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CD 0 c l o D E O B 5 c L 3 0 Research Article 3309 Distinct vSNAREs regulate direct and indirect apical delivery in polarized epithelial cells Thomas Pocard Andr Le Bivicz Thierry Gali3v4 and Chiara Zurzolo1l5z lUnit de Trafic Membranaire et Pathogenese Institut Pasteur 75724 Paris CEDEX 15 France 2UMR 6212 CNRSUniversit Aix Marseille IBDML case 907 Facult des Sciences de Luminy 13288 Marseille CEDEX 09 France 3Membrane Traffic in Neuronal and Epithelial Morphogenesis INSERM Avenir Team 75005 Paris France 4Institut Jacque Monod CNRS UMR7592 Universities Paris 6amp7 75005 Paris France 5Dipartimento di Biologia e Patologia Cellulare e Molecolare Universita degli Studi di Napoli Federico II 80131 Napoli Italy 39Author or correspondence ermall zurzolo pasteur 1r Accepted 6 July 2007 Journal of Cell Sclence 120 33093320 Publlshed by The Company of BIOoglsrs 2007 10 10 124263 007948 Summary SNARE soluble Nethylmaleimidesensitive factor NSF attachment protein SNAP receptor proteins control the membranefusion events of eukaryotic membrane traf cking pathways Speci c vesicular and target SNAREs operate in speci c trafficking routes but the degree of speci city of SNARE functions is still elusive Apical fusion requires the polarized distribution at the apical surface of the tSNARE syntaxin 3 and several v SNAREs including TIVAMP and VAMPS operate at the apical plasma membrane in polarized epithelial cells It is not known however whether speci c vSNAREs are involved in direct and indirect routes to the apical surface Here we used RNAi to assess the role of two tetanus neurotoxininsensitive vSNAREs TIVAMPVAMI 7 and VAMPS in the sorting of raft and nonraftassociated apical markers that follow either a direct or a transcytotic delivery respectively in FRT or CacoZ cells We show that TIVAMP mediates the direct apical delivery of both raft and nonraftassociated proteins By contrast sorting by means of the transcytotic pathway is not affected by TI VAMP knockdown but does appear to be regulated by VAMPS Together with the speci c role of VAMP3 in basolateral transport our results demonstrate a high degree of speci city in vSNARE function in polarized cells Supplementary material available online at httpjcsbiologistsorycgicontentjfulll20183309DC1 Key words SNAREs TlVAMP VAMPS Epithelial cells Polarized sorting Transcytosis Introduction The plasma membrane of polarized epithelial cells is divided into two domains apical and basolateral differing in their protein and lipid compositions and specialized functions Mostov 2003 RodriguezBoulan and Powell 1992 Intracellular sorting occurs at the level of the transGolgi network TGN and endosomes where proteins are segregated into distinct vesicles upon recognition of specific apical or basolateral sorting signals Folsch 2005 Matter andMellman 1994 Mostov et al 2000 The proteins are delivered to the apical or basolateral surface RodriguezBoulan and Powell 1992 Keller et al 2001 Kreitzer et al 2003 WandingerNess et 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 both cell and protein specific RodriguezBoulan et al 2005 Although direct and indirect sorting signals are quite well defined for transmembrane TM basolateral proteins this is not the case for apical proteins and in particular for GPI anchored proteins GPlAPs These proteins are anchored to membranes by a posttranslational lipid modification the glycosylphosphatidylinositol GPI anchor Bangs et al 1985 Ferguson et al 1985 lkezawa 1963 McConnell et al 1981 A relevant feature of GPlAPs is their association with lipid rafts Brown 1994 Brown and Waneck 1992 Brown 1992 Brown and Rose 1992 GPl 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 and Waneck 1992 Brown 1992 It has therefore been proposed that the GPI anchor acts as an apical sorting determinant by mediating raft association Simons and lkonen 1997 Simons and van Meer 1988 The direct sorting of GPlAPs to the apical surface of MadinDarby canine kidney MDCK cells Hua et al 2006 Paladino et al 2006 requires their oligomerization into detergentresistant membrane domains DRMs at the Golgi level Paladino et al 2004 This reinforces the hypothesis that apical sorting of GPI APs occurs intracellularly before arrival at the plasma membrane Hua et al 2006 Paladino et al 2006 However not much is known about the molecular machinery involved in the direct targeting of GPlAPs to the apical surface Various components of the exocytic machinery that transport apical and basolateral proteins have been identified but the mechanisms that regulate their localization and function have been challenged recently reviewed in RodriguezBoulan et al 2005 Polarized protein trafficking to the apical and basolateral plasma membranes requires different sets of SNARES a family of proteins specifically involved in the fusion of vesicles with their target membranes Lafont et al 1999 Low et al 1996 Low et al 1998 Steegmaier et al 2000 Weimbs et al 2003 SNAREs on vesicular cargo are called VSNARES and those on target membranes tSNAREs CD 0 c 1 o D E O 3 F c L 3 O 3310 Journal of Cell Science 120 18 Sollner et al 1992 The formation of a SNARE complex between vSNAREs and tSNAREs Schiavo et al 1997 Sollner et al 1993 mediates the specific recognition and subsequent fusion McNew et al 2000 of vesicles with their appropriate target membrane SNAREs are assisted by several partners and regulators including the small GTPase Rab proteins Grosshans et al 2006 Novick and Zerial 1997 Zerial and McBride 2001 Polarized epithelial cells represent an interesting model to study the specific function of SNAREs because plasma membrane tSNAREs are differentially localized in these cells The tSNARE syntaxin 3 Stx3 is at the apical plasma membrane whereas syntaxin 4 Stx4 is expressed predominantly at the basolateral membrane domain of MDCK cells Low et al 1996 By contrast SNAP23 ubiquitously expressed homologue of SNAP25 and syntaxin 2 Stx2 are present in both membrane domains Low et al 1998 Stx3 and SNAP23 constitute the apical tSNARE complex and they interact with the vSNARE tetanusneurotoxininsensitive vesicle associated membrane protein TIVAMP in Caco2 cells Galli et al 1998 The strong interaction between TI VAMP Stx3 and SNAP23 has been further demonstrated by yeast twohybrid assays MartinezArca et al 2003 In MDCK cells overexpression of Stx3 inhibits biosynthetic transport from the TGN to the apical membrane and the endocytic recycling pathway from apical endosomes of a mutant form of the polymeric immunoglobulin receptor pIgR signalless pIgR SL pIgR Casanova et al 1991 but not the basolateral delivery of the wildtype form of pIgR WT pIgR Low et al 1998 Furthermore overexpression of Stx3 strongly inhibits the apical delivery of TM sucrase isomaltase SI and the secreted protein uglucosidase without any effect on basolateral delivery in Caco2 cells Breuza et al 2000 Furthermore inhibition of TI VAMP with specific antibodies affects apical delivery of haemagglutinin HA but has no effect on the basolateral route Lafont et al 1999 In addition Stx3 and TI VAMP associate with DRMs in postTGN apical carriers Lafont et al 1999 These data have demonstrated that Stx3 and TI VAMP are important for apical transport of transmembrane and secretory proteins both in MDCK and Caco2 cells However apical sorting shows further levels of complexity 1 there are at least two pathways for apical delivery one raft dependent and one raft independent B enting et al 1999 Lipardi et al 2000 2 there are at least two routes followed by apical proteins direct and transcytotic Gilbert et al 1991 RodriguezBoulan et al 2005 Zurzolo et al 1992a 3 it is not clear whether apical sorting of proteins following different pathways occurs at the same intracellular site and 4 utilizes the same machinery Although the results mentioned above on the apical SNAREs indicate a clear involvement of TI VAMP in the direct sorting of transmembrane and secreted apical proteins it is not clear whether TI VAMP is also involved in the direct sorting of GPI APs and whether it participates in the transcytotic pathway for which the specific vSNARE has not yet been identified Therefore we first asked whether TI VAMP is involved in the apical sorting of GPI APs Then in order to discriminate between the direct and transcytotic pathways we used two different cell lines Fisher rat thyroid cells FRT and human colorectal cancer cells Caco2 that respectively use predominantly the direct and the transcytotic pathway to deliver apical proteins to the plasma membrane Le Bivic et al 1990 Matter et al 1990 Zurzolo et al 1992a By using an RNA interference RNAi approach targeting TI VAMP in both cell lines we found that TI VAMP is necessary for the correct localization at the apical membrane of both GPI APs and TM proteins that use a direct route to the apical membrane independently of their sorting mode ie raft dependent or raft independent Furthermore TI VAMP is not involved in apical sorting of proteins that use a transcytotic pathway Instead the apical transcytotic pathway is regulated by VAMP8 another tetanusneurotoxininsensitive vSNARE which can pair with both apical and basolateral tSNAREs Imai et al 2003 Pombo et al 2003 Wang et al 2007 Thus we demonstrate that at least two different VAMPs are involved in the regulation of the two alternative routes to the apical plasma membrane This mechanism appears to be independent of the cargo protein per se but dependent on the pathway that the protein is following in the different epithelia Results TIVAMP is necessary for the correct sorting of both GPIAP and TM apical proteins in fully polarized FFiT cells We set up a specific RNAi approach to study the role of TI VAMP in the apical sorting of a model GPI AP placental alkaline phosphatase PLAP in two different epithelial cell lines FRT and Caco2 cells that respectively use predominantly a direct or an indirect pathway to target proteins to the apical membrane Several isoforms of TI VAMP have been described MartinezArca et al 2003 but only one isoform of TI VAMP is present in FRT cells sequence NM7021659 Wang et al 2005 To decrease endogenous levels of TI VAMP in FRT cells we used a specific small interfering RNA siRNA that has been described previously Alberts et al 2003 This siRNA and an unrelated siRNA targeting human B globin used as a negative control were transiently transfected into FRT cells stably expressing PLAP FRTPLAP Lipardi et al 2000 TI VAMP expression was assayed by western blotting of total cell lysates from cells grown on filters for 4 days in conditions favoring polarization Fig 1A Compared with control cells siRNAtreated FRT cells showed a strong decrease of the western blot signal corresponding to TI VAMP 88 normalization was performed in comparison with calreticulin Fig 1A thus indicating an efficient silencing of the expression of TI VAMP in polarized FRT cells In order to analyse whether TI VAMP has a role in apical sorting of TM proteins in FRT cells we investigated the effect of TI VAMP transient knockdown on the localization of TM apical and basolateral markers endogenously expressed by these cells respectively dipeptidyl peptidase IV DPPIV and antigen 3540 kDa Ag 3540 kDa Zurzolo et al 1991 Zurzolo et al 1992a Zurzolo et al 1993 Zurzolo et al 1992b Control cells FRTPLAP transfected with siRNA targeting Bglobin and FRTPLAP cells transfected with siRNA targeting TI VAMP were grown on filters for 4 days and subjected to immunofluorescence using specific antibodies against 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 Ag 3540 kDa was localized at the basolateral membrane Fig 1B a o C 9 o 039 E O u 0 Tu C L 3 0 j Role of vSNARES in apical delivery 3311 Zurzolo et al l992a By contrast in cells transfected with TlVAMP siRNA DPPlV was missorted to the lateral membrane whereas the basolateral localization of Ag 3540 kDa was unaffected Fig 1B These results obtained by gene silencing in FRT cells confirm the specific involvement of TI VAMP in apical sorting of apical TM proteins that was previously demonstrated by antibody inhibition in MDCK cells Lafont et al 1999 and suggest that the role of Tl VAMP is conserved across different epithelial cells Next we investigated the effect of Tl VAMP transient knockdown on the localization of PLAP our model apical GPlAP Lipardi et al 2000 Fig 1C Compared with the control condition where PLAP accumulated at the apical membrane in cells transfected with siRNA targeting Tl VAMP it was also mislocalized to the lateral membrane Fig 1C These results indicate that Tl VAMP is necessary also for the correct apical localization of GPl APs The above result is particularly noteworthy because PLAP and DPPIV although both following a direct route are sorted to the apical membrane by means of two different mechanisms respectively raft dependent and nonraft dependent B enting et A siRNA siFlNA beta glob TlVAMP I 63 KDa Calretlculln 25 KDa 4 TlVAMP B siRNA beta glob siFlNA TIVAMP O SiFlNA beta glob siRNA TlVAMP PLAP Ag 3540 KDa al 1999 Lipardi et al 2000 In order to quantify and better characterize the role of TlVAMP we produced stable Tl VAMP knockdown FRT clones see Materials and Methods Selected stable knockdown clones FRT si TlVAMPPLAP which by westeni blot had a markedly decreased signal corresponding to Tl VAMP while expressing PLAP at a level similar to that of control FRT PLAP cells Fig 2A were analyzed for the localization of our apical and basolateral endogenous TM markers by indirect immunofluorescence and confocal analysis as shown in Fig 2B As expected in FRT si TlVAMPPLAP cells DPPIV lost its predominant apical localization and was mislocalized to the lateral membrane whereas the basolateral localization of Ag 3540 kDa was unchanged Fig 2B Next we examined the localization of PLAP both by indirect immunofluorescence and confocal analysis Fig 3A and by surface biotinylation Fig 3B Similar to the transient knockdown PLAP was mislocalized to the lateral membrane of stable FRT knockdown cells compare Fig 3A with Fig 1C By selective surface biotinylation we calculated that 80 of surfaceexpressed PLAP was localized at the apical membrane in control cells whereas PLAP was equally distributed to the apical and basolateral membrane 50 apical and 50 basolateral in FRT si Tl VAMPPLAP cells Fig 3B Thus both the transient and stable knockdowns showed that the apical vSNARE Tl VAMP is necessary for the correct apical localization of DPPlV and PLAP two proteins directly sorted to the apical membrane of FRT cells through two different mechanisms respectively raft independent and raft dependent Lipardi et al 2000 siFlNA beta glob siFlNA TlVAMF Fig 1 Apical but not basolateral proteins are mislocalized in fully polarized FRT cells after transient RNAi of TlVAMP FRT cells stably expressing PLAP were electroporated with 100 pmol of siRNA targeting rat TlVAMP or against human Sglobin and grown on lters for 4 days A Cell extracts 40 ug from control siRNA beta glob andTlVAMP transientknockdown siRNA TlVAMP cells were analyzed by SDSPAGE and western blotting with antibodies against calreticulin top panel internal control for protein loading and human TIVAMP bottom panel The decrease in TlVAMP expression in transient knockdown cells was calculated to be 88 in three different experiments BC Confocal Z and X Ysections top and bottom of control siRNA beta glob andTlVAMP transientknockdown siRNA TlVAMP cells B Cells were labeled with antibodies against rat DPPIV left panel and Ag 3540 kDa right panel and secondary antibodies were coupled to FITC Note that the apical localization of DPPIV in controls switches in TlVAMP transientknockdown cells to the lateral membrane Z section and X Y section bottom panel whereas the basolateral localization of Ag 3540 kDa is unchanged C Cells were labeled with antibodies against PLAP and secondary antibodies coupled to FITC Note that as for DPPIV PLAP is mislocalized to the lateral membrane Bars 10 pin 3312 Journal of Cell Science 120 18 A FRT FRT PLAP SiTIVAMF39F LAF Fig 2 DPPIV but not Ag 3540 kDa is mislocalized in fully polarized stable FRT clones 66 KDa I PLAP lacking TlVAMP FRT cells stably expressing 77 FLAP FRT FLAP and FRT cells stably expressing FLAP and a vector where the siRNA 25 KDa TI39VAMP targeting rat TlVAMP was introduced FRT si TlVAMPPLAP were grown on lters for 4 days A Cell extracts 40 ug from FRT FLAP and FRT si TlVAMPPLAP were analyzed by B FRT PLAP FRT SiTVAMPPLAP SDSPAGE and western blotting with antibodies against FLAP lanes two and four and human TlVAMP lanes one and three Note that the DPPlV Ag 3540 KDa DPPIV Ag 3540 KDa two FRT cells express similar amounts of FLAP whereas TlVAMP is constitutiver knocked down in FRT si TlVAMP PLAP compared with FRT FLAP B Confocal Z and X Ysections top and bottom of FRT FLAP and FRT si TI VAMPPLAP cells were labeled with antibodies against rat DPPlV left panel and Ag 3540 kDa right panel and secondary antibodies coupled to FITC Note that DPPlV is only mislocalized in FRT si TIVAMPPLAP cells whereas the basolateral localization of Ag 3540 kDa is unchanged in both cell lines Bars 10 um Loss of TIVAMP does not impair the development of a A FRT FRT olarized monola er Because of the efcht of TIVAMP knockdown on both raft PLAP SITI VAMP39PLAP and nonraft mediated apical sorting we asked whether the mistargeting of PLAP and DPPIV could just be a consequence of the loss of the cells overall ability to acquire a polarized phenotype Because the kinetics of formation of tight junctions has been frequently used as a measure of the ability of epithelial cells to polarize we assessed whether transient or stable knockdown of TI VAMP affected the normal development of transepithelial resistance TER which measures the ability of a monolayer to impede ion flow 7 this measure should increase with the establishment of a polarized monolayer Fig 4A The TER of FRT PLAP cells transiently transfected with siRNA targeting Bglobin versus FRT PLAP cells transiently transfected with siRNA targeting TIVAMP was monitored during a 4day period Fig 4A Both curves start at a TER between 200 and 400 0 cm 2 and reach after 4 B days 1400 0 cm 2 the TER of an empty filter is represented by the yellow curve and reaches 75 Q cm z The same observation was made for FRTFLAP cells versus the stable a o C 9 o 039 E O 0 Tu C L 3 0 j PLAP FRT FHT PLAF siTiVAMP21PLAF Biot Tot Biot Tot Fig 3 FLAP is mislocalizedin fully polarized FRT cells stably lacking TlVAMP A Confocal Z and X Y sections top and bottom of FRT cells stably expressing FLAP and FRT cells stably expressing si TIVAMPPLAP grown on filters for 4 days and 66 KDa 39 PLAP labeled w1th antibody agarnst FLAP and secondary antrbodres gG coupled to FITC FLAP 1s mrslocalized to the lateral membrane of FRT si TIVAMPPLAP right panel B FRT FLAP and FRT si TIVAMPPLAP cells were grown on filters for 4 days and labeled with LCbiotin Biot added to the apical Ap or basolateral Bl surface After lysis FLAP was imrnunoprecipitated with a speci c antibody run on SDSPAGE and revealed with HRPstreptavidin lane one and panel three One tenth of the imrnunoprecipitate Tot was run on SDSPAGE and assayed with an antibody against FLAP panels two and four Bars 10 um lgG imrnunoglobulin G AP Bl AP Bl AD 31 AP Bl a o C 9 o 039 E O 0 Tu C L 3 0 j Role of vSNARES in apical delivery 3313 knockdown clone FRT si TlVAMPPLAP cells Fig 4A The higher TER of FRT si TlVAMPPLAP cells compared with FRTPLAP suggests a clonal selection effect because both curves exhibit the same slope indicating that the two cell lines behave similarly on filters during the establishment of the polarized epithelial phenotype Fig 4A In order to confirm that the monolayer was well established we also analysed by immunofluorescence the distribution of ZOl a major protein of the tight junctional complex Fig 4B Both siRNAtreated cells and control cells grown on filters in conditions favoring polarization for 4 days exhibit the chicken wire like immunostaining characteristic of the tight junction protein Z0 1 Fig 4B confirming that the knockdown of Tl VAMP in FRT cells does not induce any significant alteration in the tight junctional complex Overall these results demonstrate that the mislocalizations A TER ol FRT cells aller lransienl ls and stablesl TirVAMP knockdown o FHT PLAP si beta glols of apical nonraft and raft associated proteins in cells transiently and stably knockdown for Tl VAMP are not due to a general perturbation of the development of the epithelium They also indicate that Tl VAMP dependent apical pathways are not involved in the events necessary for tight junction formation during the development of a polarized monolayer TIVAMP is also involved in the direct apical sorting of PLAP but not in the transcytotic delivery of DPPIV in Cac02 cells In contrast to FRT and MDCK cells which use mainly a direct pathway to sort both apical and basolateral proteins to their respective membrane domains after their intracellular sorting other epithelial cell types can use different pathways RodriguezB oulan et al 2005 A study in intestine has shown that certain apical plasma membrane proteins follow an indirect pathway to the cell surface that passes first through the basolateral domain Hauri et al 1979 These results were confirmed in intestinal cell lines eg Cac02 cells cultured FHT PLAP siTiVAMPlis on filters under conditions favoring polarization Le Bivic et al 1990 Matter et al 1990 o FHT PLAP 2000 lFFlT siTerAMP PLAPsi moo empty lilter TER ohmcmz 3 8 i a 4 Time daysl ZO1 Fig 4 Transient and stable loss of TlVAMP does not affect the establishment of a polarized monolayer on lters FRT cells stably expressing PLAP FRT cells stably expressing PLAP electroporated with 100 pmol of siRNA targeting rat TlVAMP or against human Sglobin and FRT si TIVAMPPLAP cells were grown on filters for 4 days A Transepithelial resistance TER was measured every day Note that there is no difference in the establishment of polarity between control siRNA beta glo and TlVAMP transientknockdown cells siRNA TlVAMP and between control FRT PLAP and TlVAMP stableknockdown cells FRT si TlVAMPPLAP B Confocal X Y and Z sections at the level of tightjunctions of control siRNA beta glo TlVAMP transientknockdown siRNA TIVAMP and TlVAMP stable knockdown cells show that the integrity of the tight junctions is preserved in each case Bars 10 pm Sirna beta lob sirna TlVAMP FRT siTlVAMPPLAP Because Tl VAMP is also present at the apical membrane of Cac02 cells Galli et al 1998 this appears to be a very good model to analyse its role both in the direct and indirect apical pathways To this end we followed the sorting of DPPIV and PLAP which are both endogenously expressed by Cac02 cells and are sorted respectively by means of a direct PLAP and a transcytotic DPPIV route to the apical plasma membrane under knockdown conditions for TlVAMP To decrease the endogenous levels of TI VAMP in Cac02 cells we designed a siRNA targeting human Tl VAMP siRNA 8 which was transiently transfected in Cac02 cells Tl VAMP expression levels were measured after 4 days of culture on filters by western blotting on total cell lysates in a manner similar to that described for FRT cells in Fig 1 By using a specific mouse antibody against human Tl VAMP Muzerelle et al 2003 in western blow we found a marked decrease lt70 of the Tl VAMP signal in siRNAtransfected cells compared with control cells transfected with an unrelated siRNA targeting human Bglobin normalization was done in comparison with calreticulin Fig 5A indicating that there was a significant transient knockdown of Tl VAMP in polarized Cac02 cells Similar results were obtained using a second siRNA siRNA 7 see supplementary material Fig Sl We then investigated the effect of transient Tl VAMP knockdown on the apical localization of PLAP by immuno uorescence and confocal analysis of filtergrown Cac02 cells Fig 5B Compared with control cells siRNA targeting Bglobin expression where PLAP accumulates at the a o C 9 o 039 E O u 0 To C L 3 0 j 3314 Journal of Cell Science 120 18 siFlNA siFlNA beta glob TlVAMP 68 KDa Calreticulin 25 KDa TlVAMP B siFlNA beta glob siRNA TlVAMP siRNA beta glob siFlNA Tl VAMP siFlNA beta glob siFlNA TlVAMF39 DPPIV Fig 5 Only PLAP but not DPPIV is mislocalized in Cac02 cells after transient RNAi of TIVAMP Cac02 cells were electroporated with 60 pmol of siRNA targeting human TIVAMP or against human Sglobin and grown on filters for 4 days A Cell extracts 40 ug from control siRNA beta glob and TIVAMP transient knockdown siRNA TIVAMP cells were analyzed by SDSPAGE and western blotting with antibodies against human calreticulin top panel internal control for protein loading and human TI VAMP bottom panel TIVAMP expression was decreased by more than 70 after transient RNAi of Cac02 cells on filters in three different experiments BC Confocal Z and X Y sections top and bottom of control siRNA beta glob and TIVAMP transient knockdown siRNA TIVAMP Cac02 cells B Cells were labeled with an antibody against PLAP and a secondary antibody coupled to FITC Compared with its apical localization in Cac02 cells left panel PLAP is mislocalized to the lateral membrane in Cac02 TI VAMP transientknockdown cells right panel C Cells were labeled with antibodies against human DPPIV and secondary antibodies coupled to FITC Note that compared with FRT cells the apical localization of DPPIV is unaffected by the transient knockdown ofTIVAMP as is the basolateral localization of Ag525 B right panel Bars 10 um apical membrane in Cac02 cells transfected with siRNA targeting TIVAMP PLAP was also mislocalized to the lateral membrane Fig 5B similar to our finding with FRT si TI VAMPPLAP cells Fig l and Fig 3A In order to rule out intracellular accumulation of the protein below the plasma membrane we have also performed the surface staining in non permeabilized conditions where we obtained the same results see supplementary material Fig S2A These results clearly indicate that PLAP is mislocalized to the lateral membrane in siRNAtreated Cac02 cells As expected TIVAMP knockdown did not have any effect on the basolateral localization of the basolateral marker Ag525 Fig 5B indicating that the overall polarity of the monolayer was not affected In order to eliminate the possibility of an offtarget effect of the siRNA we performed a rescue experiment by double transienttransfection of the siRNA targeting TIVAMP together with a rat cDNA encoding rat brain TIVAMP sequence NP7445983 Hibi et al 2000 that is resistant to the siRNA targeting the human sequence see supplementary material Fig S3 In approximately a third of the cells PLAP was not mislocalized to the lateral membrane like in control conditions whereas in twothirds of the monolayer the mislocalization of PLAP to the lateral membrane was less prominent compared with the one observed in the TIVAMP knockdown cells see supplementary material Fig S3 This experiment showed a partial rescue of the human TIVAMP knockdown by the rat TIVAMP cDNA demonstrating that the effects on apical sorting were specific Because PLAP is directly sorted to the apical membrane both in FRT and Cac02 cells Le Bivic et al 1990 Paladino et al 2006 these data indicate that TIVAMP is involved in direct apical sorting of TM and GPI proteins in different cells In order to test whether TIVAMP is also involved in the transcytotic apical pathway we analyzed the effect of its knockdown on the sorting of DPPIV which in Cac02 cells transcytoses through the basolateral surface before being inserted in the apical membrane Gilbert et al 1991 Matter et al 1990 Surprisingly in contrast to the results obtained in FRT cells Fig 2 we found no effect of TIVAMP knockdown on the apical localization of DPPIV Fig 5C and supplementary material Fig S2B The fact that TIVAMP knockdown has such a different effect on the same protein in two different cell lines indicates that this effect is dependent upon the pathway followed by DPPIV in the two cell lines Gilbert et al 1991 Zurzolo et al 1992a Thus our data strongly suggest that the apical vSNARE TIVAMP is involved in direct apical sorting of TM and GPIAP proteins independently of their raft association but does not function in the apical transcytotic pathway VAMPB is involved in the transcytotic pathway of DPPIV in CacoZ cells The vSNAREs that control the transcytotic pathway have not been identified In order to understand the mechanisms that control the transcytosis of DPPIV in Cac02 cells we focused our studies on two possible vSNARE candidates VAMP3 and VAMPS that have been described as being involved in endosomal recycling Advani et al 1998 Breton et al 2000 Galli et al 1994 Wong et al 1998 We reasoned that if one of these two vSNAREs functioned in apical transcytosis the localization of the vSNARE should be disturbed by the TI VAMP knockdown because of the expected crosstalk between the direct and indirect apical pathways Interestingly we observed that VAMP3 localization was not affected in TI VAMP knockdown of Cac02 cells Fig 6A left panel whereas VAMP8 partially accumulated basolaterally and was less concentrated in intracellular vesicles and on the apical surface Fig 6A right panel Quantitative analysis of fluorescence by applying a threshold in order to select intracellular objects of a specific level of intensity allowed calculation using the Image software of the number of a o C 9 o 039 E O 0 Tu C L 3 0 j Role of vSNARES in apical delivery 3315 A siRNA beta glob Fig 6 VAMP8 but not VAMP3 is mislocalized in CacoZ cells after transient RNAi of TIVAMP CacoZ cells were electroporated with 60 pmol of siRNA targeting human TlVAMP or against human Sglobin and grown on lters for 4 days A Confocal Z and X Y sections top and bottom of control siRNA beta glob and TI VAMP transientknockdown siRNA TI VAMP CacoZ cells Cells were labeled with antibodies against human VAMP3 left panel and VAMP8 right panel and secondary antibodies coupled to FITC Note that the lateral localization of VAMP3 is unaffected by the transient knockdown of TlVAMP Compared with its vesicular localization in control CacoZ cells VAMP8 seems to be less concentrated in vesicular structures and slightly mislocalized to the lateral membrane in CacoZ TIVAMP transientknockdown cells B Digitized images of confocal X Y sections of control siRNA beta glob and TlVAMP siRNA TI VAMP CacoZ cells Cells were labeled with antibody against human VAMP8 and secondary antibody coupled to FITC Fluorescent images were converted and analyzed quantitatively using Image software see Materials and Methods A statistical analysis of the area of each uorescent object was performed data not shown We found fewer objects in the case of TlVAMP knockdown compared with the control 130 versus 164 in the control We also found that there were more objects of small area less than ve pixels and fewer objects of large area greater than ten pixels in the case of the TlVAMP knockdown Bar 10 um objects and the areas occupied by each object Fig 6B This analysis showed that there are fewer and much smaller obj ects in the TI VAMP knockdown condition compared with the control Fig 6B Together with the more prominent surface staining of VAMP8 Fig 6A these experiments show that the localization of VAMP8 is altered and that it is less concentrated in vesicular structures and more at the lateral membrane in TI VAMP knockdown cells thus indicating an involvement of VAMP8 in apical sorting We therefore decided to study the effects of VAMP8 knockdown on the direct and indirect apical pathways that are followed by PLAP and DPPIV To interfere with VAMP8 expression we used a siRNA targeting human VAMP8 HsiVAMPSj HPvalidated siRNA 8102653245 from Qiagen that was transiently transfected in Caco2 cells VAMP8 expression levels were measured after 4 days of culture on filters by western blotting on total cell lysates Fig 7A By using a specific rabbit antibody against human VAMP8 in western blots we found a marked decrease gt90 of the signal corresponding to VAMP8 in knockdown cells compared with control cells transfected with human B globin siRNA Fig 7A We then investigated the effect of this knockdown on both the direct delivery of PLAP and the indirect delivery of DPPIV in filtergrown Caco2 cells Fig 7BC Interestingly while both the direct apical delivery of PLAP and direct basolateral delivery of Ag525 were not affected Fig 7B DPPIV transcytosis to the apical membrane was strongly VAMP 3 siRNA TI VAMP siRNA beta glob SiRNA TIVAMP VAMP 8 siRNA beta glob siRNA TlVAMP VAMP 8 perturbed and the protein became mislocalized to the basolateral surface Fig 7C and supplementary material Fig S2B In order to analyze the mechanism of action of VAMP8 in more detail we performed an immunofluorescence based assay to follow DPPIV internalization from the basolateral to the apical plasma membrane see supplementary material Fig S4 After adding the antibody against DPPIV from the basolateral side of cells grown on filters for 4 days we followed its internalization at different times We first labeled surface DPPIV nonpermeabilized conditions using a secondary antibody coupled to fluorescein isothiocyanate FITC After extensive washing and quenching we added a secondary antibody conjugated with TRITC in permeabilized conditions to label internal DPPIV In control conditions the basolateral surface staining of DPPIV disappeared very quickly with time followed by intracellular staining at ten minutes consistent with a transcytosis of DPPIV from the basolateral towards the apical membrane which was stained after 30 minutes see supplementary material Fig S4B In VAMP8 transientknockdown experimenm the basolateral staining of DPPIV remained unchanged with time and no signal appeared intracellularly 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 of DPPIV from the basolateral to the apical surface In summary these data indicate that VAMPS 1 does not cooperate with TI VAMP to effect direct apical delivery 2 is a o C 9 o 039 E O 0 Tu C L 3 0 j 3316 Journal of Cell Science 120 18 not responsible for the basolateral insertion of directly sorted basolateral proteins and 3 is a major player in the transcytotic apical pathway in polarized epithelial cells Discussion Virtually every intracellular membrane fusion event is mediated by a SNARE machinery Mostov et al 2003 Weimbs et al 1997 Although it has been demonstrated that only a perfect match between the vSNARE and its corresponding tSNARE leads to successful membrane fusion McNew et al 2000 Scales et al 2000 it is still debated how SNAREs contribute to specify trafficking and at which step ie targeting or fusion they regulate these events Studies of SNARE function in epithelial cells have shown a polarized distribution of tSNAREs highly conserved among epithelial cell types and support their role in the establishment and maintenance of epithelial polarity Li et al 2002 Low et al 1996 Low et al 1998 Low et al 2002 The tSNARE Stx3 localizes to the apical plasma membrane of MDCK cells Low et al 1996 and both Stx3 and the vSNARE TlVAMP operate at the apical plasma membrane of Caco2 cells Breuza et al 1999 Galli et al 1998 Overexpression of Stx3 reduces A siRNA siRNA beta glob VAMP8 63 KDa 39 Calreticulin 5 10 KDa VAMP8 B siRNA beta glob siRNA VAMP8 siRNA beta glob siRNA beta glob siRNA VAMP8 DF39PIV apical transport by a factor of 20 to 50 Low et al 1998 and does not cause mistargeting to the basolateral surface but instead results in the accumulation of apical proteins in intracellular vesicles Low et al 1998 consistent with the formation of nonproductive SNARE complexes that impair fusion However the apical pathway is rather complicated because it can be either raft dependent or raft independent and there are two ways to reach the apical surface one direct and the other indirect Paladino et al 2006 RodriguezBoulan et al 2005 Previous work has demonstrated the involvement of Stx3 in the apical sorting of two specific transmembrane proteins HA and SI both associated with DRMs that are directly sorted to the plasma membrane Breuza et al 2000 Lafont et al 1999 Low et al 1998 Consistent with these findings both apical v and tSNARE Tl VAMP and Stx3 are present in postTGN carriers in DRMs and Tl VAMP forms apical SNARE complexes with Stx3 Galli et al 1998 Although overexpression of Stx3 does not alter the transcytosis of WT plgR in MDCK cells Low et al 1998 the role of Tl VAMP in 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 whether raft and nonraftassociated apical proteins use the same exocytic mechanism at the apical surface Our data clearly demonstrate that TlVAMP is necessary for the apical localization of raft and nonraftassociated proteins implying a role for Tl VAMP in the direct apical delivery for both Fig 7 DPPIV but not PLAP is mislocalized in CacoZ cells after transient RNAi of VAMP8 CacoZ cells were electroporated with 60 pmol of siRNA targeting human VAMP8 or against human Sglobin and grown on lters for 4 days A Cell extracts 40 ug from control siRNA beta glob and VAMP8 transientknockdown siRNA VAMP8 cells were analyzedby SDSPAGE and western blotting with antibodies against human calreticulin top panel internal control for protein loading and human VAMP8 bottom panel VAMP8 expression decreased by more than 80 after transient RNAi of CacoZ cells on lters in three different experiments BC Confocal Z and X Y sections top and bottom of control siRNA beta glob and VAMP8 transient knockdown siRNA VAMP8 CacoZ cells B Cells were labeled with an antibody against PLAP and a secondary antibody coupled to FITC Compared with its apical localization in CacoZ cells left panel PLAP is unaffected by the transient knockdown of VAMP8 C Cells were labeled with antibodies against human DPPIV left panel and secondary antibodies coupled to FITC Note that compared with FRT cells the apical DPPIV is also mislocalized to the lateral membrane in CacoZ VAMP8 transient knockdown cells right panel whereas the basolateral localization of Ag525 is unaffectedB right panel Bars 10 um siRNA VAMP8 CD 0 c 1 o D E O 3 F c L 3 O Role of vSNARES in apical delivery 3317 pathways Figs 1 2 and 5 We also confirmed previous data that TI VAMP is not involved in basolateral delivery Figs 1 and 5 A quantitative and statistical analysis of these data was performed by means of a surface biotinylation assay in stable FRT clones lacking TI VAMP Fig 3 Furthermore by measuring the TER and studying the morphology of tight junctions we also showed that mislocalization of apical non raft and raftassociated 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 TIVAMP dependent apical targeu39ng pathway is not involved in the formation of tight junctions and is not essential for the development of polarized epithelia Next in order to analyse directly whether TI VAMP was also involved in the indirect pathway to the apical membrane we repeated the same knockdown experiments in Caco2 cells that unlike FRT cells target endogenous DPPIV to the apical surface via a transcytotic pathway Gilbert et al 1991 As control for the effect of TI VAMP on the direct pathway we used again PLAP which is endogenously expressed by Caco2 cells and is targeted directly to the apical membrane Le Bivic et al 1990 As in FRT cells TI VAMP knockdown impaired the direct apical delivery of PLAP however it had no effect on both the transcytotic delivery of DPPIV and as expected basolateral delivery Fig 5 These data clearly demonstrate that TI VAMP is involved only in the apical direct pathway and not in the apical indirect pathway and that this mechanism is conserved in different epithelia Interestingly by specific knockdown of TI VAMP we not only impaired the direct apical delivery but also found mislocalization of the apical proteins to the basolateral surface an effect that was not observed previously when the expression of TI VAMP was perturbed using either an overexpression or an antibody block approach Breuza et al 2000 Galli et al 1998 Low et al 1998 These data suggest that in the absence of TIVAMP apical carriers can fuse with the basolateral membrane This could be explained by two hypotheses either TIVAMP has a prominent function in the sorting of the proteins at the level of the TGN or another v SNARE allowing basolateral fusion is present on postTGN apical carriers and it is unmasked only after TIVAMP knockdown Because it is possible that this recessive VAMP would also be involved in transcytotic apical delivery we focused our attention on two vSNAREs that could be able to overcome TI VAMP loss in our knockdown experiments The first of these was VAMP3 also known as cellubrevin or Cb McMahon et al 1993 which is involved in the recycling of the transferrin T f receptor Galli et al 1994 and in early endosomal pathways such as the apical transport of H ATPase Breton et al 2000 and the other was the vSNARE VAMP8endobrevin Advani et al 1998 Wong et al 1998 which might mediate the endocytic apical recycling pathway in polarized cells Antonin et al 2000 Mullock et al 2000 Steegmaier et al 2000 Wong et al 1998 and colocalizes also with the Tf receptor Wong et al 1998 To test the possible involvement of these two vSNAREs in the apical pathway we examined their localization in TI VAMP knockdown in Caco2 cells Fig 6 Interestingly the localization of VAMP3 was not affected by TIVAMP knockdown Fig 6A left panel This is consistent with recent findings that VAMP3 coimmunoprecipitates mainly with Stx4 thus suggesting that it is not involved in the apical pathway but instead functions in the basolateral sorting of AP1Bdependent cargos as recently demonstrated Fields et al 2007 By contrast in TIVAMP knockdown cells VAMP8 appeared to be less concentrated in intracellular vesicular structures and localized more at the lateral membrane Fig 6A right panel and Fig 6B indicating that it might be involved in the apical pathway VAMP8 is the best candidate to be involved in the transcytotic pathway because it is able to interact both with the basolateral tSNARE Stx4 and with the apical t SNARE Stx3 It has been shown that VAMP8 and Stx3 can form complexes with SNAP23 Pombo et al 2003 Furthermore it has also been 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 major localization of VAMP8 is endosomal in fixed MDCK cells in live conditions it appears to be present in fine long tubular structures emanating from endosomes and to be present at the apical surface of the cell Wakabayashi et al 2007 This is consistent with the role of VAMP8 in sorting to the apical plasma membrane and because VAMP8 participates in endocytosis and apical recycling in MDCK cells Steegmaier et al 2000 but not in apical direct delivery Lafont et al 1999 all these data suggest that VAMP8 could operate in the apical transcytotic pathway To confirm this hypothesis we analysed the effect of VAMP8 knockdown on the direct and transcytotic apical delivery pathways in Caco2 cells Fig 7 As expected VAMP8 knockdown had no effect both on the direct apical delivery of PLAP and on the direct basolateral delivery of Ag525 Fig 7B However in these conditions the transcytotic apical delivery of DPPIV was remarkably affected and the protein was partially missorted to the basolateral surface Interestingly we found an increase in the total amount data not shown and of the plasma membrane fraction of DPPIV in in VAMPknockdown cells Fig 7 This could also be consistent with an impairment of the endocytic apical recycling pathway in VAMP8 knockdown However the fact that in VAMP8knockdown cells DPPIV remains on the basolateral surface supplementary material Fig S4 is consistent with a role in endocytosis from the basolateral surface to the apical membrane Overall our data indicate that VAMP8 is involved in the transcytotic pathway Furthermore because the localization of Ag525 was not impaired by VAMP8 knockdown Fig 7B we can also conclude that direct basolateral delivery and the transcytotic apical pathway use different sets of vSNAREs Fig 8 This hypothesis finds additional support from recent data showing the involvement of VAMP3 in the basolateral sorting of AP lBdependent cargos Fields et al 2007 Interestingly when we examined Stx3 and Stx4 localizations in TIVAMPknockdown Caco2 cells they were not perturbed compared with the control suggesting that tSNAREs and v SNAREs do not play the same role in apical sorting see supplementary material Fig S5 In conclusion our data reveal specificity in the sorting of apical cargos that could be explained with two different models Fig 8 We cannot exclude that each cargo vesicle is equipped with more then one vSNARE and the specificity is governed a o C 9 o 039 E O n 0 To C L 3 0 j 3318 Journal of Cell Science 120 18 39m39 rm VAMFB TerAMP r A VAMPC Rec ct39n Endosome y I g lllVAMP I TJ FLAP iDPPiv Ag525 iVAMPS iVAMPB ESyniaxin 4 nitrib VAMPS TVAMFl ESyniaxm 3 ISNAPZB Fig 8 Model for vSNARE speci city in the different pathways to the plasma membrane of polarized epithelial cells Upon sorting at the level of the TGN proteins are addressed to the apical or basolateral plasma membrane via at least three different pathways the direct apical pathway bold arrowhead the direct basolateral pathway dashed arrow and the transcytotic pathway open arrowhead PostTGN carriers could contain different vSNAREs eg TIVAMP VAMP8 VAMP3 In this case the speci city of fusion would arise from the pairing of each vSNARE with the appropriate tSNARE and from the stoichiometry of the vSNAREs present on each vesicle the predominant VAMP is indicated by underlining and alternatives are indicated by parentheses The speci city of sorting could be dependent on the functional interaction between a vSNARE and a speci c adaptor e g APl B for recycling endosomes to the basolateral plasma membrane in the direct basolateral pathway Alternatively each vSNARE might be actively sortedinto different postTGN cargos in this case only the SNARE indicated by underlining would be present in the vesicle T tightjunction by their relative amount and stoichiometry However our data support the hypothesis that there is a specific vSNARE for each postTGN cargo Fig 8 and that mistargetirig in the absence of one vSNARE arises from the loading of cargo on an alternative pathway Further work will be necessary to understand how vSNAREs and cargos are sorted together at the level of the Golgi apparatus and endosomes Materials and methods Reagents and antibodies Cell culture reagents were purchased fromInvitrogen Antibodies against PLAP and Z071 were purchased fromRockland Bioscience and Zymed The antibodies against rat 3540 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 Michel et al 2005 Quaroni and Isselbacher 1985 The antibody against TIVVAMP was used as described previously Muzerelle et al 2003 VAMP 3 VAMP 8 syntaxin 3 and syntaxin 4 were purchased from Transduction Laboratories Biotin and protein A sepharose were obtained from Pierce Chemical Co and Amersham Horseradish peroxidase HRPrlinked antibodies and streptavidin were purchased from Amersham GE Healthcare All other reagents were purchased from SigmarAldrich Cell culture and transfections FRT cells AmbesirImpiombato and Coon 1979 were grown in F12 Coon s modified medium containing 5 fetal bovine serum FBS A stable clone expressing PLAP was previously obtained Lipardi et al 2000 Cells were transiently transfected using the GeneRilserXCell BiorRad by mixing 100 pmol siRNA with 400 pl RPMI medium and 107 freshly tQpsinized cells Cells 2X105 were seeded on Transwell filters 24 mm diameter Corning NY and TER was measured each day with a MilliCell apparatus Millipore Corporation Bedford MA Cells were stably transfected using Lipofectin Invitrogen by mixing 375 ug DNA with 10 ug Lipofectin and 3 ml Optimem and selected in hygromycin 250 rigml andor G418 10 rigml for two weeks A clone of Caco2 TC7 cells was grown in DMEM containing 10 FBS Chantret et al 1994 Cells were transfected using the Amaxa device T solution and T20 program Amaxa Biosystems Germany by mixing 60 pmol siRNA with 100 pl buffer T and 18 gtlt106 freshly tQpsinized cells Cells Caco2 l8gtltlO6 and FRT 2gtlt105 were seeded on Transwell lters 24 mm diameter Corning and TER was measured with a MilliCell apparatus Millipore Corporation RNA interference Three siRNAs targeting TIVVAMP were designed one against rat 5 7 CCTCGTAGATTCGTCCGTCV3 and two against human 5 VTGCCATV TAAATI39GAAATTATAVT and 5 rCTGCCAAGACAGGATTGTATAS DNA corresponding were introduced as a hairpin behind a U6 promoter added to peGFPr N2 BD Bioscience Clontech Palo Alto CA containing a neomycinrresistance cassette using the HindIII and BglII restriction sites a kind gift from A Le Bivic Lurniny Marseilles France and sequenced The siRNA targeting rglobin bgloEl 5 VGGUGAAUGUGGAAGAAGUUttS was a kind gift from N Sauvonnet Institut Pasteur Paris France The siRNA targeting VAMP8 HsiVAMP873 HPV validated siRNA SIO2653245 was purchased from Qiagen Western blotting FRT cell extracts were prepared and analysed by western blotting as previously described Lipardi et al 2000 with primany antibodies and the corresponding secondany antibodies coupled to peroxidase Amersham GE Healthcare FRT cells were selectively biotinylated and processed as described Paladino et al 2006 Lysate were immunoprecipitated with specific antibodies and biotinylated antigens were revealed with streptavidin Caco2 cell extracts were prepared and analyzed by western blotting as described Lemmers et al 2002 with primany antibodies and the corresponding secondany antibodies coupled to peroxidase Amersham GE Healthcare Samples were run on SDSVPAGE and bands were revealed using the ECL kit Amersham GE Healthcare and quantified using the Image NIH so ware Fluorescence microscopy FRT and Caco2 cells grown on Transwell filters for 4 days were washed with PBS containing CaClz and MgClz xed with 4 PFA and quenched with 50 mM NHACI Cells were permeabilized with 0075 saponin Primany antibodies were detected with FITC or TRITCrconjugated secondany antibodies Images were collected using a laser scanning confocal microscope LSM 510 Carl Zeiss MicroImaging Inc equipped with a plan apo 63gtlt NArl 4 oilrimmersion objective lens Carl Zeiss MicroImaging Inc Fluorescent images were converted using ImageJ software to 8 bit 256 grey levels and a threshold selection was applied between 80 and 255 grey levels top panels red Measurements of the area of the uorescent objects was obtained using ImageJ software bottom panels white ellipses in Fig 6B We are grateful to Lucien Cabani for the puri cation of the mAb C11582 hybridoma and TG18 rabbit serum and to Philippe Casanova for technical help We are grateful to Veronique ProuxGillardeaux and Lydia Danglot for helpful discussion of the data This work was supported by Grants to C1 from MURST PRIN 2005 from the European Union 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