Distinct v-SNAREs regulate direct and indirect apical delivery ......apical transcytotic pathway is regulated by VAMP8, another tetanus-neurotoxin-insensitive v-SNARE, which can pair

3309Research Article

IntroductionThe plasma membrane of polarized epithelial cells is dividedinto two domains, apical and basolateral, differing in theirprotein and lipid compositions and specialized functions(Mostov, 2003; Rodriguez-Boulan and Powell, 1992).Intracellular sorting occurs at the level of the trans-Golginetwork (TGN) and endosomes where proteins are segregatedinto distinct vesicles upon recognition of specific apical orbasolateral sorting signals (Folsch, 2005; Matter and Mellman,1994; Mostov et al., 2000). The proteins are delivered to theapical or basolateral surface (Rodriguez-Boulan and Powell,1992; Keller et al., 2001; Kreitzer et al., 2003; Wandinger-Nesset al., 1990) by means of a direct or an indirect (transcytotic)route that passes first through the opposite membrane domain.The use of the direct or transcytotic pathways seems to be bothcell and protein specific (Rodriguez-Boulan et al., 2005).Although direct and indirect sorting signals are quite welldefined for transmembrane (TM) basolateral proteins, this isnot the case for apical proteins, and in particular for GPI-anchored proteins (GPI-APs). These proteins are anchored tomembranes by a posttranslational lipid modification, theglycosylphosphatidylinositol (GPI) anchor (Bangs et al., 1985;Ferguson et al., 1985; Ikezawa, 1963; McConnell et al., 1981).A relevant feature of GPI-APs is their association with lipidrafts (Brown, 1994; Brown and Waneck, 1992; Brown, 1992;Brown and Rose, 1992). GPI-APs are apically sorted in several

epithelial cell lines (Brown et al., 1989; Lisanti et al., 1989)and use their GPI anchor to associate with rafts (Brown andWaneck, 1992; Brown, 1992). It has therefore been proposedthat the GPI anchor acts as an apical sorting determinant bymediating raft association (Simons and Ikonen, 1997; Simonsand van Meer, 1988).

The direct sorting of GPI-APs to the apical surface ofMadin-Darby canine kidney (MDCK) cells (Hua et al., 2006;Paladino et al., 2006) requires their oligomerization intodetergent-resistant membrane domains (DRMs) at the Golgilevel (Paladino et al., 2004). This reinforces the hypothesis thatapical sorting of GPI-APs occurs intracellularly before arrivalat the plasma membrane (Hua et al., 2006; Paladino et al.,2006). However, not much is known about the molecularmachinery involved in the direct targeting of GPI-APs to theapical surface. Various components of the exocytic machinerythat transport apical and basolateral proteins have beenidentified, but the mechanisms that regulate their localizationand function have been challenged recently (reviewed inRodriguez-Boulan et al., 2005). Polarized protein trafficking tothe apical and basolateral plasma membranes requires differentsets of SNARES, a family of proteins specifically involved inthe fusion of vesicles with their target membranes (Lafont etal., 1999; Low et al., 1996; Low et al., 1998; Steegmaier et al.,2000; Weimbs et al., 2003). SNAREs on vesicular cargo arecalled v-SNAREs, and those on target membranes t-SNAREs

SNARE [soluble N-ethylmaleimide-sensitive factor (NSF)attachment protein (SNAP) receptor] proteins control themembrane-fusion events of eukaryotic membrane-trafficking pathways. Specific vesicular and targetSNAREs operate in specific trafficking routes, but thedegree of specificity of SNARE functions is still elusive.Apical fusion requires the polarized distribution at theapical surface of the t-SNARE syntaxin 3, and several v-SNAREs including TI-VAMP and VAMP8 operate at theapical plasma membrane in polarized epithelial cells. It isnot known, however, whether specific v-SNAREs areinvolved in direct and indirect routes to the apical surface.Here, we used RNAi to assess the role of two tetanus-neurotoxin-insensitive v-SNAREs, TI-VAMP/VAMP7 andVAMP8, in the sorting of raft- and non-raft-associated

apical markers that follow either a direct or a transcytoticdelivery, respectively, in FRT or Caco2 cells. We show thatTI-VAMP mediates the direct apical delivery of both raft-and non-raft-associated proteins. By contrast, sorting bymeans of the transcytotic pathway is not affected by TI-VAMP knockdown but does appear to be regulated byVAMP8. Together with the specific role of VAMP3 inbasolateral transport, our results demonstrate a highdegree of specificity in v-SNARE function in polarized cells.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/18/3309/DC1

Key words: SNAREs, TI-VAMP, VAMP8, Epithelial cells, Polarizedsorting, Transcytosis

Summary

Distinct v-SNAREs regulate direct and indirect apicaldelivery in polarized epithelial cellsThomas Pocard1, André Le Bivic2, Thierry Galli3,4 and Chiara Zurzolo1,5,*1Unité de Trafic Membranaire et Pathogenèse, Institut Pasteur, 75724, Paris CEDEX 15, France2UMR 6212 CNRS/Université Aix Marseille II, IBDML, case 907, Faculté des Sciences de Luminy, 13288, Marseille CEDEX 09, France3Membrane Traffic in Neuronal and Epithelial Morphogenesis, INSERM Avenir Team, 75005, Paris, France4Institut Jacque Monod, CNRS UMR7592, Universities Paris 6&7, 75005, Paris, France5Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università degli Studi di Napoli Federico II, 80131 Napoli, Italy*Author for correspondence (e-mail: [email protected])

Accepted 6 July 2007Journal of Cell Science 120, 3309-3320 Published by The Company of Biologists 2007doi:10.1242/jcs.007948

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(Sollner et al., 1992). The formation of a SNARE complexbetween v-SNAREs and t-SNAREs (Schiavo et al., 1997;Sollner et al., 1993) mediates the specific recognition andsubsequent fusion (McNew et al., 2000) of vesicles with theirappropriate target membrane. SNAREs are assisted by severalpartners and regulators including the small GTPase Rabproteins (Grosshans et al., 2006; Novick and Zerial, 1997;Zerial and McBride, 2001).

Polarized epithelial cells represent an interesting model tostudy the specific function of SNAREs because plasmamembrane t-SNAREs are differentially localized in these cells.The t-SNARE syntaxin 3 (Stx3) is at the apical plasmamembrane, whereas syntaxin 4 (Stx4) is expressedpredominantly at the basolateral membrane domain of MDCKcells (Low et al., 1996). By contrast, SNAP23 (ubiquitouslyexpressed homologue of SNAP25) and syntaxin 2 (Stx2) arepresent in both membrane domains (Low et al., 1998). Stx3and SNAP23 constitute the apical t-SNARE complex, and theyinteract with the v-SNARE tetanus-neurotoxin-insensitivevesicle-associated membrane protein (TI-VAMP) in Caco2cells (Galli et al., 1998). The strong interaction between TI-VAMP, Stx3 and SNAP23 has been further demonstrated byyeast two-hybrid assays (Martinez-Arca et al., 2003). InMDCK cells, overexpression of Stx3 inhibits biosynthetictransport from the TGN to the apical membrane and theendocytic recycling pathway from apical endosomes of amutant form of the polymeric immunoglobulin receptor(pIgR), signal-less pIgR (SL-pIgR) (Casanova et al., 1991), butnot the basolateral delivery of the wild-type form of pIgR (WT-pIgR) (Low et al., 1998). Furthermore, overexpression of Stx3strongly inhibits the apical delivery of TM sucrase isomaltase(SI) and the secreted protein �-glucosidase without any effecton basolateral delivery in Caco2 cells (Breuza et al., 2000).Furthermore, inhibition of TI-VAMP with specific antibodiesaffects apical delivery of haemagglutinin (HA) but has noeffect on the basolateral route (Lafont et al., 1999). In addition,Stx3 and TI-VAMP associate with DRMs in post-TGN apicalcarriers (Lafont et al., 1999). These data have demonstratedthat Stx3 and TI-VAMP are important for apical transport oftransmembrane and secretory proteins both in MDCK andCaco2 cells. However, apical sorting shows further levels ofcomplexity: (1) there are at least two pathways for apicaldelivery, one raft dependent and one raft independent (Bentinget al., 1999; Lipardi et al., 2000); (2) there are at least tworoutes followed by apical proteins, direct and transcytotic(Gilbert et al., 1991; Rodriguez-Boulan et al., 2005; Zurzoloet al., 1992a); (3) it is not clear whether apical sorting ofproteins following different pathways occurs at the sameintracellular site and (4) utilizes the same machinery.

Although the results mentioned above on the apical SNAREsindicate a clear involvement of TI-VAMP in the direct sortingof transmembrane and secreted apical proteins, it is not clearwhether TI-VAMP is also involved in the direct sorting of GPI-APs and whether it participates in the transcytotic pathway, forwhich the specific v-SNARE has not yet been identified.Therefore, we first asked whether TI-VAMP is involved in theapical sorting of GPI-APs. Then, in order to discriminatebetween the direct and transcytotic pathways, we used twodifferent cell lines: Fisher rat thyroid cells (FRT) and humancolorectal cancer cells (Caco2) that, respectively, usepredominantly the direct and the transcytotic pathway to

deliver apical proteins to the plasma membrane (Le Bivic etal., 1990; Matter et al., 1990; Zurzolo et al., 1992a). By usingan RNA interference (RNAi) approach targeting TI-VAMP inboth cell lines, we found that TI-VAMP is necessary for thecorrect localization at the apical membrane of both GPI-APsand TM proteins that use a direct route to the apical membraneindependently of their sorting mode (i.e., raft dependent or raftindependent). Furthermore, TI-VAMP is not involved in apicalsorting of proteins that use a transcytotic pathway. Instead, theapical transcytotic pathway is regulated by VAMP8, anothertetanus-neurotoxin-insensitive v-SNARE, which can pair withboth apical and basolateral t-SNAREs (Imai et al., 2003;Pombo et al., 2003; Wang et al., 2007). Thus, we demonstratethat at least two different VAMPs are involved in the regulationof the two alternative routes to the apical plasma membrane.This mechanism appears to be independent of the cargo proteinper se but dependent on the pathway that the protein isfollowing in the different epithelia.

ResultsTI-VAMP is necessary for the correct sorting of bothGPI-AP and TM apical proteins in fully polarized FRTcellsWe set up a specific RNAi approach to study the role of TI-VAMP in the apical sorting of a model GPI-AP, placentalalkaline phosphatase (PLAP), in two different epithelial celllines, FRT and Caco2 cells, that respectively usepredominantly a direct or an indirect pathway to target proteinsto the apical membrane. Several isoforms of TI-VAMP havebeen described (Martinez-Arca et al., 2003), but only oneisoform of TI-VAMP is present in FRT cells [sequenceNM_021659 (Wang et al., 2005)].

To decrease endogenous levels of TI-VAMP in FRT cells,we used a specific small interfering RNA (siRNA) that hasbeen described previously (Alberts et al., 2003). This siRNAand an unrelated siRNA targeting human �-globin, used as anegative control, were transiently transfected into FRT cellsstably expressing PLAP (FRT-PLAP) (Lipardi et al., 2000). TI-VAMP expression was assayed by western blotting of total celllysates from cells grown on filters for 4 days in conditionsfavoring polarization (Fig. 1A). Compared with control cells,siRNA-treated FRT cells showed a strong decrease of thewestern blot signal corresponding to TI-VAMP [~88%;normalization was performed in comparison with calreticulin;(Fig. 1A)], thus indicating an efficient silencing of theexpression of TI-VAMP in polarized FRT cells.

In order to analyse whether TI-VAMP has a role in apicalsorting of TM proteins in FRT cells, we investigated the effectof TI-VAMP transient knockdown on the localization of TMapical and basolateral markers endogenously expressed bythese cells, respectively dipeptidyl peptidase IV (DPPIV) andantigen 35/40 kDa (Ag 35/40 kDa) (Zurzolo et al., 1991;Zurzolo et al., 1992a; Zurzolo et al., 1993; Zurzolo et al.,1992b). Control cells (FRT-PLAP transfected with siRNAtargeting �-globin) and FRT-PLAP cells transfected withsiRNA targeting TI-VAMP were grown on filters for 4 daysand subjected to immunofluorescence using specific antibodiesagainst the two TM proteins [A. Quaroni, personal gift(Zurzolo et al., 1992a)] (Fig. 1B). As expected, in control cells,DPPIV was localized at the apical membrane, whereas Ag35/40 kDa was localized at the basolateral membrane (Fig. 1B)

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(Zurzolo et al., 1992a). By contrast, in cells transfected withTI-VAMP siRNA, DPPIV was mis-sorted to the lateralmembrane, whereas the basolateral localization of Ag 35/40kDa was unaffected (Fig. 1B). These results obtained by genesilencing in FRT cells confirm the specific involvement of TI-VAMP in apical sorting of apical TM proteins that waspreviously demonstrated by antibody inhibition in MDCK cells(Lafont et al., 1999) and suggest that the role of TI-VAMP isconserved across different epithelial cells. Next, weinvestigated the effect of TI-VAMP transient knockdown on thelocalization of PLAP, our model apical GPI-AP (Lipardi et al.,2000) (Fig. 1C). Compared with the control condition wherePLAP accumulated at the apical membrane, in cells transfectedwith siRNA targeting TI-VAMP it was also mislocalized to thelateral membrane (Fig. 1C). These results indicate that TI-VAMP is necessary also for the correct apical localization ofGPI-APs.

The above result is particularly noteworthy because PLAPand DPPIV, although both following a direct route, are sortedto the apical membrane by means of two different mechanisms,respectively raft dependent and non-raft dependent (Benting et

al., 1999; Lipardi et al., 2000). In order to quantify and bettercharacterize the role of TI-VAMP, we produced stable TI-VAMP knockdown FRT clones (see Materials and Methods).Selected stable knockdown clones (FRT si TI-VAMP::PLAP),which by western blot had a markedly decreased signalcorresponding to TI-VAMP while expressing PLAP at a levelsimilar to that of control FRT PLAP cells (Fig. 2A), wereanalyzed for the localization of our apical and basolateralendogenous TM markers by indirect immunofluorescence andconfocal analysis, as shown in Fig. 2B. As expected, in FRT siTI-VAMP::PLAP cells, DPPIV lost its predominant apicallocalization and was mislocalized to the lateral membrane,whereas the basolateral localization of Ag 35/40 kDa wasunchanged (Fig. 2B). Next, we examined the localization ofPLAP both by indirect immunofluorescence and confocalanalysis (Fig. 3A) and by surface biotinylation (Fig. 3B).Similar to the transient knockdown, PLAP was mislocalized tothe lateral membrane of stable FRT knockdown cells (compareFig. 3A with Fig. 1C). By selective surface biotinylation, wecalculated that ~80% of surface-expressed PLAP was localizedat the apical membrane in control cells, whereas PLAP wasequally distributed to the apical and basolateral membrane(~50% apical and ~50% basolateral) in FRT si TI-VAMP::PLAP cells (Fig. 3B).

Thus, both the transient and stable knockdowns showed thatthe apical v-SNARE TI-VAMP is necessary for the correctapical localization of DPPIV and PLAP, two proteins directlysorted to the apical membrane of FRT cells through twodifferent mechanisms [respectively raft independent and raftdependent (Lipardi et al., 2000)].

Fig. 1. Apical but not basolateral proteins aremislocalized in fully polarized FRT cells aftertransient RNAi of TI-VAMP. FRT cells stablyexpressing PLAP were electroporated with 100pmol of siRNA targeting rat TI-VAMP or againsthuman �-globin and grown on filters for 4 days.(A) Cell extracts (40 �g) from control (siRNA betaglob) and TI-VAMP transient-knockdown (siRNATI-VAMP) cells were analyzed by SDS-PAGE andwestern blotting with antibodies againstcalreticulin (top panel, internal control for proteinloading) and human TI-VAMP (bottom panel). Thedecrease in TI-VAMP expression in transient-knockdown cells was calculated to be ~88% inthree different experiments. (B,C) Confocal Z andX-Y sections (top and bottom) of control (siRNAbeta glob) and TI-VAMP transient-knockdown(siRNA TI-VAMP) cells. (B) Cells were labeledwith antibodies against rat DPPIV (left panel) andAg 35/40 kDa (right panel), and secondaryantibodies were coupled to FITC. Note that theapical localization of DPPIV in controls switchesin TI-VAMP transient-knockdown cells to thelateral membrane (Z section and X-Y sectionbottom panel), whereas the basolateral localizationof Ag 35/40 kDa is unchanged. (C) Cells werelabeled with antibodies against PLAP andsecondary antibodies coupled to FITC. Note that,as for DPPIV, PLAP is mislocalized to the lateralmembrane. Bars, 10 �m.

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Loss of TI-VAMP does not impair the development of apolarized monolayerBecause of the effect of TI-VAMP knockdown on both raft-and non-raft-mediated apical sorting, we asked whether themistargeting of PLAP and DPPIV could just be a consequenceof the loss of the cells’ overall ability to acquire a polarizedphenotype. Because the kinetics of formation of tight junctionshas been frequently used as a measure of the ability ofepithelial cells to polarize, we assessed whether transient orstable knockdown of TI-VAMP affected the ‘normal’development of transepithelial resistance (TER), whichmeasures the ability of a monolayer to impede ion flow – thismeasure should increase with the establishment of a polarizedmonolayer (Fig. 4A). The TER of FRT PLAP cells transientlytransfected with siRNA targeting �-globin versus FRT PLAPcells transiently transfected with siRNA targeting TI-VAMPwas monitored during a 4-day period (Fig. 4A). Both curvesstart at a TER between 200 and 400 � cm–2 and reach after 4days 1400 � cm–2 (the TER of an empty filter is representedby the yellow curve and reaches 75 � cm–2). The sameobservation was made for FRT-PLAP cells versus the stable


Fig. 2. DPPIV but not Ag 35/40 kDa ismislocalized in fully polarized stable FRT cloneslacking TI-VAMP. FRT cells stably expressingPLAP (FRT PLAP), and FRT cells stablyexpressing PLAP and a vector where the siRNAtargeting rat TI-VAMP was introduced (FRT siTI-VAMP::PLAP), were grown on filters for 4days. (A) Cell extracts (40 �g) from FRT PLAPand FRT si TI-VAMP::PLAP were analyzed bySDS-PAGE and western blotting with antibodiesagainst PLAP (lanes two and four) and humanTI-VAMP (lanes one and three). Note that thetwo FRT cells express similar amounts of PLAP,whereas TI-VAMP is constitutively knocked-down in FRT si TI-VAMP::PLAP compared withFRT PLAP. (B) Confocal Z and X-Y sections (topand bottom) of FRT PLAP and FRT si TI-VAMP::PLAP cells were labeled with antibodiesagainst rat DPPIV (left panel) and Ag 35/40 kDa(right panel) and secondary antibodies coupled toFITC. Note that DPPIV is only mislocalized inFRT si TI-VAMP::PLAP cells, whereas thebasolateral localization of Ag 35/40 kDa isunchanged in both cell lines. Bars, 10 �m.

Fig. 3. PLAP is mislocalized in fully polarized FRT cells stablylacking TI-VAMP. (A) Confocal Z and X-Y sections (top and bottom)of FRT cells stably expressing PLAP, and FRT cells stablyexpressing si TI-VAMP::PLAP, grown on filters for 4 days andlabeled with antibody against PLAP and secondary antibodiescoupled to FITC. PLAP is mislocalized to the lateral membrane ofFRT si TI-VAMP::PLAP (right panel). (B) FRT PLAP and FRT siTI-VAMP::PLAP cells were grown on filters for 4 days and labeledwith LC-biotin (Biot) added to the apical (Ap) or basolateral (Bl)surface. After lysis, PLAP was immunoprecipitated with a specificantibody, run on SDS-PAGE and revealed with HRP-streptavidin(lane one and panel three). One tenth of the immunoprecipitate (Tot)was run on SDS-PAGE and assayed with an antibody against PLAP(panels two and four). Bars, 10 �m. IgG, immunoglobulin G.

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knockdown clone FRT si TI-VAMP::PLAP cells (Fig. 4A). Thehigher TER of FRT si TI-VAMP::PLAP cells compared withFRT-PLAP suggests a clonal selection effect because bothcurves exhibit the same slope, indicating that the two cell linesbehave similarly on filters during the establishment of thepolarized epithelial phenotype (Fig. 4A). In order to confirmthat the monolayer was well established, we also analysed byimmunofluorescence the distribution of ZO-1, a major proteinof the tight junctional complex (Fig. 4B). Both siRNA-treatedcells and control cells grown on filters in conditions favoringpolarization for 4 days exhibit the ‘chicken wire’-likeimmunostaining characteristic of the tight junction protein ZO-1 (Fig. 4B), confirming that the knockdown of TI-VAMP inFRT cells does not induce any significant alteration in the tightjunctional complex.

Overall, these results demonstrate that the mislocalizations

of apical non-raft and raft associated proteins in cellstransiently and stably knockdown for TI-VAMP are not due toa general perturbation of the development of the epithelium.They also indicate that TI-VAMP dependent apical pathwaysare not involved in the events necessary for tight junctionformation during the development of a polarized monolayer.

TI-VAMP is also involved in the direct apical sorting ofPLAP but not in the transcytotic delivery of DPPIV inCaco2 cellsIn contrast to FRT and MDCK cells, which use mainly a directpathway to sort both apical and basolateral proteins to theirrespective membrane domains after their intracellular sorting,other epithelial cell types can use different pathways(Rodriguez-Boulan et al., 2005). A study in intestine has shownthat certain apical plasma membrane proteins follow an

indirect pathway to the cell surface that passesfirst through the basolateral domain (Hauri etal., 1979). These results were confirmed inintestinal cell lines (e.g. Caco2 cells) culturedon filters under conditions favoringpolarization (Le Bivic et al., 1990; Matter etal., 1990).

Because TI-VAMP is also present at theapical membrane of Caco2 cells (Galli et al.,1998), this appears to be a very good modelto analyse its role both in the direct andindirect apical pathways. To this end, wefollowed the sorting of DPPIV and PLAP,which are both endogenously expressed byCaco2 cells and are sorted, respectively, bymeans of a direct (PLAP) and a transcytotic(DPPIV) route to the apical plasma membraneunder knockdown conditions for TI-VAMP.

To decrease the endogenous levels of TI-VAMP in Caco2 cells, we designed a siRNAtargeting human TI-VAMP (siRNA 8), whichwas transiently transfected in Caco2 cells. TI-VAMP expression levels were measured after4 days of culture on filters by western blottingon total cell lysates in a manner similar to thatdescribed for FRT cells in Fig. 1. By using aspecific mouse antibody against human TI-VAMP (Muzerelle et al., 2003) in westernblots, we found a marked decrease (<70%) ofthe TI-VAMP signal in siRNA-transfectedcells compared with control cells transfectedwith an unrelated siRNA targeting human�-globin (normalization was done incomparison with calreticulin) (Fig. 5A),indicating that there was a significant transientknockdown of TI-VAMP in polarized Caco2cells. Similar results were obtained using asecond siRNA (siRNA 7) (see supplementarymaterial Fig. S1). We then investigated theeffect of transient TI-VAMP knockdownon the apical localization of PLAP byimmunofluorescence and confocal analysis offilter-grown Caco2 cells (Fig. 5B). Comparedwith control cells (siRNA targeting �-globinexpression) where PLAP accumulates at the

Fig. 4. Transient and stable loss of TI-VAMP does not affect the establishment of apolarized monolayer on filters. FRT cells stably expressing PLAP, FRT cells stablyexpressing PLAP electroporated with 100 pmol of siRNA targeting rat TI-VAMP oragainst human �-globin, and FRT si TI-VAMP::PLAP cells, were grown on filters for4 days. (A) Transepithelial resistance (TER) was measured every day. Note that thereis no difference in the establishment of polarity between control (siRNA beta glo) andTI-VAMP transient-knockdown cells (siRNA TI-VAMP) and between control (FRTPLAP) and TI-VAMP stable-knockdown cells (FRT si TI-VAMP::PLAP).(B) Confocal X-Y and Z sections at the level of tight junctions of control (siRNA betaglo), TI-VAMP transient-knockdown (siRNA TIVAMP) and TI-VAMP stable-knockdown cells show that the integrity of the tight junctions is preserved in eachcase. Bars, 10 �m.

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apical membrane, in Caco2 cells transfected with siRNAtargeting TI-VAMP, PLAP was also mislocalized to the lateralmembrane (Fig. 5B), similar to our finding with FRT si TI-VAMP::PLAP cells (Fig. 1 and Fig. 3A). In order to rule outintracellular accumulation of the protein below the plasmamembrane, we have also performed the surface staining in non-permeabilized conditions where we obtained the same results(see supplementary material Fig. S2A). These results clearlyindicate that PLAP is mislocalized to the lateral membrane insi-RNA-treated Caco2 cells. As expected, TI-VAMP

knockdown did not have any effect on the basolaterallocalization of the basolateral marker Ag525 (Fig. 5B),indicating that the overall polarity of the monolayer was notaffected. In order to eliminate the possibility of an off-targeteffect of the siRNA, we performed a rescue experiment bydouble transient-transfection of the siRNA targeting TI-VAMPtogether with a rat cDNA encoding rat brain TI-VAMP[sequence NP_445983 (Hibi et al., 2000)] that is resistant tothe siRNA targeting the human sequence (see supplementarymaterial Fig. S3). In approximately a third of the cells, PLAPwas not mislocalized to the lateral membrane like in controlconditions, whereas in two-thirds of the monolayer themislocalization of PLAP to the lateral membrane was lessprominent compared with the one observed in the TI-VAMPknockdown cells (see supplementary material Fig. S3). Thisexperiment showed a partial rescue of the human TI-VAMPknockdown by the rat TI-VAMP cDNA, demonstrating that theeffects on apical sorting were specific.

Because PLAP is directly sorted to the apical membraneboth in FRT and Caco2 cells (Le Bivic et al., 1990; Paladinoet al., 2006), these data indicate that TI-VAMP is involved indirect apical sorting of TM and GPI proteins in different cells.In order to test whether TI-VAMP is also involved in thetranscytotic apical pathway, we analyzed the effect of itsknockdown on the sorting of DPPIV, which in Caco2 cellstranscytoses through the basolateral surface before beinginserted in the apical membrane (Gilbert et al., 1991; Matter etal., 1990). Surprisingly, in contrast to the results obtained inFRT cells (Fig. 2), we found no effect of TI-VAMP knockdownon the apical localization of DPPIV (Fig. 5C andsupplementary material Fig. S2B). The fact that TI-VAMPknockdown has such a different effect on the same protein intwo different cell lines indicates that this effect is dependentupon the pathway followed by DPPIV in the two cell lines(Gilbert et al., 1991; Zurzolo et al., 1992a). Thus our datastrongly suggest that the apical v-SNARE TI-VAMP isinvolved in direct apical sorting of TM and GPI-AP proteinsindependently of their raft association but does not function inthe apical transcytotic pathway.

VAMP8 is involved in the transcytotic pathway of DPPIVin Caco2 cellsThe v-SNAREs that control the transcytotic pathway have notbeen identified. In order to understand the mechanisms thatcontrol the transcytosis of DPPIV in Caco2 cells, we focusedour studies on two possible v-SNARE candidates (VAMP3 andVAMP8) that have been described as being involved inendosomal recycling (Advani et al., 1998; Breton et al., 2000;Galli et al., 1994; Wong et al., 1998). We reasoned that, if oneof these two v-SNAREs functioned in apical transcytosis, thelocalization of the v-SNARE should be disturbed by the TI-VAMP knockdown because of the expected crosstalk betweenthe direct and indirect apical pathways. Interestingly, weobserved that VAMP3 localization was not affected in TI-VAMP knockdown of Caco2 cells (Fig. 6A, left panel),whereas VAMP8 partially accumulated basolaterally and wasless concentrated in intracellular vesicles and on the apicalsurface (Fig. 6A, right panel). Quantitative analysis offluorescence by applying a threshold in order to selectintracellular objects of a specific level of intensity allowedcalculation (using the ImageJ® software) of the number of


Fig. 5. Only PLAP but not DPPIV is mislocalized in Caco2 cellsafter transient RNAi of TI-VAMP. Caco2 cells were electroporatedwith 60 pmol of siRNA targeting human TI-VAMP or againsthuman �-globin and grown on filters for 4 days. (A) Cell extracts(40 �g) from control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA TI-VAMP) cells were analyzed by SDS-PAGEand western blotting with antibodies against human calreticulin(top panel, internal control for protein loading) and human TI-VAMP (bottom panel). TI-VAMP expression was decreased bymore than 70% after transient RNAi of Caco2 cells on filters inthree different experiments. (B,C) Confocal Z and X-Y sections (topand bottom) of control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA TI-VAMP) Caco2 cells. (B) Cells were labeledwith an antibody against PLAP and a secondary antibody coupledto FITC. Compared with its apical localization in Caco2 cells (leftpanel), PLAP is mislocalized to the lateral membrane in Caco2 TI-VAMP transient-knockdown cells (right panel). (C) Cells werelabeled with antibodies against human DPPIV and secondaryantibodies coupled to FITC. Note that, compared with FRT cells,the apical localization of DPPIV is unaffected by the transientknockdown of TI-VAMP, as is the basolateral localization of Ag525(B, right panel). Bars, 10 �m.

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objects and the areas occupied by each object (Fig. 6B). Thisanalysis showed that there are fewer and much smaller objectsin the TI-VAMP knockdown condition compared with thecontrol (Fig. 6B). Together with the more prominent surfacestaining of VAMP8 (Fig. 6A), these experiments show that thelocalization of VAMP8 is altered and that it is less concentratedin vesicular structures and more at the lateral membrane in TI-VAMP knockdown cells, thus indicating an involvement ofVAMP8 in apical sorting. We therefore decided to study theeffects of VAMP8 knockdown on the direct and indirect apicalpathways that are followed by PLAP and DPPIV.

To interfere with VAMP8 expression, we used a siRNAtargeting human VAMP8 [Hs_VAMP8_3 HP-validated siRNA(SI02653245) from Qiagen] that was transiently transfected inCaco2 cells. VAMP8 expression levels were measured after 4days of culture on filters by western blotting on total celllysates (Fig. 7A). By using a specific rabbit antibody againsthuman VAMP8 in western blots, we found a marked decrease(>90%) of the signal corresponding to VAMP8 in knockdowncells compared with control cells (transfected with human �-globin siRNA) (Fig. 7A). We then investigated the effect of thisknockdown on both the direct delivery of PLAP and theindirect delivery of DPPIV in filter-grown Caco2 cells (Fig.7B,C).

Interestingly, while both the direct apical delivery of PLAPand direct basolateral delivery of Ag525 were not affected (Fig.7B), DPPIV transcytosis to the apical membrane was strongly

perturbed and the protein became mislocalized to thebasolateral surface (Fig. 7C and supplementary material Fig.S2B). In order to analyze the mechanism of action of VAMP8in more detail, we performed an immunofluorescence-basedassay to follow DPPIV internalization from the basolateral tothe apical plasma membrane (see supplementary material Fig.S4). After adding the antibody against DPPIV from thebasolateral side of cells grown on filters for 4 days, wefollowed its internalization at different times. We first labeledsurface DPPIV (non-permeabilized conditions) using asecondary antibody coupled to fluorescein isothiocyanate(FITC). After extensive washing and quenching, we added asecondary antibody conjugated with TRITC in permeabilizedconditions to label internal DPPIV. In control conditions, thebasolateral surface staining of DPPIV disappeared very quicklywith time, followed by intracellular staining at ten minutes,consistent with a transcytosis of DPPIV from the basolateraltowards the apical membrane, which was stained after 30minutes (see supplementary material Fig. S4B). In VAMP8transient-knockdown experiments, the basolateral staining ofDPPIV remained unchanged with time, and no signal appearedintracellularly after ten minutes (supplementary material Fig.S4) or at the plasma membrane at later times (data not shown),thus demonstrating a role forVAMP8 in the endocytosis ofDPPIV from the basolateral to the apical surface.

In summary, these data indicate that VAMP8: (1) does notcooperate with TI-VAMP to effect direct apical delivery, (2) is

Fig. 6. VAMP8 but not VAMP3 ismislocalized in Caco2 cells after transientRNAi of TI-VAMP. Caco2 cells wereelectroporated with 60 pmol of siRNAtargeting human TI-VAMP or against human�-globin and grown on filters for 4 days.(A) Confocal Z and X-Y sections (top andbottom) of control (siRNA beta glob) and TI-VAMP transient-knockdown (siRNA TI-VAMP) Caco2 cells. Cells were labeled withantibodies against human VAMP3 (left panel)and VAMP8 (right panel) and secondaryantibodies coupled to FITC. Note that thelateral localization of VAMP3 is unaffectedby the transient knockdown of TI-VAMP.Compared with its vesicular localization incontrol Caco2 cells, VAMP8 seems to be lessconcentrated in vesicular structures andslightly mislocalized to the lateral membranein Caco2 TI-VAMP transient-knockdowncells. (B) Digitized images of confocal X-Ysections of control (siRNA beta glob) and TI-VAMP (siRNA TI-VAMP) Caco2 cells. Cells were labeled with antibody against humanVAMP8 and secondary antibody coupled to FITC. Fluorescentimages were converted and analyzed quantitatively using ImageJ®

software (see Materials and Methods). A statistical analysis of thearea of each fluorescent object was performed (data not shown). Wefound fewer objects in the case of TI-VAMP knockdown comparedwith the control (130 versus 164 in the control). We also found thatthere were more objects of small area (less than five pixels) andfewer objects of large area (greater than ten pixels) in the case of theTI-VAMP knockdown. Bar, 10 �m.

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not responsible for the basolateral insertion of directly sortedbasolateral proteins and (3) is a major player in the transcytoticapical pathway in polarized epithelial cells.

DiscussionVirtually every intracellular membrane fusion event ismediated by a SNARE machinery (Mostov et al., 2003;Weimbs et al., 1997). Although it has been demonstrated thatonly a perfect match between the v-SNARE and itscorresponding t-SNARE leads to successful membrane fusion(McNew et al., 2000; Scales et al., 2000), it is still debated howSNAREs contribute to specify trafficking and at which step(i.e. targeting or fusion) they regulate these events.

Studies of SNARE function in epithelial cells have shown apolarized distribution of t-SNAREs highly conserved amongepithelial cell types and support their role in the establishmentand maintenance of epithelial polarity (Li et al., 2002; Low etal., 1996; Low et al., 1998; Low et al., 2002). The t-SNAREStx3 localizes to the apical plasma membrane of MDCK cells(Low et al., 1996), and both Stx3 and the v-SNARE TI-VAMPoperate at the apical plasma membrane of Caco2 cells (Breuzaet al., 1999; Galli et al., 1998). Overexpression of Stx3 reduces

apical transport by a factor of 20 to 50 (Low et al., 1998) anddoes not cause mistargeting to the basolateral surface but,instead, results in the accumulation of apical proteins inintracellular vesicles (Low et al., 1998), consistent with theformation of non-productive SNARE complexes that impairfusion.

However, the apical pathway is rather complicated becauseit can be either raft dependent or raft independent and there aretwo ways to reach the apical surface, one direct and the otherindirect (Paladino et al., 2006; Rodriguez-Boulan et al., 2005).Previous work has demonstrated the involvement of Stx3 in theapical sorting of two specific transmembrane proteins HA andSI (both associated with DRMs) that are directly sorted to theplasma membrane (Breuza et al., 2000; Lafont et al., 1999;Low et al., 1998). Consistent with these findings, both apicalv- and t-SNARE TI-VAMP and Stx3 are present in post-TGNcarriers in DRMs, and TI-VAMP forms apical SNAREcomplexes with Stx3 (Galli et al., 1998). Althoughoverexpression of Stx3 does not alter the transcytosis of WT-pIgR in MDCK cells (Low et al., 1998), the role of TI-VAMPin the transcytotic pathway followed by apical proteins,especially in intestinal cells, has not been directly established(Breuza et al., 2000). Furthermore, it was not known whetherraft- and non-raft-associated apical proteins use the sameexocytic mechanism at the apical surface. Our data clearlydemonstrate that TI-VAMP is necessary for the apicallocalization of raft- and non-raft-associated proteins, implyinga role for TI-VAMP in the direct apical delivery for both


Fig. 7. DPPIV but not PLAP is mislocalizedin Caco2 cells after transient RNAi ofVAMP8. Caco2 cells were electroporatedwith 60 pmol of siRNA targeting humanVAMP8 or against human �-globin andgrown on filters for 4 days. (A) Cell extracts(40 �g) from control (siRNA beta glob) andVAMP8 transient-knockdown (siRNAVAMP8) cells were analyzed by SDS-PAGEand western blotting with antibodies againsthuman calreticulin (top panel, internalcontrol for protein loading) and humanVAMP8 (bottom panel). VAMP8 expressiondecreased by more than 80% after transientRNAi of Caco2 cells on filters in threedifferent experiments. (B,C) Confocal Z andX-Y sections (top and bottom) of control(siRNA beta glob) and VAMP8 transient-knockdown (siRNA VAMP8) Caco2 cells.(B) Cells were labeled with an antibodyagainst PLAP and a secondary antibodycoupled to FITC. Compared with its apicallocalization in Caco2 cells (left panel), PLAPis unaffected by the transient knockdown ofVAMP8. (C) Cells were labeled withantibodies against human DPPIV (left panel)and secondary antibodies coupled to FITC.Note that, compared with FRT cells, theapical DPPIV is also mislocalized to thelateral membrane in Caco2 VAMP8 transient-knockdown cells (right panel), whereas thebasolateral localization of Ag525 isunaffected (B, right panel). Bars, 10 �m.

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pathways (Figs 1, 2 and 5). We also confirmed previous datathat TI-VAMP is not involved in basolateral delivery (Figs 1and 5). A quantitative and statistical analysis of these data wasperformed by means of a surface biotinylation assay in stableFRT clones lacking TI-VAMP (Fig. 3). Furthermore, bymeasuring the TER and studying the morphology of tightjunctions, we also showed that mislocalization of apical non-raft and raft-associated proteins in knockdown cells for TI-VAMP was not due to a general perturbation of the epithelia(Fig. 4B). These results also suggest that the TI-VAMP-dependent apical targeting pathway is not involved in theformation of tight junctions and is not essential for thedevelopment of polarized epithelia.

Next, in order to analyse directly whether TI-VAMP wasalso involved in the indirect pathway to the apical membrane,we repeated the same knockdown experiments in Caco2 cellsthat, unlike FRT cells, target endogenous DPPIV to the apicalsurface via a transcytotic pathway (Gilbert et al., 1991). Ascontrol for the effect of TI-VAMP on the direct pathway, weused again PLAP, which is endogenously expressed by Caco2cells and is targeted directly to the apical membrane (Le Bivicet al., 1990). As in FRT cells, TI-VAMP knockdown impairedthe direct apical delivery of PLAP; however, it had no effecton both the transcytotic delivery of DPPIV and, as expected,basolateral delivery (Fig. 5). These data clearly demonstratethat TI-VAMP is involved only in the apical direct pathway andnot in the apical indirect pathway and that this mechanism isconserved in different epithelia.

Interestingly, by specific knockdown of TI-VAMP, we notonly impaired the direct apical delivery but also foundmislocalization of the apical proteins to the basolateralsurface, an effect that was not observed previously when theexpression of TI-VAMP was perturbed using either anoverexpression or an antibody block approach (Breuza et al.,2000; Galli et al., 1998; Low et al., 1998). These data suggestthat, in the absence of TI-VAMP, apical carriers can fuse withthe basolateral membrane. This could be explained by twohypotheses: either TI-VAMP has a prominent function in thesorting of the proteins at the level of the TGN, or another v-SNARE allowing basolateral fusion is present on post-TGNapical carriers and it is unmasked only after TI-VAMPknockdown. Because it is possible that this ‘recessive’ VAMPwould also be involved in transcytotic apical delivery, wefocused our attention on two v-SNAREs that could be able toovercome TI-VAMP loss in our knockdown experiments. Thefirst of these was VAMP3 (also known as cellubrevin or Cb)(McMahon et al., 1993), which is involved in the recycling ofthe transferrin (Tf) receptor (Galli et al., 1994) and in earlyendosomal pathways such as the apical transport of H+-ATPase (Breton et al., 2000), and the other was the v-SNAREVAMP8/endobrevin (Advani et al., 1998; Wong et al., 1998),which might mediate the endocytic apical recycling pathwayin polarized cells (Antonin et al., 2000; Mullock et al., 2000;Steegmaier et al., 2000; Wong et al., 1998) and colocalizesalso with the Tf receptor (Wong et al., 1998). To test thepossible involvement of these two v-SNAREs in the apicalpathway, we examined their localization in TI-VAMPknockdown in Caco2 cells (Fig. 6). Interestingly, thelocalization of VAMP3 was not affected by TI-VAMPknockdown (Fig. 6A, left panel). This is consistent with recentfindings that VAMP3 co-immunoprecipitates mainly with

Stx4, thus suggesting that it is not involved in the apicalpathway but instead functions in the basolateral sorting ofAP1B-dependent cargos, as recently demonstrated (Fields etal., 2007). By contrast, in TI-VAMP knockdown cells,VAMP8 appeared to be less concentrated in intracellularvesicular structures and localized more at the lateralmembrane (Fig. 6A, right panel and Fig. 6B), indicating thatit might be involved in the apical pathway.

VAMP8 is the best candidate to be involved in thetranscytotic pathway because it is able to interact both with thebasolateral t-SNARE Stx4 and with the apical t-SNARE Stx3.It has been shown that VAMP8 and Stx3 can form complexeswith SNAP23 (Pombo et al., 2003). Furthermore, it has alsobeen proposed that VAMP8, Stx4 and SNAP23 act together(Wang et al., 2007). Finally VAMP8, Stx3 and Stx4 were co-immunoprecipitated in parotid acinar cells (Imai et al., 2003).In addition, it was recently shown that, although the majorlocalization of VAMP8 is endosomal in fixed MDCK cells, inlive conditions it appears to be present in fine, long tubularstructures emanating from endosomes and to be present at theapical surface of the cell (Wakabayashi et al., 2007). This isconsistent with the role of VAMP8 in sorting to the apicalplasma membrane, and, because VAMP8 participates inendocytosis and apical recycling in MDCK cells (Steegmaieret al., 2000), but not in apical direct delivery (Lafont et al.,1999), all these data suggest that VAMP8 could operate in theapical transcytotic pathway.

To confirm this hypothesis, we analysed the effect ofVAMP8 knockdown on the direct and transcytotic apicaldelivery pathways in Caco2 cells (Fig. 7). As expected,VAMP8 knockdown had no effect both on the direct apicaldelivery of PLAP and on the direct basolateral delivery ofAg525 (Fig. 7B). However, in these conditions, the transcytoticapical delivery of DPPIV was remarkably affected and theprotein was partially mis-sorted to the basolateral surface.Interestingly, we found an increase in the total amount (datanot shown) and of the plasma membrane fraction of DPPIV inin VAMP-knockdown cells (Fig. 7). This could also beconsistent with an impairment of the endocytic apical recyclingpathway in VAMP8 knockdown. However, the fact that, inVAMP8-knockdown cells, DPPIV remains on the basolateralsurface (supplementary material Fig. S4) is consistent with arole in endocytosis from the basolateral surface to the apicalmembrane.

Overall, our data indicate that VAMP8 is involved in thetranscytotic pathway. Furthermore, because the localization ofAg525 was not impaired by VAMP8 knockdown (Fig. 7B), wecan also conclude that direct basolateral delivery and thetranscytotic apical pathway use different sets of v-SNAREs(Fig. 8). This hypothesis finds additional support from recentdata showing the involvement of VAMP3 in the basolateralsorting of AP-1B-dependent cargos (Fields et al., 2007).Interestingly, when we examined Stx3 and Stx4 localizationsin TI-VAMP-knockdown Caco2 cells, they were not perturbedcompared with the control, suggesting that t-SNAREs and v-SNAREs do not play the same role in apical sorting (seesupplementary material Fig. S5).

In conclusion, our data reveal specificity in the sorting ofapical cargos that could be explained with two different models(Fig. 8). We cannot exclude that each cargo vesicle is equippedwith more then one v-SNARE and the specificity is governed

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by their relative amount and stoichiometry. However, our datasupport the hypothesis that there is a specific v-SNARE foreach post-TGN cargo (Fig. 8) and that mistargeting in theabsence of one v-SNARE arises from the loading of cargo onan alternative pathway. Further work will be necessary tounderstand how v-SNAREs and cargos are sorted together atthe level of the Golgi apparatus and endosomes.

Materials and methodsReagents and antibodiesCell culture reagents were purchased from Invitrogen. Antibodies against PLAP andZO-1 were purchased from Rockland Bioscience and Zymed. The antibodies againstrat 35/40 kDa and DPPIV were gifts from A. Quaroni (Dept of Biomedical Sciences,VRT 8004, Cornell University, Ithaca, NY). The antibodies against human Ag525,DPPIV and Patj were used as described previously (Le Bivic et al., 1988; Michelet al., 2005; Quaroni and Isselbacher, 1985). The antibody against TI-VAMP wasused as described previously (Muzerelle et al., 2003). VAMP 3, VAMP 8, syntaxin3 and syntaxin 4 were purchased from Transduction Laboratories. Biotin and proteinA sepharose were obtained from Pierce Chemical Co. and Amersham. Horseradishperoxidase (HRP)-linked antibodies and streptavidin were purchased fromAmersham GE Healthcare. All other reagents were purchased from Sigma-Aldrich.

Cell culture and transfectionsFRT cells (Ambesi-Impiombato and Coon, 1979) were grown in F12 Coon’smodified medium containing 5% fetal bovine serum (FBS). A stable cloneexpressing PLAP was previously obtained (Lipardi et al., 2000). Cells weretransiently transfected using the GenePulserXCell (Bio-Rad) by mixing 100 pmol

siRNA with 400 �l RPMI medium and 107 freshly trypsinized cells. Cells (2�106)were seeded on Transwell filters (24 mm diameter, Corning, NY) and TER wasmeasured each day with a MilliCell apparatus (Millipore Corporation, Bedford,MA). Cells were stably transfected using Lipofectin® (Invitrogen) by mixing 3-5�g DNA with 10 �g Lipofectin® and 3 ml Optimem, and selected in hygromycin(250 �g/ml) and/or G418 (10 �g/ml) for two weeks. A clone of Caco2 (TC7 cells)was grown in DMEM containing 10% FBS (Chantret et al., 1994). Cells weretransfected using the Amaxa device, T solution and T20 program (AmaxaBiosystems, Germany) by mixing 60 pmol siRNA with 100 �l buffer T and 1.8�106

freshly trypsinized cells. Cells (Caco2, 1.8�106 and FRT, 2�106) were seeded onTranswell filters (24 mm diameter, Corning) and TER was measured with aMilliCell apparatus (Millipore Corporation).

RNA interferenceThree siRNAs targeting TI-VAMP were designed: one against rat (5�-CCTCGTAGATTCGTCCGTC-3�) and two against human (5�-TGCCAT -TAAATTGAAATTATA-3� and 5�-CTGCCAAGACAGGATTGTATA-3�). DNAcorresponding were introduced as a hairpin behind a U6 promoter added to peGFP-N2 (BD Bioscience Clontech, Palo Alto, CA) containing a neomycin-resistancecassette, using the HindIII and BglII restriction sites (a kind gift from A. Le Bivic,Luminy, Marseilles, France) and sequenced. The siRNA targeting �-globin (bgloE1;5�-GGUGAAUGUGGAAGAAGUUtt-3�) was a kind gift from N. Sauvonnet(Institut Pasteur, Paris, France). The siRNA targeting VAMP8 [Hs_VAMP8_3 HP-validated siRNA (SI02653245)] was purchased from Qiagen.

Western blottingFRT cell extracts were prepared and analysed by western blotting as previouslydescribed (Lipardi et al., 2000), with primary antibodies and the correspondingsecondary antibodies coupled to peroxidase (Amersham, GE Healthcare). FRT cellswere selectively biotinylated and processed as described (Paladino et al., 2006).Lysate were immunoprecipitated with specific antibodies and biotinylated antigenswere revealed with streptavidin. Caco2 cell extracts were prepared and analyzed bywestern blotting as described (Lemmers et al., 2002) with primary antibodies andthe corresponding secondary antibodies coupled to peroxidase (Amersham, GEHealthcare). Samples were run on SDS-PAGE and bands were revealed using theECL kit (Amersham, GE Healthcare) and quantified using the ImageJ (NIH)software.

Fluorescence microscopyFRT and Caco2 cells grown on Transwell filters for 4 days were washed with PBScontaining CaCl2 and MgCl2, fixed with 4% PFA, and quenched with 50 mMNH4Cl. Cells were permeabilized with 0.075% saponin. Primary antibodies weredetected with FITC or TRITC-conjugated secondary antibodies. Images werecollected using a laser scanning confocal microscope (LSM 510; Carl ZeissMicroImaging Inc.) equipped with a plan apo 63� NA-1.4 oil-immersion objectivelens (Carl Zeiss MicroImaging Inc.). Fluorescent images were converted usingImageJ® software to 8 bit (256 grey levels) and a threshold selection was appliedbetween 80 and 255 grey levels (top panels, red). Measurements of the area of thefluorescent objects was obtained using ImageJ software (bottom panels, whiteellipses in Fig. 6B).

We are grateful to Lucien Cabanié for the purification of the mAbCl158.2 hybridoma and TG18 rabbit serum and to Philippe Casanovafor technical help. We are grateful to Véronique Proux-Gillardeauxand Lydia Danglot for helpful discussion of the data. This work wassupported by Grants to C.Z. from MURST (PRIN 2005), from theEuropean Union (LSH-2004-1.2.2-4) and from Agence Nationale dela Recherche ANR (05-BLAN 296-01) and to T.P. from theWeizmann-Pasteur Foundation. Work in the T.G. group was supportedin part by grants from INSERM (Avenir Program), the EuropeanCommission (‘Signalling and Traffic’ STREP 503229), theAssociation pour la Recherche sur le Cancer, and the Fondation pourla Recherche Médicale. A.L.B. is supported by Ligue Nationale contrele Cancer and UMR 6216 CNRS/Université Aix Marseille II.

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Fig. 8. Model for v-SNARE specificity in the different pathways tothe plasma membrane of polarized epithelial cells. Upon sorting atthe level of the TGN, proteins are addressed to the apical orbasolateral plasma membrane via at least three different pathways:the direct apical pathway (bold arrowhead), the direct basolateralpathway (dashed arrow) and the transcytotic pathway (openarrowhead). Post-TGN carriers could contain different v-SNAREs(e.g. TI-VAMP, VAMP8, VAMP3). In this case, the specificity offusion would arise from the pairing of each v-SNARE with theappropriate t-SNARE and from the stoichiometry of the v-SNAREspresent on each vesicle (the predominant VAMP is indicated byunderlining and alternatives are indicated by parentheses). Thespecificity of sorting could be dependent on the functionalinteraction between a v-SNARE and a specific adaptor (e.g. AP-1Bfor recycling endosomes to the basolateral plasma membrane in thedirect basolateral pathway). Alternatively, each v-SNARE might beactively sorted into different post-TGN cargos (in this case, only theSNARE indicated by underlining would be present in the vesicle). T-J, tight junction.

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