully DAPI indicate with arrows Host stained White (green). were (B) DNA Sec8 parasite or and (A) Exo70 against mAbs anti- with stained and with infected cells invasion. for cell required host to are recruited and are vacuoles Sec8 parasitophorous and Exo70 1. Fig. otema ftilcts( correspond triplicates data of The mean h. the 1 for to infected Sec8-specific and or siRNA (D) (E) Exo70-specific control, for with transfected h cells 72 HeLa in parasites intracellular 10 parasites. bars: internalized Scale indicate arrows anti- White with (blue). stained and for min trypomastigotes 10 to exposed periods, indicated 2+ , emaiie el i o reseal, not did cells permeabilized , 0n Tee,2005). (Tweten, nm 30 m .(,)Qatfcto of Quantification (D,E) m. .cruzi T. AB ofcliae fHeLa of images Confocal (A,B) .cruz T. rpmsioe o 0min 30 for trypomastigotes 6 ˚ Cto37 ..;*** s.d.); niois(e)and (red) antibodies i , .cruz T. 0 ncdw was knockdown 80% t ts) nesshow Insets -test). .cruzi T. 2+ ˚ rd n DAPI and (red) i o the for C P o plasma for ) # 0.0004 m .The m. antibodies .cruzi T. 2+ ,as

Journal of Cell Science noeosEo0 one el eeietfe by with pattern, intracellular identified punctate non-wounded typical In a wounding. were showed during Exo70 medium fluorescent cells, the cells membrane-impermeant to added a against dextran with Wounded antibodies staining intracellular Exo70. with immunofluorescence endogenous performed and ls ed ntepeec rasneo xrclua Ca extracellular of with absence contact or by presence cells the injured in we Exo70, beads of glass wounding distribution mechanical of the effect on the examine To repair. membrane HR REPORT SHORT rpdu oieifu ntepeec fCa of presence the in influx iodide propidium ae)rsle nasrn eeti Ca in defect of right strong lower silencing 3C, a (Fig. in that Sec8 or resulted showing panel) right panel) result, lower 3B, this (Fig. cytometric scraping Exo70 confirmed Flow after 3A). 3B,C) cells (Fig. (Fig. propidium-iodide-positive repair of membrane quantification plasma in defect 2+ dpnetplasma -dependent 2+ elciga reflecting , 2+ , iia soito ihtepam ebae n h presence Ca the of and absence membrane, or plasma the with association minimal ugot Mrh ta. 03,aplrzdpoesta involves that process neurite polarized a in 2003), exocyst al., et the remodeling. (Murthy implicated outgrowth membrane studies plasma previous for Interestingly, necessary events trafficking erto,cnas euaeCa constitutive regulate to the linked also that previously suggests This can been parasites. has the secretion, by which invasion exocyst, cell of and for wounding and mechanical of sites cruzi to plasma recruited the is to complex redistributed was Exo70 min, 4E–M). 3 glass (Fig. with or membrane wounded s were 30 cells for the beads when However, 4A–D). (Fig. olciey u eut hwta h xcs tethering exocyst the that show results our Collectively, nr,adta hsi eurdfrpam ebaerepair membrane plasma for required is this that and entry, ora fCl cec 21)18 73 doi:10.1242/jcs.150573 27–32 128, (2015) Science Cell of Journal 2+ rae ih30n/lSOfr5mno c n shifted and ice on min 37 5 for to SLO ng/ml were 300 siRNA with Exo70-specific treated or control with transfected bec fCa or of presence the absence after in permeabilization or SLO panels) panels) (upper (lower Exo70- before or siRNA, (black) (red) control specific with transfected cells imaged. HeLa and DAPI 20 with bars: stained Scale fixed, for were (PI) iodide cells propidium min, with 1 incubation After min. 3 for Ca Generalized 2. Fig. aes L emaiiaini h rsneor Ca presence of the absence (lower in after permeabilization or SLO panels) panels) (upper Sec8-specific before or siRNA, (black) (red) control with transfected cells *** of secretion the and Exo70-specific siRNA, or KD) Ctrl) (Exo70 (Neg control with transfected cells components. in exocyst impaired in not deficient are SLO with permeabilized xctssadCa and exocytosis orsodt h eno rpiae ( triplicates data of The mean dish. the the to from correspond of scraping concentration (B) indicated or the SLO with treatment supernatant (A) the after in measured was hexosaminidase a oipc nti taysaedistribution steady-state this on impact no had P # ˚ oidc oefrainwt rwtotCa without or with formation pore induce to C .02(Student’s 0.0002 2+ 2+ m E lwctmti nlsso HeLa of analysis cytometric Flow (E) . .()Fo yoercaayi of analysis cytometric Flow (D) m. . 2+ dpnetrsaigo cells of resealing -dependent 2+ t 2+ ts) C eacells HeLa (C) -test). dpnetlysosomal -dependent dpnetmembrane -dependent R el were cells NRK 6 s.d.); b - 2+ 29 T.

Journal of Cell Science ta. 1987). al., et HR REPORT SHORT 30 in grown 37 were at cells supplemented FBS (NRK) (Gibco) 10% (DMEM) kidney medium with rat Eagle’s normal modified Dulbecco’s and CCL-2.1 HeLa parasites and cells Host METHODS AND MATERIALS Ca lysosomal VAMP7) the as and known 2000) (also al., TI-VAMP et SNARE (Martinez-Arca lysosomal the eas hyivleeet htaersrce ospecific to restricted are that delivery events sites. membrane membrane involve the plasma exocyst-mediated they Thus, polarized because SLO. and require wounds toxin both mechanical cholesterol-binding of the resealing pores by distributed homogenously generated smaller localized findings and large of our wounds the repair mechanical vesicles, the of between secretory differences non- Regardless functional exocyst-regulated reveal that manner. the of exocyst-dependent however, these origin an to out, localized, contribute in membrane rule highly processes intracellular for cannot of sources need We lysosomal of a process outgrowth. the to to neurite similar exocytosis, attributed lysosomal regulated be spatially might of role wounds the in Thus, 2006). exocyst Andrews, the and (Arantes 7 synaptotagmin netdmnlyr fLLC-MK of monolayers infected cruzi Trypanosoma .cruzi T. tanwr bandfo h uentn of supernatant the from obtained were strain Y ˚ ne %CO 5% under C nainadi eeln fmechanical of resealing in and invasion 2 el sdsrbdpeiul (Andrews previously described as cells 2 rpmsioe rmthe from Trypomastigotes . .cruzi T. nainmight invasion 2+ sensor N nalsmlswssandwt 10 with stained was samples all in DNA o i.Frfo yoer sas 1 assays, cytometry flow For min. 1 for elmnlyr a 0 ofune eewse he ie t4 at times three washed were confluence) 60% (at monolayers Cell assays exocytosis and Resealing 63 a a antibodies on h, with 2009) acquired 1 secondary SPX5 were al., for Images Leica et anti-mouse-IgG (Invitrogen). PA) confocal 488 (Liu laser-scanning Pennsylvania, Fluor with Sec8 Alexa of to incubation or University conjugated 2006) Guo, by Wei al., followed Dr et by (Zuo antibodies monoclonal (provided Exo70 mouse with against 0.2% incubated and with (mAb) min permeabilized 5 were for cells X-100 100 components, Triton a exocyst of with staining microscope the E200 Nikon a for using processed Intracellular in and 1992). quantified min were al., 15 parasites et for (Tardieux PBS immunofluorescence in inside/outside (PFA) paraformaldehyde 4% with 1.8 immunofluorescence and assays Invasion nuae ihSOfr5mna 4 at min 5 for SLO with incubated ihhsiietge L R wee,Uiest fOlhm,O)in OK) Oklahoma, of University Ca Tweeten, (R. SLO histidine-tagged with xeiet n nuae ih1 with incubated and experiments fDE cnann %FS e elfrvrosproso ieat time of periods various for well per FBS) 37 2% (containing DMEM of fe i,clswr tie ih50 with stained were cells min, 3 After olwdb nuaina 37 at incubation by followed 37 with medium the replacing ihCa with ˚ 6 2+ ,wse ietmswt B ormv xrclua aaie,fixed parasites, extracellular remove to PBS with times five washed C, fe MMfr5mna 4 at min 5 for DMEM -free 10 5 2+ el eepae n3-mwlso ls oesis2 before h 24 coverslips glass on wells 35-mm in plated were cells fe MM(otiigMg (containing DMEM -free ora fCl cec 21)18 73 doi:10.1242/jcs.150573 27–32 128, (2015) Science Cell of Journal cace ihanel ntepeec rasneof absence or presence the in Ca needle were a siRNA with Exo70-specific scratched or control repair with to transfected fail Sec8 or Exo70 wounds. of mechanical depleted Cells 3. Fig. iN ntepeec rasneo Ca of absence or presence (red) the Sec8-specific in or siRNA cells (black) HeLa control scraped with of transfected analysis cytometric levels. Flow repair (C) reflects wounding; staining after iodide min propidium 3 reduced iodide cells propidium show with panels incubated Lower cell wounded. whole was the population high that iodide; indicate propidium levels of fluorescence presence the in scraped rsneo bec fCa the of in absence siRNA or (red) presence control Exo70-specific with or transfected black) cells (Ctrl, HeLa scraped of analysis and 20 DAPI bars: with Scale stained imaged. fixed, (PI), iodide propidium oiea i fe wounding. after min propidium 3 with at incubation iodide show propidium panels of presence Lower the iodide. in scraped cells show panels 2+ n,atr3mna 37 at min 3 after and, , 0 otclsprcvrlpi rpiae by triplicate, in coverslip per cells host 300 ˚ ˚ MMwt rwtot18m Ca mM 1.8 without or with DMEM C nDE iho ihu Ca without or with DMEM in C 6 ˚ .Pr omto a rgee by triggered was formation Pore C. 10 ˚ n250 in C 8 2+ .cruzi T. m A R elmonolayers cell NRK (A) n 0m GA n treated and EGTA) mM 10 and /lpoiimidd (Sigma) iodide propidium g/ml m m 6 AI(Sigma). DAPI M .()Fo cytometric Flow (B) m. 6 10 2+ ˚ ,wr nuae with incubated were C, A13olojcie For objective. oil 1.3 NA pe aesso cells show panels Upper . 6 6 m rpmsioe n2ml 2 in trypomastigotes lofCa rpiie el were cells trypsinized A14olobjective. oil 1.4 NA 2+ 2+ fe DMEM, -free Upper . 2+ and 2+ ˚ C .

Journal of Cell Science FCCno DBocecs eeaaye sn lwo6.3 Ca to without or FlowJo attached with DMEM monolayers in using assays, needle a scratch analyzed with For scratched were Inc.). were coverslips Star, (Tree Biosciences) software BD (FACSCanto, HR REPORT SHORT R el eeclue ngascvrlp ocnlec nuae in incubated confluency to coverslips glass on 500 cultured were cells NRK beads glass with Wounding from removed were cells 37 assays, at scrape For dish min. the 4 for iodide propidium xhne o 500 for exchanged ecie rvosy(Rodrı previously described imaged. and DAPI, oiewsadddrn caigo fe i t37 at min 4 after or scraping during added was iodide ons ahdi c-odPSwtotCa without PBS ice-cold in washed points, nuae o i t37 at min 3 for incubated oia uehl maoetewl.Clswr okdtretmsand Ca times without three rocked or were with Cells 1-ml DMEM well. a the in from above incubated coverslip) cm per 1 500- g held fixable; tube (0.05 using conical sprinkled lysine wounded (Sigma) beads mechanically Da, glass 10,000 were washed cells of and mass Invitrogen) (molecular Texas-Red–dextran ml rasneo Ca of absence or m fDE iho ihu Ca without or with DMEM of l ˚ iharbe oiea B isine) Propidium Biosciences). (BD policeman rubber a with C b m 2+ hxsmnds erto saswr efre as performed were assays secretion -hexosaminidase fDE iho ihu Ca without or with DMEM of l lwctmtydt from data cytometry Flow . ˚ ,sandfr1mnwt rpdu oieand iodide propidium with min 1 for stained C, ´ uze l,1997). al., et guez 2+ t37 at 2+ rdxrnfrvrostime- various for dextran or 2+ ˚ eoetemdu was medium the before C n ie n4 PFA. 4% in fixed and 2+ ˚ ntepresence the in C otiig5mg/ 5 containing , 000cells 10,000 m acid- m 2+ , olwn muotiigfrEo0 el eeiae sn a using 1.42 imaged NA an were with cells equipped 60 microscope Exo70, deconvolution Elite for Deltavision immunostaining Following CACUCAA-3 ecie rvosy(omee l,20)wr lcda 39 at placed were 2000) al., et as (Toomre VSVG-ts045–YFP encoding previously adenovirus with described transduced cells HeLa assay trafficking VSVG–YFP (1.4 cells HeLa blotting western and RNAi ltigfrEo0o e8wspromdo ellststa were that lysates antibodies. cell anti-Sec8 on mouse with or Western performed incubated and anti-Exo70 post-transfection. was nitrocellulose mouse to h Sec8 transferred 72 SDS-PAGE, or by at separated Exo70 assays for repair blotting membrane plasma or 5 Invitrogen), UGACUGACUA-3 content; were UCUACCUGUGUU-3 GC used to duplexes according GUAUCAAGG-3 (medium The duplexes siRNA (Invitrogen). control stealth instructions of manufacturer’s pmol the 160 and Lipofectamine 6 objective. ora fCl cec 21)18 73 doi:10.1242/jcs.150573 27–32 128, (2015) Science Cell of Journal 9 treigrtSc) el eeue for used were Cells Sec8). rat (targeting 9 6 9 treighmnSc) 5 Sec8), human (targeting 10 treigrtEo0 r5 or Exo70) rat (targeting 5 9 h lsammrn fclswuddi the in Ca wounded of cells presence of membrane plasma the AB o-one el nuae nCa in incubated cells Non-wounded imaged. (A,B) and and (blue) (green), DAPI antibodies with anti-Exo70 stained fluorescent fixed, of (red), presence dextran the or in beads unwounded glass left with wounded either were wounding. cells mechanical NRK plasma of the sites to at recruited membrane is Exo70 4. Fig. one o 0si Ca in s Cells 30 (E,F) for D. wounded in outlined region the of magnification ftergo ulndi .Saebr:10 bars: H Scale in M. arrows in outlined region the of (H eim (K medium. staining). one o 0si Ca in s 30 Cells (G,H) for F. wounded in outlined region the of magnification eim (B medium. .(,)Clswuddfr3mni Ca in min 3 for wounded Cells (J,K) H. ulndi .(,)Clswuddfr3mnin min 3 for wounded Cells Ca (L,M) K. in outlined ulndi .(,)Nnwuddclsincubated cells Ca Non-wounded in (C,D) B. in outlined n3-mwlswr rnfce with transfected were wells 35-mm in ) treighmnEo0,5 Exo70), human (targeting 9 2+ ihrmgiiaino h einotie in outlined region the of magnification Higher ) cnann eim (M medium. -containing 2+ cnann eim (D medium. -containing 9 9 9 ihrmgiiaino h region the of magnification Higher ) ihrmgiiaino h region the of magnification Higher ) n M and 2+ ietfe yrddextran red by (identified 9 on oEo0rlclzto to relocalization Exo70 to point 2+ 2+ 9 fe eim (F medium. -free cnann medium. -containing -UCGCAGAGAAGAA- 9 -AUUCCUUGAUGC- 9 ihrmagnification Higher ) 9 9 -AUAGUGAGAG- Higher ) 9 -GGCUAAAGG- .cruzi T. 2+ -free 2+ 9 m invasion Higher ) .The m. -free 31 ˚ C

Journal of Cell Science e .adGo W. P. Guo, Novick, and and B. S. He, Ferro-Novick, J., Barrowman, M., Sacher, W., Guo, P. Novick, and M. Pypaert, T., Hughes, C., W.Boyd, N. Andrews, and M. R. Arantes, Uiest fMrln)frassac ihcnoa irsoyadflow and microscopy Class confocal K. with Guo respectively. and assistance W. cytometry, Beaven for Dr A. Maryland) construct, and of VSVG antibodies (University the for for Pennsylvania) University) of (Yale Toomre (University D. Dr thank We Acknowledgements 37 to shifted overnight, REPORT SHORT 32 W. Guo, and M. Abraham, D., TerBush, C., S. Hsu, M. Munson, W. and Guo, R. M. and Heider, X. Zhang, D., TerBush, J., Zhang, F., Xi, B., He, P. Novick, and A. Grant, W., Guo, V. Nussenzweig, and S. E. Robbins, S., K. Hong, W., N. Andrews, 12 References after release for PMC R37 in numbers Deposited [grant N.W.A. Health to months. of GM064625] Institutes R01 National and the AI34867 by supported was work The paper. Funding the wrote N.W.A. the and performed M.C.F. M.C. experiments; the experiments; wounding performed repair bead C.T. PM glass and and D.C.M. exocytosis M.C.F, invasion, project; parasite the designed N.W.A. and M.C.F. contributions Author interests. competing no declare authors The interests Competing rpmsioe o 0mn ie n rcse o h uniiainof quantification the for processed parasites. and fixed intracellular min, 10 for trypomastigotes enne,M . otz . lney .R,Tm . otr,R .adAndrews, and A. R. Mortara, C., Tam, R., A. Flannery, M., Cortez, C., M. Fernandes, rti opee ntasotvscetargeting. vesicle transport in complexes Protein primary from outgrowth neurite in lysosomes neurons. of sympathetic exocytosis regulated nplrzdexocytosis. polarized in cell the of stages 898-907. early at growth. vesicles cell exocytic polarized specific for of cycle secretion the mediates Biol. Cell Opin. secretion. for Sec3p subunits, two remaining the by Exo70p. marked and sites exocytic to subunits exocyst of morphogenesis the during cruzi. Trypanosoma expressed of forms antigens vertebrate surface Stage-specific lsammrn earptwyfrcl invasion. cell for pathway repair membrane plasma W. N. 21) rpnsm rz uvrstesphingomyelinase-mediated the subverts cruzi Trypanosoma (2011). .Cl Biol. Cell J. .Bo.Chem. Biol. J. 21 537-542. , 20) h xcs ope nplrzdexocytosis. polarized in complex exocyst The (2009). .Neurosci. J. n.Rv Cytol. Rev. Int. 167 ˚ o 5 0 5ad6 i,epsdto exposed min, 60 and 45 30, 15, for C 274 21) xriigteeoytcomplex. exocyst the Exorcising (2012). 889-901. , .Cl Biol. Cell J. 23558-23564. , 19) x8pi neoytpoenessential protein exocyst an is Exo84p (1999). 26 4630-4637. , 20) oefrsnpoamnVII- synaptotagmin for role A (2006). 233 x.Parasitol. Exp. 176 243-265. , 771-777. , rnsCl Biol. Cell Trends .Ep Med. Exp. 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