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Human syntaxin 3 is localized apically in human intestinal cells
Abstract
To understand the molecular mechanisms underlying sorting of apical and basolateral membrane components in human intestinal epithelial cells, we have cloned the human homolog of rat syntaxin 3 and looked for its subcellular localization. Endogenous human syntaxin 3 was found to be localized at the apical membrane of colon epithelial and Caco-2 cells. This apical localization was confirmed by confocal microscopy after transfection of the cDNA coding for either full length or N-terminally truncated human syntaxin 3 in Caco-2 cells. Furthermore the signal(s) and machinery targeting human syntaxin 3 to the apical membrane of epithelial cells are conserved between species since human syntaxin 3 was also localized at the apical membrane of canine MDCK cells and of epithelial cells in transgenic Drosophila melanogaster.
... The diversity of syntaxins and their cognate SNARE binding partners are thought to contribute to the overall fidelity and specificity of membrane trafficking (Jahn and Scheller, 2006;Rodriguez-Boulan et al., 2005). Others and we have previously reported that Syntaxin 3 (Stx3) localizes to the apical plasma membrane in a wide variety of polarized epithelial cells (Delgrossi et al., 1997;Li et al., 2002;Low et al., 1996;Low et al., 1998;Low et al., 2006;Weimbs et al., 1997a). Apical targeting of Stx3 is governed by an apical targeting signal in its N-terminal domain, and its mutation causes mislocalization of Stx3, improper trafficking of apical membrane proteins, and cell polarity defects ter Beest et al., 2005). ...
... In addition to the apical plasma membrane, a fraction of Stx3 also localizes to late endosomal/lysosomal compartments (Delgrossi et al., 1997;Low et al., 1996). It is unknown how Stx3 traffics to these organelles or what its function there may be. ...
... While the bulk of Stx3 resides on the apical plasma membrane in polarized epithelial cells, a portion of Stx3 also localizes to a late endosomal population (Delgrossi et al., 1997;Low et al., 1996). We hypothesized that ubiquitination may be involved in the targeting of endosomal Stx3. ...
Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes / lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono - ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral – but not the apical - plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non - ubiquitinatable mutant of Stx3 (Stx3 - 5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV / exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3 - 5R mutant acts as a dominant - negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3 - 5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.
... The diversity of syntaxins and their cognate SNARE binding partners are thought to contribute to the overall fidelity and specificity of membrane trafficking (Rodriguez-Boulan et al., 2005;Jahn and Scheller, 2006). Others and we have previously reported that syntaxin 3 (Stx3) localizes to the apical plasma membrane in a wide variety of polarized epithelial cells (Low et al., 1996Delgrossi et al., 1997;Weimbs et al., 1997a;Li et al., 2002). Apical targeting of Stx3 is governed by an apical targeting signal in its N-terminal domain, and its mutation causes mislocalization of Stx3, improper trafficking of apical membrane proteins, and cell polarity defects (ter Beest et al., 2005;Sharma et al., 2006). ...
... Although the bulk of Stx3 resides on the apical plasma membrane in polarized epithelial cells, a portion of Stx3 also localizes to a late endosomal population (Low et al., 1996;Delgrossi et al., 1997). We hypothesized that ubiquitination may be involved in the targeting of endosomal Stx3. ...
... Monoubiquitinated Stx3 can be detected as a band with an increased molecular weight of ∼9 kDa ( Figure 1B). Using Madin-Darby canine kidney In addition to the apical plasma membrane, a fraction of Stx3 also localizes to late endosomal/lysosomal compartments (Low et al., 1996;Delgrossi et al., 1997). It is unknown how Stx3 traffics to these organelles or what its function there may be. ...
Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono-ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral - but not the apical - plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non-ubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.
... Consistent with these data, Caco-2 cells under the conditions of our study did not show expression of STX1A (data not shown). Syntaxin 3 (STX3), the exocytic protein involved in vesicle fusion has been shown to be abundantly expressed apically in IECs [26]. To examine whether STX3 interacts with SERT, co-IP studies were performed. ...
... STX1A expression, however, is detected to a much lesser extent than brain in intestinal epithelial cells such as in colonic HT-29-CL109 cells [42] and is not detected in Caco-2 cells [23]. In contrast to STX1A, STX3 has been shown to be abundantly expressed in IECs [23] and plays important role in membrane polarity, apical targeting, recycling and vesicle fusion in the intestine [26]. Syntaxin 3 protein has been precisely localized to the apical cell surface in epithelial cells such as MDCK and Caco2 cells [26]. ...
... In contrast to STX1A, STX3 has been shown to be abundantly expressed in IECs [23] and plays important role in membrane polarity, apical targeting, recycling and vesicle fusion in the intestine [26]. Syntaxin 3 protein has been precisely localized to the apical cell surface in epithelial cells such as MDCK and Caco2 cells [26]. Previous studies have implicated STX3 in regulated exocytosis of CFTR [43] and in cAMP induced acid secretion [44]. ...
TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer's or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM) attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders.
... SNAP-23 binds to syntaxins 3 and 4 in vivo (Galli et al., 1998; St-Denis et al., 1999) and is involved in biosynthetic and endocytic recycling and transcytotic pathways to both plasma membrane domains in MDCK cells (Leung et al., 1998; Low et al., 1998a). The subcellular localization of these SNAREs is generally very similar in other epithelial cell lines and tissues, although variations have been reported (Gaisano et al., 1996; Delgrossi et al., 1997; Weimbs et al., 1997a; Fujita et al., 1998; Galli et al., 1998; Riento et al., 1998). Temporary or permanent loss of cell polarity is a common phenomenon during the development of epithelial tissues (Sorokin and Ekblom, 1992; Birchmeier et al., 1996) as well as in a number of pathological conditions (Louvard et al., 1992; Fish and Molitoris, 1994; Birchmeier et al., 1996). ...
... Syntaxins 2 and 11 as well as SNAP-23 are found at both the basolateral and the apical plasma membrane in addition to some remaining intracellular labeling. Syntaxin 3 is absent from the basolateral domain but localizes to the apical domain in addition to intracellular lysosomes, as established previously (Low et al., 1996; Delgrossi et al., 1997). Syntaxin 4, in turn, is absent from the apical domain but has partially relocalized to the basolateral domain. ...
... The human colon carcinoma cell line Caco-2 was grown in LCM as described above and stained for endogenously expressed syntaxin 3 and the microvillar protein villin. While syntaxin 3 and villin are localized at the apical plasma membrane in fully polarized Caco-2 cells (Delgrossi et al., 1997; Galli et al., 1998; Riento et al., 1998; our unpublished results), they are strongly enriched in VACs in nonpolarized cells (Figure 5). This result indicates that the localization of syntaxin 3 in VACs is a general phenomenon of nonpolarized epithelial cells and not an artifact of syntaxin overexpression.Figure 3. ...
In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.
... This cell type has been selected for two main reasons. First, polarized epithelial cells have been extensively used to characterize the sorting mechanisms of many plasma membrane proteins (including the syntaxins; Low et al., 1996;Delgrossi et al., 1997;Galli et al., 1998;Lafont et al., 1999;Riento et al., 1998;Riento et al., 2000) and to investigate the targeting of neuronal plasma membrane proteins, since their apical and basolateral domains are thought to parallel the axonal and dendritic compartments of neurons (Winckler and Mellman, 1999;Mostov et al., 2000). Secondly, the neuronal specific t-SNARE syn1A is also expressed in certain epithelial cells (e.g. ...
... In order to analyze the expression levels of the various exogenous proteins, total cell extracts from transfected or non-transfected MDCK cells and, as a control, rat brain were separated by SDS-PAGE and analyzed by immunoblotting (Fig. 2). Syn3 is endogenously expressed in wild type MDCK and Caco-2 cells (Fig. 2, lane 1; see also Low et al., 1996;Delgrossi et al., 1997;Galli et al., 1998) and its level in transfected cells increased about six times in comparison with the untransfected cells. After cDNA electroporation, a band corresponding to syn1A was clearly detected in the cell extracts (Fig. 2, lane 3). ...
... Under these conditions, and in the presence of munc-18-1, syn1A was found at both the basolateral and apical cell surfaces of polarized cells as observed in the horizontal sections collected at different heights throughout the cells (Fig. 5, Fig. 6A,B) and vertical sections (Fig. 6C). In contrast, the endogenous syn3 was mainly found at the apical domain of Caco-2 cells (Fig. 6D) as previously described (Delgrossi et al., 1997;Galli et al., 1998). ...
SNARE (Soluble N-ethyl-maleimide sensitive factor Attachment protein Receptor) proteins assemble in tight core complexes, which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and are distributed in distinct subcellular compartments. The molecular mechanisms underlying sorting of SNAREs to their physiological sites of action are still poorly understood. Here have we analyzed the transport of syntaxin1A in epithelial cells. In line with previous data we found that syntaxin1A is not transported to the plasma membrane, but rather is retained intracellularly when overexpressed in MDCK and Caco-2 cells. Its delivery to the cell surface is recovered after munc-18-1 cotransfection. Furthermore, overexpression of the ubiquitous isoform of munc-18, munc-18-2, is also capable of rescuing the transport of the t-SNARE. The interaction between syntaxin 1A and munc-18 occurs in the biosynthetic pathway and is required to promote the exit of the t-SNARE from the Golgi complex. This enabled us to investigate the targeting of syntaxin1A in polarized cells. Confocal analysis of polarized monolayers demonstrates that syntaxin1A is delivered to both the apical and basolateral domains independently of the munc-18 proteins used in the cotranfection experiments. In search of the mechanisms underlying syntaxin 1A sorting to the cell surface, we found that a portion of the protein is included in non-ionic detergent insoluble complexes. Our results indicate that the munc-18 proteins represent limiting but essential factors in the transport of syntaxin1A from the Golgi complex to the epithelial cell surface. They also suggest the presence of codominant apical and basolateral sorting signals in the syntaxin1A sequence.
... The net result of these molecular interactions is an approximately 75% reduction in the turn-over rate of GAT1, while surface expression is increased at the same time (33). Syntaxin 1A paralogs are also involved in membrane protein regulation in epithelial cells (50,51) and syntaxin isoforms in kidney and small intestinal epithelium localise in a manner which suggests a potential role in apical and baso-lateral cell polarity (52,53). Although it is often inferred from evidence of interactions with SNARE proteins that collectrin may mediate vesicular fusion events and formation of SNARE complexes, no direct evidence for this has been shown (23,54,55). ...
... While our data suggest competition for a common binding site between collectrin and syntaxin-1A, the cellular expression of both proteins does not overlap (38). Database searches comparing tissue-specific E.S.T. expression data (http://www.ncbi.nlm.nih.gov/unigene) and literature searches (52,53), however, identified several syntaxin isoforms (3, 4, 7 and 17) with overlapping expression to B 0 AT1 and/or collectrin. Co-expression experiments showed strong inhibition of B 0 AT1 + collectrin activity by syntaxin 3, while syntaxin 7 had no effect (Fig. 6E). ...
Many Solute Carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B(0)AT1 and B(0)AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters is modulated by interaction with syntaxin 1A. Utilising monocarboxylate MCT-B(0)AT1/3 fusion constructs we discovered that collectrin is also necessary for B(0)AT1 and B(0)AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B(0)AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5 and 7 on one face of B(0)AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions: plasma membrane expression of B(0)AT3, catalytic activation or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... This led the authors to propose a mechanism of membrane fusion at the apical plasma membrane of epithelial cells independent of v-and t-SNAREs, although they had found HA transport to be sensitive to NEM (Simons and Ikonen, 1997). On the contrary, several groups have localized syntaxin isoforms at the apical plasma membrane of epithelial cells (Gaisano et al., 1996; Low et al., 1996; Mandon et al., 1996; Delgrossi et al., 1997) and proposed a role for these SNAREs in apical docking and fusion (Weimbs et al., 1997). We have searched for a TeNT-insensitive v-SNARE that could be a partner of apical t-SNAREs in epithelial cells. ...
... In the case of syntaxin 3, we were unable to detect any intracellular or basolateral plasma membrane staining either by confocal microscopy or by immunoelectron microscopy on fully polarized and differentiated cells. This is in contrast to two studies on MDCK (Low et al., 1996) and CaCo-2 cells (Delgrossi et al., 1997) overexpressing syntaxin 3 in which significant intracellular labeling was detected , including lysosome labeling (Low et al., 1996). In our case, we observed syntaxin 3-positive vesicular structures only in CaCo-2 cells that were not confluent and not yet fully polarized (our unpublished observations ). ...
The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.
... Localization of Syn3TfR was performed by double labeling with apical and basolateral markers in transfected cells and confocal microscopy analysis (Fig. 2). Syn3TfR labeled with anti-Syn3 antibody colocalized with SI (Fig. 2C), suggesting that it behaved like endogenous Syn3, which we localized at the apical membrane in a previous study (4). As for transfected Syn3 ( Fig. 2A), we could observe some subapical staining in Syn3TfR-transfected cells. ...
... These data raise the possibility that syntaxins may control the expression of a subset of plasma membrane proteins with a tight physiological regulation of their polarized expression in a tissue-specific manner. In our case, we showed that Syn3 is localized apically both in Caco-2 and in normal colonic cells in vivo (4). ...
To follow the transport of human syntaxin (Syn) 3 to the apical surface of intestinal cells, we produced and expressed in Caco-2 cells a chimera made of the entire Syn3 coding sequence and the extracellular domain of the human transferrin receptor (TfR). This chimera (Syn3TfR) was localized to the apical membrane and was transported along the direct apical pathway, suggesting that this is also the case for endogenous Syn3. To test the potential role of Syn3 in apical transport, we overexpressed it in Caco-2 cells and measured the efficiency of apical and basolateral delivery of several endogenous markers. We observed a strong inhibition of apical delivery of sucrase-isomaltase (SI), an apical transmembrane protein, and of alpha-glucosidase, an apically secreted protein. No effect was observed on the basolateral delivery of Ag525, a basolateral antigen, strongly suggesting that Syn3 is necessary for efficient delivery of proteins to the apical surface of intestinal cells.
... Reporter constructs bearing the tails of syntaxin 3 and 4 are sorted differently in MDCK cells From the results described above, no differences in post-Golgi sorting between the 22-and 25-residue TMD TA constructs were detected, and apical delivery was induced only by addition of an artificial glycosylated lumenal tag. However, some TA proteins, without lumenal domains, do have a polarised surface distribution in epithelial cells (Low et al., 1996;Delgrossi et al., Journal of Cell Science 115 (8) 1997). To investigate whether the TMDs of these endogenous TA proteins are involved in apical/basolateral sorting, we produced fusion constructs in which GFP was attached to the tail region of syntaxin 3 or 4, two TA target-Soluble Nethylmaleimide sensitive factor Attachment protein Receptors (t-SNAREs), which are sorted respectively to the apical and basolateral domain in MDCK cells (Low et al., 1996). ...
... Sorting of GFP-syntaxin tail fusion proteins in polarised epithelial cells Syntaxins are t-SNAREs that play a central role in recognition and fusion of vesicles and target membrane (for a review, see Chen and Scheller, 2001). In polarised epithelial cells, syntaxins 3 and 4 are enriched at the apical and basolateral membrane, respectively (Low et al., 1996;Fujita et al., 1998;Delgrossi et al., 1997); this different localisation is thought to contribute to the specificity of vesicle fusion with these surface domains (Galli et al., 1998). However, the mechanism of specific targeting of the two syntaxin isoforms themselves is at present poorly understood. ...
Tail-anchored (TA) proteins, which are defined by an N-terminal cytosolic region and a C-terminal transmembrane domain (TMD), provide useful models for studying the role of the TMD in sorting within the exo-endocytic system. Previous work has shown that a short TMD is required to keep ER-resident TA proteins from escaping to downstream compartments of the secretory pathway. To investigate the role of the TMD in TA protein sorting, we used model constructs, which consisted of GFP linked at its C-terminus to the tail region of cytochrome b(5) with TMDs of differing length or hydrophobicity. Expression of these constructs in CV-1 cells demonstrated that the feature determining exit from the ER is hydrophobicity and that if exit occurs, at least a part of the protein reaches the cell surface. To investigate which pathway to the surface is followed by plasma-membrane-directed TA constructs, we expressed the TA constructs in polarised Madin Darby Canine Kidney (MDCK) cells. The constructs with 22 and 25 residue TMDs were localised basolaterally, but addition at the C-terminus of a 20-residue peptide containing an N-glycosylation site resulted in glycosylation-dependent relocation of approximately 50% of the protein to the apical surface. This result suggests that TA proteins may reach the basolateral surface without a signal or that our constructs contain a weak basolateral determinant that is recessive to the apical information carried by the glycan. To assess the effect of the TMDs of endogenous TA proteins, GFP was linked to the tails of syntaxin 3 and 4, which localise to the apical and basolateral surface, respectively, of MDCK cells. The two GFP fusion proteins showed a different surface distribution, which is consistent with a role for the two syntaxin TMDs in polarised sorting.
... In this context, it is interesting to note that syntaxin 3B shares a greater (67%) sequence homology with syntaxin 1A than does syntaxin 3A [10,12], and both syntaxin 1A and syntaxin 3B form functional SNARE complexes with SNAP-25 and VAMP2 [5, 10,14]. By contrast, syntaxin 3A is expressed in polarized epithelial cells and non-neuronal secretory cells throughout the body, including in the gastrointestinal, urinary, and immune systems, where syntaxin 3A is typically involved in basal to apical trafficking and regulated secretion [15][16][17][18]. In addition, syntaxin 3 has been postulated to be present in the postsynaptic spines of conventional synapses and to have an important role in some types of synaptic There are also two minor isoforms of syntaxin 3 whose roles and localization are not well elucidated. ...
Syntaxin 3 is a member of a large protein family of syntaxin proteins that mediate fusion between vesicles and their target membranes. Mutations in the ubiquitously expressed syntaxin 3A splice form give rise to a serious gastrointestinal disorder in humans called microvillus inclusion disorder, while mutations that additionally involve syntaxin 3B, a splice form that is expressed primarily in retinal photoreceptors and bipolar cells, additionally give rise to an early onset severe retinal dystrophy. In this review, we discuss recent studies elucidating the roles of syntaxin 3B and the regulation of syntaxin 3B functionality in membrane fusion and neurotransmitter release in the vertebrate retina.
... The delay in patients from our case series is mild and might be largely attributable to the extensive hospitalizations, immobility caused by dependency on parenteral nutrition and severe visual impairment. Our examination of human brain samples did not indicate significant levels of STX3 expression overall, which is consistent with previous studies that have found the lowest level of STX3 expression in brain compared to other tissues in humans and rodents (Bennett et al. 1993;Curtis et al. 2008;Delgrossi et al. 1997). However, we cannot exclude the possibility that STX3 may be expressed in a small, specific subset of human brain cells, where it may impact human development or cognition. ...
Biallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.
... The human genome encodes at least 16 syntaxins that localize to their specific membrane domains or organelles to mediate membrane fusion reactions (Hong, 2005). For example, Stx1A and Stx1B are involved in synaptic vesicle fusion during neurotransmitter release (Sudhof, 2014) while Stx3 localizes to the apical plasma membrane domains of polarized epithelial cells (Delgrossi et al., 1997;Low et al., 1996;Low et al., 2002), functions in polarized trafficking pathways and is essential to the correct establishment of cell polarity (Kreitzer et al., 2003;Low et al., 1998;Sharma et al., 2006;Weimbs et al., 1997a). ...
Syntaxins - a conserved family of SNARE proteins - contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that syntaxin 3 (Stx3) unexpectedly also functions as a nuclear regulator of gene expression. Alternative splicing leads to a soluble isoform, termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5, targets to the nucleus and interacts physically and functionally with several transcription factors, including ETV4 and ATF2. Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer vs. normal breast tissue. Inhibition of endogenous Stx3S expression leads to changes in the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified suggesting that nuclear signaling is a conserved, novel function common among these membrane trafficking proteins.
... STX3 is a plasma membrane-localized SNARE (Delgrossi et al., 1997) with a myriad of secretory functions (Brochetta et al., 2014;Frank et al., 2011;D. Zhu et al., 2013). ...
Members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family mediate membrane fusion processes associated with vesicular trafficking and autophagy. SNAREs mediate core membrane fusion processes essential for all cells, but some SNAREs serve cell/tissue type-specific exocytic/endocytic functions, and are therefore critical for various aspects of embryonic development. Mutations or variants of their encoding genes could give rise to developmental disorders, such as those affecting the nervous system and immune system in humans. Mutations to components in the canonical synaptic vesicle fusion SNARE complex (VAMP2, STX1A/B, and SNAP25) and a key regulator of SNARE complex formation MUNC18-1, produce variant phenotypes of autism, intellectual disability, movement disorders, and epilepsy. STX11 and MUNC18-2 mutations underlie 2 subtypes of familial hemophagocytic lymphohistiocytosis. STX3 mutations contribute to variant microvillus inclusion disease. Chromosomal microdeletions involving STX16 play a role in pseudohypoparathyroidism type IB associated with abnormal imprinting of the GNAS complex locus. In this short review, I discuss these and other SNARE gene mutations and variants that are known to be associated with a variety developmental disorders, with a focus on their underlying cellular and molecular pathological basis deciphered through disease modeling. Possible pathogenic potentials of other SNAREs whose variants could be disease predisposing are also speculated upon.
... Stx-1A is sensitive to botulinium neurotoxin C (BoNT/C) evidenced by completel abolishment of insulin release from secretory granules in ß-cells upon application of BoNT/C ( Figure 4) (Land et al., 1997). Stx-2 and 3 were present in polarized epithelial cells specifically localized to the apical membrane (Delgrossi et al., 1997;Fujita et al., 1998), while Stx-4 was found to localize at the apical membrane of the renal collecting duct cells (Mandon et al., 1996). Other Stx family members are possibly playing role in the early stages of the secretory pathway such as endoplasmic reticulum (ER)-Golgi transport, or endosomal transport (Prekeris et al., 1999). ...
... Mice that are deficient in both Rab8a and Rab11a show a severe enteropathy and extensive formation of basolateral microvilli (Feng et al., 2017). Syntaxin 3 is also known to localize apically in epithelial cells where it facilitates fusion of vesicles to the plasma membrane (Delgrossi et al., 1997;Sharma et al., 2006). In summary, appropriate apical morphology and physiology requires an intimate relationship between cargo trafficking, recycling and local assembly of delivered cargo into a functional membrane domain. ...
Cells of transporting epithelia are characterized by the presence of abundant F-actin-based microvilli on their apical surfaces. Likewise, auditory hair cells have highly reproducible rows of apical stereocilia (giant microvilli) that convert mechanical sound into an electrical signal. Analysis of mutations in deaf patients has highlighted the critical components of tip links between stereocilia, and related structures that contribute to the organization of microvilli on epithelial cells have been found. Ezrin/radixin/moesin (ERM) proteins, which are activated by phosphorylation, provide a critical link between the plasma membrane and underlying actin cytoskeleton in surface structures. Here, we outline recent insights into how microvilli and stereocilia are built, and the roles of tip links. Furthermore, we highlight how ezrin is locally regulated by phosphorylation, and that this is necessary to maintain polarity. Localized phosphorylation is achieved through an intricate coincidence detection mechanism that requires the membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and the apically localized ezrin kinase, lymphocyte-oriented kinase (LOK, also known as STK10) or Ste20-like kinase (SLK). We also discuss how ezrin-binding scaffolding proteins regulate microvilli and how, despite these significant advances, it remains to be discovered how the cell polarity program ultimately interfaces with these processes.
... The human genome encodes at least 16 syntaxins that localize to their specific membrane domains or organelles to mediate membrane fusion reactions (Hong, 2005). For example, Stx1A and Stx1B are involved in synaptic vesicle fusion during neurotransmitter release (Sudhof, 2014) while Stx3 localizes to the apical plasma membrane domains of polarized epithelial cells (Delgrossi et al., 1997;Li et al., 2002;Low et al., 1996;Low et al., 2002), functions in polarized trafficking pathways and is essential to the correct establishment of cell polarity Low et al., 1998;Sharma et al., 2006;Weimbs et al., 1997a). ...
Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that syntaxin 3 (Stx3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform, we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RAN-binding protein 5 (RanBP5), targets to the nucleus and interacts physically and functionally with several transcription factors, including ETS variant 4 (ETV4) and activating transcription factor 2 (ATF2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified suggesting that nuclear signaling is a conserved, novel function common among these membrane trafficking proteins.
... T-SNAREs consist of syntaxins and synaptosomal-associated proteins (SNAP), whereas v-SNAREs are a family of vesicleassociated membrane proteins (VAMPs). In IECs, vesicle tethering and fusion is controlled by the v-SNAREs synaptotagmin-like protein 4a (SLP4A) and vesicle-associated membrane protein 7 (VAMP7), in conjunction with RAB27A/RAB3/RAB8A/RAB11A proteins and the t-SNARE syntaxin-3 (STX3) (Breuza et al., 2000;Delgrossi et al., 1997;Galli et al., 1998;Pocard et al., 2007;Riento et al., 1998;Vogel et al., 2015;Wiegerinck et al., 2014). Furthermore, the Sec1-related protein syntaxin binding protein 2 (STXBP2), also known as Sec1/ Munc18-like protein (MUNC18-2), mediates binding of SLP4A with STX3 at the apical plasma membrane of IECs, where it regulates the accessibility of its SNARE partners (Riento et al., 1998;Vogel et al., 2017b). ...
The intestinal epithelium is a highly organized tissue. The establishment of epithelial cell polarity, with distinct apical and basolateral plasma membrane domains, is pivotal for both barrier formation and for the uptake and vectorial transport of nutrients. The establishment of cell polarity requires a specialized subcellular machinery to transport and recycle proteins to their appropriate location. In order to understand and treat polarity-associated diseases, it is necessary to understand epithelial cell-specific trafficking mechanisms. In this Review, we focus on cell polarity in the adult mammalian intestine. We discuss how intestinal epithelial polarity is established and maintained, and how disturbances in the trafficking machinery can lead to a polarity-associated disorder, microvillus inclusion disease (MVID). Furthermore, we discuss the recent developments in studying MVID, including the creation of genetically manipulated cell lines, mouse models and intestinal organoids, and their uses in basic and applied research.
... While our data suggest competition for a common binding site between collectrin and syntaxin-1A, the cellular expression of both proteins does not overlap [391]. Database searches comparing tissue-specific E.S.T. expression data (http://www.ncbi.nlm.nih.gov/unigene) and literature searches [545,546], however, identified several syntaxin isoforms (3, 4, 7 and 17) with overlapping expression to B 0 AT1 and/or collectrin. Co-expression experiments showed strong inhibition of B 0 AT1 + collectrin activity by syntaxin 3, while syntaxin 7 had no effect (Fig. 4.6F). ...
Neutral amino acids are essential for protein synthesis, energy homeostasis, and many vital biochemical pathways. As several neutral amino acids are essential and cannot be synthesised by humans in vivo, their uptake and reuptake by the body’s absorbing epithelial layers is of major importance for systemic amino acid homeostasis and human health. Two of the major pathways for the absorption of neutral amino acids in the small intestine and kidney are the Broad Neutral Amino acid Transporters 1 and 3 (B0AT1 and B0AT3). B0AT1 is of medical interest as an indirect regulator of blood glycemia via cellular amino acid starvation response pathways. Absence of B0AT1 results in improved glycemic control and other metabolic effects, revealing it as a potential pharmacological target to treat type II diabetes. Essential to understanding the role of B0AT1 as a global metabolic regulator is its requirement for plasma membrane expression with heteromeric protein partners: collectrin in the kidney and Angiotensin Converting Enzyme 2 (ACE2) in the small intestine. Both proteins are membrane-anchored by a single trans-membrane domain and are homologous. B0AT3 also requires collectrin or ACE2 for membrane expression. Beyond this requirement for plasma membrane expression, however, little is known about the underlying mechanisms of the interaction. This thesis demonstrates additional protein partners of B0AT1, namely Aminopeptidase N (APN), syntaxin 1A, and syntaxin 3. These proteins facilitate changes in the kinetic parameters of neutral amino acid transport, in addition to regulating membrane expression. Moreover, I demonstrate that collectrin is required for catalytic activation of both B0AT1 and B0AT3, while both APN and ACE2 increase B0AT1 and B0AT3 substrate affinity, respectively. ACE2 and APN also form large complexes with B0AT1 at the small intestine brush border membrane. A binding site in both transporters for collectrin is identified as lying in the hydrophobic pocket between TM 5 and 7 of the transporters. Using a mixture of experimental and bioinformatics tools, I was able to map the interacting domains of collectrin, which involve multiple regions of the protein, including the TM domain and large regions of the N-terminus. The ability of collectrin and B0AT1 orthologs to cross-react with each other and the homologous region of ACE2, suggests a highly conserved structure-function relationship between them. In conclusion, B0AT1 and B0AT3 form large, stable protein complexes with collectrin, ACE2 and other proteins, the binding site and function of which are highly conserved. The necessity of these interactions mean these complexes should be thought of as heteromultimeric transporter metabolons – multimeric protein units facilitating efficient neutral amino acid transport in epithelial cells. There is increasing evidence that the formation of stable heteromeric membrane transport complexes is a common theme underlying the function of many transporters initially studied in isolation. Understanding the physiology and structure-function relationships of transporters in their native multimeric states is fundamental to understanding relevant biological roles. Especially as membrane transporters are heavily over-represented as causative agents of human disease and as therapeutic drug targets. Targeting stable, intrinsic protein-protein interactions presents a still unexplored field of drug treatment in biomedical science.
... The SNARE protein Syntaxin 3 (Stx3) is known to establish apical-basolateral polarity in polarized epithelial cells (Low et al., 1996;Delgrossi et al., 1997;Weimbs et al., 1997;Low et al., 1998;Li et al., 2002;Low et al., 2006). Apical localization of Stx3 depends on a conserved targeting motif near the N-terminus of the protein (ter Beest et al., 2005;Sharma et al., 2006). ...
The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports,e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.
... Das subzelluläre Verteilungsmuster der SNARE-Proteine in den verschiedenen Epithelzellen ist sehr heterogen und läßt kein einheitliches Schema erkennen (Gaisano et al., 1996;Delgrossi et al., 1997;Fujita et al., 1998). Einige SNARE-Proteine wie das in Epithelzellen verbreitete SNAP23 (ein Homolog von SNAP25), kommt sowohl basolateral vor (Azinuszellen des Pankreas), aber auch in einer hauptsächlich apikalen Verteilung (MDCK-Zellen, Low et al., 1998). ...
Die in der vorliegenden Habilitationsschrift zusammengefaßten Publikationen stellen Untersuchungen zu zwei Themenschwerpunkten dar: 1. Verankerungsmechanismen von Membranproteinen der basolateralen und der apikalen Plasmamembrandomäne der Parietalzellen mit dem Membranzytoskelett und 2. die regulierte Fusion von zytoplasmatischen Vesikeln mit der apikalen Plasmamembran dieser Zellen. Die strukturell und molekular sehr unterschiedlich gestaltete apikale und basolaterale Membrandomäne der Parietalzellen sollte funktionell charakterisiert und die Mechanismen der Membranumbauvorgänge aufgeklärt werden, die nach Aktivierung der Zellen im apikalen Membrankompartiment ablaufen. Für die strukturelle Stabilität der basolateralen Domäne spielt wahrscheinlich die Verankerung von AE2 über das Verknüpfungsprotein Ankyrin mit dem Membranzytoskelett eine wichtige Rolle. Die apikale Membrandomäne der Parietalzellen kann in drei Kompartimente unterteilt werden. Die freie apikale Membran, die canalikuläre Membran und die Membranen der tubulären Vesikel. Entlang der freien apikalen und der canaliculären Plasmamembran kommen wie auf der basolateralen Seite die Zytoskelett-Proteine Actin und Spectrin vor. Nach unseren Untersuchungen könnte es während der Sekretionsphase zu einer temporären Verbindung von H+,K+-ATPase Molekülen mit dem Membranzytoskelett kommen. Diese Verbindung wird wahrscheinlich durch das Verknüpfungsprotein Ezrin vermittelt. Der Mechanismus des Fusionsvorgangs der tubulären Vesikel mit der canaliculären Membran war bisher nicht bekannt. In Parietalzellen konnten die neuronalen SNARE-Proteine Synaptobrevin 2, Syntaxin 1 und SNAP25 sowie das zur Familie der kleinen G-Proteine gehörende Protein Rab3A und die Regulatorproteine NSF und alpha/beta SNAP nachgewiesen werden. Das in Parietalzellen gefundene Verteilungsmuster der SNARE-Proteine entspricht nicht der klassischen Vorstellung einer heterotypischen Membranfusion, vielmehr entspricht diese Verteilung einer homotypischen Fusion, wie sie für Vakuolen in Hefezellen beschrieben wurde. Die Bedeutung der SNARE-Proteine für die Fusion der tubulären Vesikel mit der canaliculären Membran und damit für die Steigerung der HCl-Sekretion konnte durch Inkubation der Zellen mit Tetanus Neurotoxin (TeNt) gezeigt werden. Die Behandlung der Parietalzellen mit TeNt führte zum vollständigen Ausbleiben der, nach Stimulation mit cAMP bei Kontrollzellen beobachteten Erhöhung, der Säuresekretion
... Interestingly, some SNAREs have been shown to play a role in targeted trafficking [70,100] or even as morphogenic signals in various cell types [101]. Indeed, Stx2 and/or 3 are mainly localized at the APM in polarized epithelial cells, such as Caco-2, MDCK and acinar cells [73,102,103]. Strikingly, Stx2 was initially called epimorphin (EPM) because of its morphogenic properties. This membrane-anchored protein was first characterized as a stromal cell-surface molecule involved in embryonic epithelial morphogenesis [104]. ...
During lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones.
... Without exception, syntaxin 3 localizes to the apical and sjTitaxin 4 to the basolateral domain. SjTitaxin 3 has also previously been found to be apical specific in the Caco-2 colon epithelial cell line (6,12,17,54) and in hepatocytes (15). Syntaxin 4 has been foimd on the basolateral plasma membrane of pancreatic acinar cells (16). ...
The specific purposes of this project are to investigate how the expression and localization of membrane SNAREs in prostate carcinomas, and what the relationship to the degree of malignancy and metastasis. 15 transgenic mice of prostate cancer (Tramp) with different stages of carcingensis, and 5 cases of human prostatic carcinomas were used. Paraffin sections were immuno-stained with anti-t-SNARE antibodies, syntaxin 3 and syntaxin 4. The localizations of t-SNAREs were observed with a confocal microscope. Cell polarity was preserved in most prostatic lesions both in Tramp mice and human prostatic cancers as shown by basolateral E-cadherin, except one metastatic lesion in the kidney of one Tramp mouse. Down-regulation of syntaxin 3 was observed in 7 of 15 tramp mice, and in 4 of 5 human prostate cancers. This is correlated with the low degrees of differentiation, down-expression of E-cadherin and p27. But the intracellular localization of syntaxin 3 and 4 was only observed in the metastatic lesions, suggesting that down-regulation and mislocalization of t-SNARE play a role in progression of prostate cancer.
... Data supporting vesicle-dependent trafficking of the H + -K + -ATPase was provided by the identification of two SNARE proteins in H + -K + -ATPase-rich membranes, syntaxin 3 and vesicle-associated membrane protein (VAMP) 2 (Calhoun & Goldenring, 1997;Peng et al. 1997;Calhoun et al. 1998). Syntaxin 3 and VAMP 2 have been shown to be associated with the apical membrane trafficking pathway in other epithelial cells (Braun et al. 1994;Gaisano et al. 1994;Low et al. 1996;Delgrossi et al. 1997;Riento et al. 2000). ...
Gastric HCl secretion by the parietal cell involves the secretagogue-regulated re-cycling of the H+–K+-ATPase at the apical membrane. The trafficking of the H+–K+-ATPase and the remodelling of the apical membrane during this process are likely to involve the co-ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H+–K+-ATPase and of components of the actin-based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein- protein interactions that regulate apical membrane trafficking in many other epithelial cells.
... The porcine kidney epithelial cell line LLC-PK1 produces both of the syntaxins in significant amounts (III,Fig.7). Endogenous syntaxin 3 is localised on apical membranes in Caco-2 cells (Delgrossi et al., 1997) where syntaxin 2A is also located (Quinones et al., 1999). In stably transfected MDCK II cells, syntaxin 3 was found in the apical domain whereas syntaxin 4 was in the basolateral part, showing no overlap with syntaxin 3 staining. ...
... Exocytosis involves SNARE-mediated protein-protein interactions between transport vesicles and plasma membranes (for review see Jahn and Südhof, 1999). In polarized epithelial cells, evidence that t-SNAREs are localized to different plasma membrane domains has been presented (Gaisano et al., 1996;Low et al., 1996;Delgrossi et al., 1997;Fujita et al., 1998), and it is likely that different SNAREs are involved in exocytosis to apical and basal-lateral membrane domains (Ikonen et al., 1995;Low et al., 1998). However, during directed membrane growth in budding yeast and developing neurons there is no spatial correlation between the distribution of t-SNAREs and sites of exocytosis (Brennwald et al., 1994;Garcia et al., 1995). ...
Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane–bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.
... This t-SNARE, which is not transmembrane but is bound to membranes by palmitoylation (Gonzalo and Linder, 1998), was shown to accumulate neither in the ER nor in the Golgi complex, but to proceed freely to its major physiological location, the plasmalemma, even in PC12-27 cells, as revealed by confocal microscopy (Fig. 8). In order to verify whether the results reported so far with syn1A are specific for the neuronal isoform or whether similar effects can be observed with other members of the family, microinjection and transfection experiments were repeated in all cell types examined using the cDNAs encoding two other members of the syntaxin family: syntaxin 3, which is delivered apically in polarized cells and is believed to be involved in constitutive exocytosis (Hay and Scheller, 1997; Low et al., 1996 Low et al., , 1998 Delgrossi et al., 1997; Galli et al., 1998) and a yeast homologue of syntaxin, Sso2p (Aalto et al., 1993) which is transported to the plasma membrane when transfected in BHK cells (Jäntti et al., 1994). The results shown inFig. ...
The t-SNAREs syntaxin1A and SNAP-25, i.e. the members of the complex involved in regulated exocytosis at synapses and neurosecretory cells, are delivered to their physiological site, the plasma membrane, when transfected into neurosecretion-competent cells, such as PC12 and AtT20. In contrast, when transfection is made into cells incompetent for neurosecretion, such as those of a defective PC12 clone and the NRK fibroblasts, which have no endogenous expression of these t-SNAREs, syntaxin1A (but neither two other syntaxin family members nor SNAP-25) remains stuck in the Golgi-TGN area with profound consequences to the cell: blockade of both membrane (SNAP-25, GAT-1) and secretory (chromogranin B) protein transport to the cell surface; progressive disassembly of the Golgi complex and TGN; ultimate disappearance of the latter structures, with intermixing of their markers (mannosidase II; TGN-38) with those of the endoplasmic reticulum (calreticulin) and with syntaxin1A itself. When, however, syntaxin 1A is transfected together with rbSec1, a protein known to participate in neurosecretory exocytosis via its dynamic interaction with the t-SNARE, neither the blockade nor the alterations of the Golgi complex take place. Our results demonstrate that syntaxin1A, in addition to its role in exocytosis at the cell surface, possesses a specific potential to interfere with intracellular membrane transport and that its interaction with rbSec1 is instrumental to its physiological function not only at the plasma membrane but also within the cell. At the latter site, the rbSec1-induced conversion of syntaxin1A into a form that can be transported and protects the cell from the development of severe structural and membrane traffic alterations.
... In contrast to syntaxin 1A, syntaxin 3 protein was more precisely localized to the apical cell surface. A very similar pattern has been reported for syntaxin 3 in other epithelial cells such as MDCK cells (34) and CaCo2 colonic epithelial cells (35), although there is 1 report that this syntaxin isoform is localized to the basolateral surfaces of renal collecting duct cells (36). Interestingly, syntaxin 3 does not appear to directly interact with CFTR or to regulate CFTR Clcurrents (Figure 6a), in spite of the fact that it is more abundant than syntaxin 1A and is more precisely localized to the apical membranes of airway and gut epithelial cells. ...
The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.
... However, one should not draw detailed conclusions from this data since the amino acid identity between yeast Sec1p and Munc18-2 is only 29%. The E467K mutant tentatively corresponding to yeast SLY1-20 (53) displayed mildly increased binding to syntaxin 3. The implications of this finding are, however, unclear since Grabowski and Gallwitz (54) (TI-VAMP) (47,55). We have previously shown that overexpression of Munc18-2 modifies the quantity of the other SNAREs complexed with syntaxin 3 (9). ...
The Sec1-related proteins bind to syntaxin family t-SNAREs with high affinity, thus controlling the interaction of syntaxins
with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin
3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions
in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro andin vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their
ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do
not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the
sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical
delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the
HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of
HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.
... Data supporting vesicle-dependent trafficking of the H + -K + -ATPase was provided by the identification of two SNARE proteins in H + -K + -ATPase-rich membranes, syntaxin 3 and vesicle-associated membrane protein (VAMP) 2 (Calhoun & Goldenring, 1997;Peng et al. 1997;Calhoun et al. 1998). Syntaxin 3 and VAMP 2 have been shown to be associated with the apical membrane trafficking pathway in other epithelial cells (Braun et al. 1994;Gaisano et al. 1994;Low et al. 1996;Delgrossi et al. 1997;Riento et al. 2000). ...
Gastric HCl secretion by the parietal cell involves the secretagogue-regulated re-cycling of the H+-K+-ATPase at the apical membrane. The trafficking of the H+-K+-ATPase and the remodelling of the apical membrane during this process are likely to involve the co-ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H+-K+-ATPase and of components of the actin-based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein- protein interactions that regulate apical membrane trafficking in many other epithelial cells.
Apicobasal epithelial polarity controls the functional properties of most organs. Thus, there has been extensive research on the molecular intricacies governing the establishment and maintenance of cell polarity. Whereas loss of apicobasal polarity is a well-documented phenomenon associated with multiple diseases, less is known regarding another type of apicobasal polarity alteration – the inversion of polarity. In this Review, we provide a unifying definition of inverted polarity and discuss multiple scenarios in mammalian systems and human health and disease in which apical and basolateral membrane domains are interchanged. This includes mammalian embryo implantation, monogenic diseases and dissemination of cancer cell clusters. For each example, the functional consequences of polarity inversion are assessed, revealing shared outcomes, including modifications in immune surveillance, altered drug sensitivity and changes in adhesions to neighboring cells. Finally, we highlight the molecular alterations associated with inverted apicobasal polarity and provide a molecular framework to connect these changes with the core cell polarity machinery and to explain roles of polarity inversion in health and disease. Based on the current state of the field, failure to respond to extracellular matrix (ECM) cues, increased cellular contractility and membrane trafficking defects are likely to account for most cases of inverted apicobasal polarity.
Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death worldwide. Circular RNAs (circRNAs), a novel class of non-coding RNA, have been reported to be involved in the etiology of various malignancy. However, the underlying cellular mechanisms of circRNAs implicated in the pathogenesis of HCC remains unknown. Here, we identified a functional RNA - hsa_circ_0000384 (circMRPS35) from public tumor databases using a set of computational analyses, and we further identified that circMRPS35 was highly expressed in 35 pairs of HCC from patients. Moreover, knockdown the expression of circMRPS35 in Huh-7 and HCC-LM3 cells suppressed their proliferation, migration, invasion, clone formation and cell cycle in vitro, and suppressed tumor growth in vivo as well. Mechanically, circMRPS35 sponged microRNA-148a-3p (miR-148a) regulating the expression of Syntaxin 3 (STX3), which modulated the ubiquitination and degradation of Phosphatase and tensin homolog (PTEN). Unexpectedly, we detected a peptide encoded by circMRPS35 (circMRPS35-168aa), which was significantly induced by chemotherapeutic drugs and promoted cisplatin resistance in HCC. These results demonstrated that circMRPS35 might be a novel mediator in HCC progress, and raise the potential as a new biomarker for HCC diagnosis and prognosis, as well as a novel therapeutic target for HCC patients.
Herein, we investigated the functional association of the serotonin transporter (SERT) with syntaxin-3 (STX3). We first overexpressed SERT and STX3 in various cells and examined their interaction, localization, and functional association. Immunoprecipitation studies revealed that STX3 interacted with SERT when expressed in COS-7 cells. Immunocytochemical studies revealed that SERT and STX3 were colocalized in the endoplasmic reticulum (ER) and Golgi apparatus. STX3 overexpression significantly reduced the uptake activity of SERT by attenuating its plasma membrane expression, suggesting that overexpressed STX3 anchors SERT in the ER and Golgi apparatus. STX3 knockdown did not affect the uptake activity of SERT but altered its glycosylation state. To elucidate the association of STX3 with SERT under physiological conditions, rather than overexpressing cells, we investigated this interaction in polarized Caco-2 cells, which endogenously express both proteins. Immunocytochemical studies revealed that SERT and STX3 were localized in microvilli-like structures at the apical plasma membrane. STX3 knockdown marginally but significantly decreased the serotonin uptake activity of Caco-2 cells, suggesting that STX3 positively regulates SERT function in Caco-2 cells, as opposed to SERT regulation by STX3 in overexpressing cells. Collectively, STX3 may colocalize with SERT during SERT membrane trafficking and regulate SERT function in an STX3-expressing lesion-dependent manner.
Luminal uterine epithelial cells (UEC) have a surge in vesicular activity during early uterine receptivity. It has been predicted these vesicles exit the UEC via exocytosis resulting in secretion and membrane trafficking. The present study investigated the changes in SNARE proteins VAMP2 (v-SNARE) and syntaxin 3 (t-SNARE) localisation, and abundance in UECs during early pregnancy in the rat. We found VAMP2 and syntaxin 3 are significantly higher on day 5.5 compared to day 1 of pregnancy. On day 5.5, VAMP2 is perinuclear and syntaxin 3 is concentrated in the apical cytoplasm compared to a cytoplasmic localisation on day 1. This change in localisation and abundance show VAMP2 and syntaxin 3 are involved in vesicular movement and membrane trafficking in UECs during early pregnancy. This study also investigated the influence of cytoskeletal disruption of microtubules and actin filaments on VAMP2 and syntaxin 3 in UECs grown in vitro since microtubules and actin influence vesicle trafficking. As expected, this study found disruption to microtubules with colchicine and actin with cytochalasin D impacted VAMP2 and syntaxin 3 localisation. These results suggest VAMP2 and syntaxin 3 are involved in the timely trafficking of vesicular membranes to the apical surface in UECs during early pregnancy, as are of microtubules and actin.
] The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.
The transport functions of epithelial cells in the gastrointestinal (GI) tract, and elsewhere, are made possible by their remarkable apical-basolateral plasma membrane asymmetry. This property, commonly called membrane polarity, is achieved by a combination of intracellular trafficking operations, including sorting, vectorial delivery and membrane-specific docking, fusion and retention mechanisms. Here we provide a review of this rapidly evolving research field within a historically relevant context.
Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.
Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID revealed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in CaCo2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.
The asymmetric distribution of proteins and lipids in two distinct domains of the plasma membrane (PM) of polarized epithelial cells is accomplished by continuous sorting of newly synthesized components and their regulated internalization. Here we discuss the mechanisms of apical sorting of glycosylphosphatidilinositol-anchored proteins (GPI-APs) especially focusing on their biosynthetic pathway. Recent evidences indicate that this event depends on both association to specialized lipid domains (rafts) and clustering in high-molecular-weight complexes at the level of the Golgi apparatus. We discuss the evidences present in literature pointing toward the involvement of the protein ectodomain, the GPI anchor, the lipid environment, and the presence of a putative receptor in this process. We also examine the role of the cytoskeleton and other molecular components of the exocytic pathway in post-TGN transport of GPI-APs to the PM.
We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37 degreesC by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5'nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.
Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2(BB)e cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2(BB)e cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.
The docking/fusion of transport vesicles mediated by the soluble NSF attachment protein receptors (SNAREs) is thought to be regulated by Sec1-related proteins. Munc-18-2, a member of this family, is predominantly expressed in the epithelial cells of several tissues. We demonstrate here that Munc-18-2 colocalizes with syntaxin 3 at the apical plasma membrane of intestinal epithelium and Caco-2 cells. The presence of a physical complex of the two proteins is verified by 2-way coimmunoprecipitation. The quantity of the complex is reduced by treatment of Caco-2 cells with the alkylating agent N-ethylmaleimide which also has an inhibitory effect on the ability of Munc-18-2 to associate with syntaxin 3 in vitro. The amount of Munc-18-2 in the complex increases upon treatment of the cells with the protein kinase C activator phorbol myristate acetate, indicating a functional connection between the complex and cell signalling. Increasing the amount of Munc-18-2 bound to syntaxin 3 by overexpression results in a marked decrease in the SNARE proteins SNAP-23 and cellubrevin bound to the syntaxin. These results define a novel functional complex of Munc-18-2 and syntaxin 3 involved in the regulation of apical membrane transport.
Polarized epithelial cells form barriers that separate biological compartments and regulate homeostasis by controlling ion and solute transport between those compartments. Receptors, ion transporters and channels, signal transduction proteins, and cytoskeletal proteins are organized into functionally and structurally distinct domains of the cell surface, termed apical and basolateral, that face these different compartments. This review is about mechanisms involved in the establishment and maintenance of cell polarity. Previous reports and reviews have adopted a Golgi-centric view of how epithelial cell polarity is established, in which the sorting of apical and basolateral membrane proteins in the Golgi complex is a specialized process in polarized cells, and the generation of cell surface polarity is a direct consequence of this process. Here, we argue that events at the cell surface are fundamental to the generation of cell polarity. We propose that the establishment of structural asymmetry in the plasma membrane is the first, critical event, and subsequently, this asymmetry is reinforced and maintained by delivery of proteins that were constitutively sorted in the Golgi. We propose a hierarchy of stages for establishing cell polarity.
We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin- Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.
Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid-cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid-cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.
Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles. This apparatus comprises the secretory (or 'protein export') pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles. Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another. Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion. These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e. the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion. SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events. In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g. 'SNARE-masters') in mediating events leading to membrane fusion. A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented.
Differentiation of epithelial cells involves the assembly of polarized membrane transport machineries necessary for the generation and maintenance of the apical and basolateral membrane domains characteristic of this cell type. We have analyzed the expression patterns of vesicle-docking proteins of the syntaxin family in mouse kidney, focusing on syntaxin 3 and its interaction partner, the Sec1-related Munc-18-2.
Expression patterns were studied by in situ hybridization and immunocytochemistry and the complex formation of syntaxin 3 and Munc-18-2 by coimmunoprecipitation and Western blotting.
We have previously shown by in situ hybridization that Munc-18-2 is present in the proximal tubules and collecting ducts of embryonic day 17 mouse kidney. We compared this with the expression patterns of syntaxin 1A, 2, 3, 4, and 5, and found that syntaxin 3 was enriched in the same epithelial structures in which Munc-18-2 was abundant. By immunocytochemistry, the two proteins colocalized at the apical plasma membrane of proximal tubule and collecting duct epithelial cells, and they were shown to form a physical complex in the kidney. The expression of both proteins was up-regulated during kidney development. The most prominent changes in expression levels coincided with the differentiation of proximal tubules, suggesting a role in the generation of the highly active reabsorption machinery characterizing this segment of the nephron.
The results show that Munc-18-2 and syntaxin 3 form a complex in vivo and suggest that they participate in epithelial cell differentiation and targeted vesicle transport processes in the developing kidney.
Previous results from our laboratory have indicated a requirement for CK intermediate filaments (IF) for the organization of the apical domain in polarized epithelial cells in culture. The results seemed to be challenged by the phenotype of cytokeratin (CK) 8-deficient mice, which comprises only colorectal hyperplasia, female sterility and a weaker hepatocyte integrity. In this work localization with anti-CK antibodies indicated that many Ck8-/- epithelia still form IF in CK8-deficient mice, perhaps because of the expression of the promiscuous CK7. In the small intestine, only villus enterocytes lacked IFs. These cells appeared to lose syntaxin 3, and three apical membrane proteins (alkaline phosphatase, sucrase isomaltase and cystic fibrosis transmembrane conductance regulator) as they progressed along the villus. At the distal third of the villi, gamma-tubulin was found scattered within the cytoplasm of enterocytes, in contrast to its normal sub-apical localization, and the microtubules were disorganized. These results could not be attributed to increased numbers of apoptotic or necrotic cells. The only other cell type we found without IFs in CK8 null mice, the hepatocyte, displayed increased basolateral levels of one apical marker (HA4), indicating a correlation between the lack of intermediate filaments and an apical domain phenotype. These data suggest a novel function for intermediate filaments organizing the apical pole of simple polarized epithelial cells.
1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to exist in three pools in the brush border (BB), including a population in lipid rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-soluble fractions and the other approximately 50% with Triton X-100-insoluble fractions; approximately 33% of the detergent-insoluble NHE3 was present in cholesterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activity with epidermal growth factor (EGF). Both EGF and clonidine treatments were associated with a rapid increase in the total amount of BB NHE3. This EGF- and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the total detergent-insoluble fraction, but there was no change in the detergent-soluble pool. In agreement with the rapid increase in the amount of NHE3 in the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activity. 3. Disrupting rafts by removal of cholesterol with methyl-beta-cyclodextrin (MbetaCD) or destabilizing the actin cytoskeleton with cytochalasin D decreased the amount of NHE3 in early endosomes isolated by OptiPrep gradient fractionation. Specifically, NHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibody-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and MbetaCD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and to a lesser extent, the cytoskeleton, but not the detergent-soluble NHE3 pool, are involved in the EGF- and clonidine-induced acute increase in amount of BB NHE3; (iv) lipid rafts and the actin cytoskeleton play important roles in the basal endocytosis of BB NHE3.
Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.
We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.
A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.
The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.
The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.
We have reconstituted polarized protein transport in streptolysin O-permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross-linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin-2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.
In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.
Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.
The N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs) appear to be essential components of the intracellular membrane fusion apparatus. An affinity purification procedure based on the natural binding of these proteins to their targets was used to isolate SNAP receptors (SNAREs) from bovine brain. Remarkably, the four principal proteins isolated were all proteins associated with the synapse, with one type located in the synaptic vesicle and another in the plasma membrane, suggesting a simple mechanism for vesicle docking. The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicle or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesicle-to-target specificity.
The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.
Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle.
Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.
Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3, and 4 were found to be endogenously present in Madin-Darby canine kidney cells. The localization of syntaxins 1A, 1B, 2, 3, and 4 in stably transfected Madin-Darby canine kidney cell lines was studied with confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures, with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, whereas the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity.
Transport between Golgi dsternae in a cell-free system is blocked hy cither nonhydrolyzable analogue ol'GTP (GTPyS) or by the sulfhydryl reagenl N-cIhylmaleimide (NEM). With GTPyS, vesicles accumulate encased in a coat which is fro/en in place because GTP cannot he hydrnlyzed. With NEM, uncoated vesicles accumulate ducked to their target cisterna. but tail to fuse; NEM inactivates a protein required tor fusion, termed NSF, for NEM Sensitive Fusion protein. The coal is composed uf many copies of a protein termed coatomcr (short for coat protomer), an assembly of seven distinct subunils. termed COP proteins. The coated vesicles also contain many copies of ;i GTPase, ARF. Cytosohc ARF binds GTP, binds to the Golgi, and then recruits cytosolic coatomer lo join the assembling coat. Later, after budding, ARF hydrolyzes its bound GTP, releasing coatomer; this explains why non-hydrolyzable GTP blocks transport and accumulates coated transport vesicles. Similar or identical COPI-coated vesicles mediate antcrograde and retrograde transport both in isolated Golgi stacks ;md in whole cells.
Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.
The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.
We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.
A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.
Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.
The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.
Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.
We have reconstituted polarized protein transport in streptolysin O-permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross-linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin-2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.
A Summary of Drosophila Embryogenesis.- Stages of Drosophila Embryogenesis.- Mesoderm Development.- Musculature.- Circulatory System and Fat Body.- Macrophages.- The Gut and its Annexes.- Epidermis.- Peripheral Nervous System.- The Peripheral Nervous System.- Central Nervous System.- Tracheal Tree.- The Gonads.- The Pattern of Embryonic Cell Divisions.- Morphogenetic Movements.- Cephalogenesis.- Some Aspects of Segmentation.- A Fate Map of the Blastoderm.
The intestinal epithelium is a heterogeneous cell monolayer that undergoes continuous renewal and differentiation along the crypt-villus axis. We have used transgenic mice to examine the compartmentalization of a regulated endocrine secretory protein, human growth hormone (hGH), in the four exocrine cells of the mouse intestinal epithelium (Paneth cells, intermediate cells, typical goblet cells, and granular goblet cells), as well as in its enteroendocrine and absorptive (enterocyte) cell populations. Nucleotides -596 to +21 of the rat liver fatty acid binding protein gene, when linked to the hGH gene (beginning at nucleotide +3) direct efficient synthesis of hGH in the gastrointestinal epithelium of transgenic animals (Sweetser, D. A., D. W. McKeel, E. F. Birkenmeier, P. C. Hoppe, and J. I. Gordon. 1988. Genes & Dev. 2:1318-1332). This provides a powerful in vivo model for analyzing protein sorting in diverse, differentiating, and polarized epithelial cells. Using EM immunocytochemical techniques, we demonstrated that this foreign polypeptide hormone entered the regulated basal granules of enteroendocrine cells as well as the apical secretory granules of exocrine Paneth cells, intermediate cells, and granular goblet cells. This suggests that common signals are recognized by the "sorting mechanisms" in regulated endocrine and exocrine cells. hGH was targeted to the electron-dense cores of secretory granules in granular goblet and intermediate cells, along with endogenous cell products. Thus, this polypeptide hormone contains domains that promote its segregation within certain exocrine granules. No expression of hGH was noted in typical goblet cells, suggesting that differences exist in the regulatory environments of granular and typical goblet cells. In enterocytes, hGH accumulated in dense-core granules located near apical and lateral cell surfaces, raising the possibility that these cells, which are known to conduct constitutive vesicular transport toward both apical and basolateral surfaces, also contain a previously unrecognized regulated pathway. Together our studies indicate that transgenic mice represent a valuable system for analyzing trafficking pathways and sorting mechanisms of secretory proteins in vivo.
We have Isolated a population of vesicular carriers involved in the transport (transcytosis) of proteins from the basolateral to the apical plasma membrane of hepatocytes. The obtained fraction was enriched in compartments containing known transcytosed proteins and depleted in elements of the secretory pathway, Golgi elements, basolateral plasma membrane, as well as early endosomal components. The fraction was analyzed by biochemical and Immunological procedures. Antibodies raised against the proteins in the fraction recognized a single 108K antigen. Based on its subcellular distribution, the 108K antigen may represent a novel marker for transcytotic vesicular carriers.
Partial sequences corresponding to eleven novel Rab proteins and one new Rho protein have been isolated using a PCR-based cloning approach. These results confirm that the overall diversity of the Rab and Rho protein subfamilies account for more than thirty different members in mammalian cells.
A novel 150 kd protein expressed on the surface of mesenchymal cells of mouse embryonic tissues was identified. A monoclonal antibody to this molecule inhibited various processes of epithelial morphogenesis, such as hair follicle growth and lung epithelial tubular formation, in organ cultures of these tissues. Sequence analysis of cDNA encoding this protein revealed that it had 289 amino acids with a hydrophobic stretch at the C-terminus. NIH 3T3 cells transfected with the cDNA of this protein expressed the exogenous 150 kd protein on their surface. When lung epithelial cells were cocultured with these transfected cells, they showed normal tubular morphogenesis, but not with untransfected NIH 3T3 cells. These results indicate that this protein, termed epimorphin, plays a central role in epithelial-mesenchymal interactions.
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We studied the surface delivery pathways followed by newly synthesized plasma membrane proteins in intestinal cells. To this end, we developed an assay and characterized an epithelial cell line (SK-CO-15) derived from human colon adenocarcinoma. Polarized confluent monolayers (2000 omega.cm2), grown on polycarbonate filter chambers, were pulsed with radioactive methionine/cysteine and, at different times of chase, the protein fraction reaching the apical or basolateral surface was recovered by domain-selective biotinylation, immunoprecipitation, and immobilized streptavidin precipitation. Both an apical and a basolateral marker were found to be delivered vectorially to the respective surface, with a sorting efficiency of 50:1 for the basolateral marker and 14:1 for the apical marker.
Selective biotinylation of the apical or basolateral domains of confluent MDCK monolayers grown on polycarbonate filters with a water soluble biotin analog, sulfo-NHS-biotin, was employed to reveal strikingly distinct patterns of endogenous "peripheral" and "integral" membrane proteins. "Peripheral" proteins were found to be approximately fivefold more abundant with this procedure than "integral" membrane proteins, both on the apical and on the basolateral surface. The distinct apical and basal patterns were shown to depend upon the integrity of the monolayer; when the tight junctions were disrupted by preincubation in calcium-depleted medium, the patterns appeared practically indistinguishable. Two-dimensional gel electrophoresis demonstrated that only a very small percentage of the biotinylated proteins were found in similar amounts on both apical and basolateral domains. These results indicate that the sorting mechanisms that segregate apical and basolateral epithelial proteins are very strict. The simple procedure described here has clear advantages over other methods available to label apical and basal epithelial surface domains, namely, higher accessibility of the biotin probe to the basolateral membrane, possibility of purifying biotinylated proteins via immobilized streptavidin and minimal exposure of the researcher to isotopes. It should be very useful in characterizing the apical and basolateral protein compositions of other epithelial cells and in studies on the development of epithelial cell polarity.
A protein from the basolateral domain of adult human intestinal epithelial cells has been identified and characterized by the monoclonal antibody technique in combination with immunocytochemical and biochemical methods. The protein is found preferentially on the lateral surface of the cells. Extraction of intestinal membranes with Triton X-114 or their treatment with hydrolases indicated that the protein is an integral membrane glycoprotein of apparent molecular weight 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In the adult this protein is restricted to intestine, pancreas and gallbladder, consistent with their common embryonic origin. It was also expressed in the intestine of a 24-week-old human embryo and was found as a basolateral marker in cultured cell lines originating from human colonic adenocarcinomas.
The fidelity of membrane trafficking is mediated by specific interactions between transport vesicles and their target membranes. SNAREs (SNAP receptors) are cytoplasmically oriented membrane proteins that form complexes implicated in transport vesicle targeting. Advances in the past year have provided insight into the fundamental mechanisms underlying the formation, regulation, and function of SNARE complexes.
cDNAs for four different forms of syntaxin 3 were cloned from a mouse brain cDNA library, and the proteins encoded by these clones were named syntaxin 3A (previous syntaxin 3), 3B, 3C and 3D. Syntaxin 3B contained a different sequence in the carboxyl terminal region from that of syntaxin 3A. The amino terminal region of syntaxin 3C contained an 18 amino acid sequence instead of a 34 amino acid sequence present in syntaxins 3A and 3B. Syntaxin 3D consisted of only 86 amino acids and lacked any putative transmembrane segments. These forms of syntaxin 3 are probably generated by alternative splicing of the primary transcript of syntaxin 3 gene. Cytoplasmic portions of syntaxins 3A and 3B but not of syntaxin 3C or 3D bound to Munc-18/n-sec1.
We have examined the process of membrane protein targeting in the polarized cells of the developing Drosophila melanogaster embryo. Human placental alkaline phosphatase (PLAP) is a glycosylphosphatidyl inositol-linked protein that accumulates at the apical membranes of mammalian epithelial cells. A chimeric construct composed of the transmembrane and cytosolic portions of the vesicular stomatitis virus G protein coupled to the ectodomain of PLAP, termed PLAPG, has been found to behave as a basolateral protein (D. A. Brown, B. Crise, and J. K. Rose. Science Wash. DC 232: 34-47, 1989). The subcellular distributions of these proteins were examined in the epithelial and neuronal tissues of transgenic Drosophila embryos. In the surface ectoderm, both PLAP and PLAPG were restricted to the basolateral membranes throughout development. Internal epithelia derived from the surface ectoderm accumulated PLAP at their apical surfaces, whereas PLAPG retained its basolateral distribution. The redistribution of PLAP from the basolateral to the apical plasma membrane was found to be coincident with the invagination of the surface epithelium to form internal structures, suggesting that the sorting pathways that function in the epithelium of the Drosophila embryo are developmentally regulated.