Mechanisms and molecules regulating transcytosis
- Transcytosis: Crossing Cellular Barriers


In this section, we discuss what is known about the mechanisms and molecules that regulate transcytosis in polarized epithelial cells. Are there unique mechanisms/molecules required? Do different cell types require specific molecules or combinations of molecules to perform their specialized transcytotic functions? How does the cell discriminate between transcytotic delivery and self use? Surprisingly, there is little direct evidence for the involvement of specific molecules in transcytosis, but many promising candidates are emerging. We will describe some of these candidates, emphasizing those involved in vesicle transport, targeting, and consumption along the transcytotic route. However, a cautionary flag must be raised. Much of what is discussed is based on studies performed only in tissue culture models of polarized cells (especially MDCK cells) and has not yet been tested in a physiological context. Furthermore, many candidates have been named solely upon their subcellular location; the functional studies have not been performed. Nonetheless, there are exciting possibilities and the groundwork is clearly set to direct future investigations.

A. Targeting Machinery

1. The SNARE hypothesis

How do cargo-bearing vesicles deliver their contents to the correct target domain? Morphological, biochemical, and genetic approaches have successfully identified many molecules involved in "vesicle targeting," a complex series of molecular events that minimally includes docking and fusion. These approaches indicate that the basic mechanisms are conserved among the membrane compartments throughout the biosynthetic and endocytic pathways and in organisms ranging from yeast to humans. The players so far identified fall into two broad categories: some are used repeatedly throughout the pathways, others belong to discrete protein families, where one or a few members act at one or a few transport sites. Members of three protein families are central players in vesicle targeting and fusion (216, 262, 335, 443). They are as follows: 1) the SNAREs which, in general, are a group of cytoplasmically oriented integral membrane proteins that are present on vesicles (v-SNAREs) or target membranes (t-SNAREs) (also referred to as Q- and R-SNAREs; see Refs. 613 and 144); 2) the Sec1/Munc18 proteins; and 3) small-molecular-weight GTP-binding proteins, the rabs.

The "SNARE hypothesis" emerged to describe the mechanism by which the SNAREs promote membrane docking and fusion through interactions with an ATPase, N-ethylmaleimide sensitive factor (NSF), and its "receptor," {alpha}-SNAP (547), but has undergone considerable revision as more is learned about these and other essential proteins and the cyclical nature of the process is better appreciated (262). For vesicle trafficking to continue past one round, some of the machinery must continually cycle between vesicle and target membranes, making the order of events difficult to define. Also, the t- and v-SNAREs, whose pairwise coupling was originally thought to confer targeting specificity, show promiscuity in their binding in vitro, suggesting that other factors are required for the high fidelity of membrane targeting observed in vivo. Furthermore, the ATPase, NSF, which was originally postulated to function in the fusion reaction itself, most likely functions as a chaperone to disassemble the SNARE pairs (204, 216). Whether this occurs when the pairs are in "trans" (on the cognate membranes) or in "cis" (on the same membrane after vesicle consumption) may differ depending on the cell type or transport step examined.

It is also important to note that the "SNARE hypothesis" developed mainly from studies examining vesicle docking and fusion at the PM where SNAP-25 family members (t-SNARE isoforms) are required. Because these molecules are predominantly PM associated, the mechanisms regulating intracellular vesicle transport are likely different. Consistent with this is the finding that two t-SNAREs (both syntaxins) and two v-SNAREs are required during late endosome fusion, whereas at the PM, two different t-SNAREs (a syntaxin and SNAP-25 protein) and one v-SNARE participate (15). Interestingly, in both cases the molecules form core complexes that are structurally similar, indicating that the mechanisms, albeit different, are conserved. Newer models have integrated the enormous and confusing body of information and have identified common steps in membrane targeting which minimally include 1) SNARE activation (which likely includes NSF chaperone activity) and rab recruitment to proper organelle sites; 2) cognate membrane attachment; and 3) membrane fusion and bilayer mixing (262). The order of events and the mechanistic details are not clear, yet there is considerable evidence that the SNAREs, rabs, and Sec1/Munc18 proteins are key players in the process. In the following sections, we discuss the possible roles of selected members of the transport machinery in regulating transcytosis in polarized epithelial cells. Table 5 summarizes what we currently know.

2. NSF and {alpha}-SNAP

NSF was initially implicated as a regulator of polarized PM targeting from studies that examined the effects of NEM on protein transport. Addition of NEM reduced IgA transcytosis by ~70% and its basolateral targeting by ~90% in permeabilized MDCK cells (17, 332). It also inhibited fusion (as assayed by pIgA-R processing) of transcytotic carrier vesicles with the apical PM in a hepatic cell-free system reconstituting the final step of transcytosis (564). Because NSF ATPase activity is inhibited by alkylating (617), it was the likely target for NEM. In support of that, addition of recombinant NSF restored much of the activity lost in MDCK cells or hepatic cell-free systems treated with NEM or anti-NSF antibodies (17, 253, 332, 564). Interestingly, direct apical PM targeting was insensitive to NEM (253, 332), indicating that it requires an unidentified NSF homolog or does not require NSF-like activity at all. Other key factors, the SNAP family of NSF receptors ({alpha}-, {beta}-, and {gamma}-isoforms), recruit NSF to organelles and activate its ATPase activity (617). {alpha}-SNAP has been identified in polarized epithelial cells and its role in TGN to PM targeting examined in MDCK cells. Addition of {alpha}-SNAP antibodies and treatment of cells with botulinum E (an {alpha}-SNAP-specific toxin) inhibited direct transport to both domains (17, 253, 300). Unfortunately, its role in transcytosis was not examined.

NSF has also been implicated as an important regulator of transcytosis in endothelial cells. Treatment of myocardial or rat lung endothelium with NEM significantly inhibited transcytosis in situ (80 and 50%, respectively) (455, 505). NSF and its receptor {alpha}-SNAP were also found associated with caveolae, supporting the role of this vesicle type as the transcytotic carrier in endothelial cells (509). Furthermore, immunoprecipitation of exogenously added recombinant myc-tagged NSF from rat lung endothelial cell extracts revealed the presence of large, ATP-dependent, NEM-sensitive complexes that contained many members of the transport machinery including {alpha}- and {gamma}-SNAP, cellubrevin, syntaxin, and rab5 as well as caveolin and dynamin (457). The complexes were also found to contain cholesterol, the ganglioside GM1, and other unidentified lipids. As predicted, these complexes were immunoprecipitated from membrane extracts, but they were also surprisingly recovered from cytosol preparations. How these supramolecular protein-lipid complexes function in membrane transport is not yet known but supports a role for both caveolae (the complexes contained caveolin) and the molecules of the SNARE hypothesis in endothelial transcytosis. Do the complexes present in the cytosol or on membranes serve the same function? Are similar complexes present in other polarized epithelial cell types?

3. t-SNARES and v-SNAREs

There are two families of t-SNARE proteins: the syntaxins and the SNAP-25 family. To date, at least 18 syntaxin family members have been identified in mammalian cells of which 5 (syntaxins 1A, 1B, 2–4) are PM-specific isoforms (262). The SNAP-25 family is much smaller, with only three identified members: SNAP-25, SNAP-23, and SNAP-29 (262). Like the syntaxins, these proteins are relatively small (23–29 kDa), cytoplasmically oriented molecules. Although not integral membrane proteins as are the PM syntaxins, SNAP-25 proteins associate with the bilayer through cysteine-linked palmitoyl chains located near the middle of the protein. Biochemically, SNAP-25 proteins bind PM-associated syntaxins and v-SNAREs in vitro to form the four-stranded {alpha}-helical ternary complexes required for vesicle docking and fusion (262).

The distributions of syntaxins 2, 3, and 4 at the PM have been examined in different epithelial cells. In particular, we found that rat hepatocytes express three endogenous PM-associated syntaxin isoforms (syntaxins 2, 3, and 4) and SNAP-23 (163). Quantitative immunoblotting revealed that all four t-SNAREs are relatively abundant in liver (~11–668 nM corresponding to 0.5–28 x 105 molecules/cell). Biochemically, each of the t-SNAREs was observed predominantly in hepatocyte PM sheets with overlapping but distinct expression patterns among the PM domains. Both syntaxin 4 and SNAP-23 are restricted to the basolateral PM while syntaxins 2 and 3 are more apically distributed, with greater enrichment of syntaxin 3 in this domain. Despite the biochemical abundance of the molecules, we were able to detect only syntaxins 2 and 4 in rat liver sections in situ. However, the distributions did not fit with our biochemical data; we found syntaxin 4 in both domains. Similar discrepancies were observed in WIF-B cells. Like in liver hepatocytes, syntaxin 3 was at the apical domain, but unlike liver, syntaxin 2 was restricted to the apical domain. Also, syntaxin 4 and SNAP-23 were found in both domains. These varied distributions likely reflect important differences in regulation of PM dynamics between the in vivo and in vitro systems and may point to interesting features of the cellular itineraries and functions of the t-SNAREs. Little is known how these putative targeting molecules are themselves targeted to the correct PM domain, and once delivered, how and if they are retained there.

Interestingly, the PM syntaxins display remarkable variability in their domain distributions in other polarized cells (see Table 5). Only syntaxin 3 appears to be consistent with apical distributions observed in all polarized cells examined (117, 163, 170, 331, 438). SNAP-23 distributes to both PM domains in all but two epithelial cell types (see Table 5). Since it is thought to be required for vesicle docking and fusion, SNAP-23's uniform PM distribution fits with the current model of its ubiquitous involvement in t- and v-SNARE ternary complex formation. However, its absence at the apical PM in hepatocytes and pancreatic cells is somewhat paradoxical. Either these cells have unique mechanisms for regulating transport at their apical surfaces, or other SNAP-25 isoforms are yet to be identified.

To date, the roles of the t-SNAREs in polarized PM targeting have been tested directly mainly in MDCK cells that were stably expressing pIgA-R and overexpressing wild-type syntaxins 2, 3, or 4 (332). Neither transcytosis nor basolateral transport of pIgA-R was affected in these cells. Similarly, in Caco-2 cells overexpressing syntaxin 3, no changes in basolateral protein targeting were observed (59). Together, these results suggest that the PM-associated syntaxins do not regulate these transport pathways or that other yet-to-be isoforms are involved. Alternatively, overexpression was not high enough to be inhibitory or does not negatively regulate these processes. However, overexpression of syntaxin 3 in MDCK and Caco-2 cells did lead to alterations in apical PM dynamics. In MDCK cells, a slight impairment (~20%) of direct TGN to apical PM delivery of a chimeric pIgA-R molecule and IgA was observed as well as apical recycling (also ~20%). Likewise in Caco-2 syntaxin 3 overexpressors, the direct apical targeting of sucrase-isomaltase and the apical secretion of {alpha}-glucosidase was significantly impaired. Furthermore, anti-syntaxin 3 antibodies inhibited direct targeting of hemagglutinin (HA) in MDCK cells, confirming a role for this syntaxin in apical delivery (300). Unfortunately, the effects on transcytosis of anti-syntaxin 3 or syntaxin 3 overexpression in Caco-2 cells were not examined. However, the role of SNAP-23 in transcytosis has been examined in MDCK cells by treating with SNAP-23/25-specific neurotoxins. In these cells, basolateral to apical transport of pIgA was inhibited by 30% (17), as was basolateral targeting of the receptor, but the TGN to apical targeting of HA was not affected (17, 253). More recent studies confirmed this result and found that toxin activity also impairs transferrin recycling (314).

The direct involvement of v-SNARES in transcytosis has not been explored, but the obvious prediction is that they are required. VAMP 1, 2 and VAMP 3/cellubrevin are ubiquitously expressed, and the presence of VAMP 3/cellubrevin on endosomal structures in hepatocytes and VAMP 2 on endothelial caveolae has been reported (75, 371). VAMP 8/endobrevin is enriched in epithelial tissues and has been localized to the apical pole in kidney epithelium (622). Interestingly, in hepatocytes, this VAMP species was found to be enriched in basolateral early endosomal fractions, whereas in MDCK cells, it was localized to both apical endosomes and the apical PM (554). Whether this protein functions in basolateral or apical PM targeting (or both) is not yet known. In Caco-2 cells, another VAMP isoform, VAMP 7/TI-VAMP, is also localized to both the apical PM and in subapically located structures where it has been proposed to function in the later steps of apical PM delivery (171). Interestingly, this VAMP species has a long NH2-terminal extension that resembles a region in annexin XIIIb, another protein implicated in regulating apical vesicle delivery (150). This sequence in annexin XIIIb encodes a lipid-binding domain, but whether VAMP 7/TI-VAMP shares this biochemical property is not yet known.

A possible role for VAMP 2 in transcytosis has been suggested from studies in SLO-permeabilized rat lung endothelial cells using VAMP-specific neurotoxins (371). In cells treated with botulinum D and F, VAMP 2 cleavage occurred concomitant with the impairment of caveolae-mediated cholera toxin B endocytosis. At the ultrastructural level, large, aberrant subplasmalemmal organelles were observed in treated cells, indicating that delivery of cholera toxin to intracellular intermediates (endosomes?) was impaired. Unfortunately, transcytosis was not examined in toxin-treated cells to determine whether VAMP 2 is a general regulator of caveolae-mediated internalization in endothelial cells. These findings also expand the function of VAMP 2 to include regulation of endocytic transport, whereas previously, this v-SNARE was thought to function in exocytic membrane docking and fusion. Whether VAMP 2 functions in PM vesicle docking and fusion in endothelial cells has not yet been tested. Likewise, a possible role for VAMP 2 in endocytosis in other cell types may warrant further investigation.

4. Munc18

Munc18 homologs have been identified in systems from yeast to neurons and are thought to participate in multiple vesicle transport steps (262, 442). The 68-kDa mammalian Munc18 proteins peripherally associate with the PM through interactions with syntaxins; in vitro, they bind syntaxins 1, 2, and 3 with nanomolar affinity. Interestingly, Munc18 binding to syntaxins cannot occur when the syntaxins are bound to SNAP-25 proteins, suggesting that the different complexes form reciprocally. However, it is presently not known whether Munc18 isoforms play a positive or negative regulatory role in PM targeting. Mutational analysis of related proteins in yeast, Drosophila, and Caenorhabditis elegans all implicate Munc18 species as positive regulators whereas in vitro assays suggest the opposite (262, 442). As for most of the SNARE molecules, no direct evidence for the involvement of Munc18 proteins in polarized PM targeting exists. However, the Munc18–2 isoform is primarily limited to polarized epithelial cells (472). Furthermore, its expression seems restricted to the apical PM where it forms complexes with syntaxin 3 (471), a characteristic that suggests a unique function for Munc18–2 in vesicle delivery to the apical PM.

5. The rab proteins

The rab proteins belong to the largest family of small molecular mass (20–30 kDa) GTP binding proteins. There are 11 known yeast isoforms and at least 60 rabs in mammalian cells (195, 354, 500). Examination of transfected cells either overexpressing wild-type or dominant negative mutant (usually the GDP-bound conformer) forms of various rabs either stimulate or inhibit protein transport and in some cases alter organelle morphology. Although their precise roles are not known, they have been proposed to function in one of three ways: 1) facilitating vectorial traffic via associations with the cytoskeleton; 2) regulating vesicle docking by recruiting effector molecules, thereby promoting the formation of "molecular tethers"; and 3) "priming" docking and fusion by activating SNARE molecules (195, 500).

Given the large number of mammalian rabs and their varied distributions, it is likely that transcytosis in epithelial cells is regulated by multiple isoforms, but which ones? Rabs 3B, 13, 17, 18, 20, and 25 are preferentially expressed in epithelial cells (see Table 5), suggesting a unique function in polarized membrane transport. Although also expressed in nonpolarized cells, rabs 1, 2, 3D, 4, 5, 6, and 11 have also been implicated in regulating polarized PM transport. Of these 13 rabs, 9 have been localized to the apical pole: at the apical PM (rab3D), the tight junction (rabs 3B and 13), or in subapical structures (rabs 5, 11, 17, 18, 20, and 25). The multiple rab proteins in the apical region may point to the complexity of membrane transport events at this PM domain both in terms of specific transport steps as well as organellar intermediates.

Rab5 is the most extensively studied isoform, and much is known about the relationship between its catalytic activity and function in membrane transport (478). In all nonpolarized cells examined to date, rab5 is localized to the PM, clathrin-coated vesicles, and/or early endosomes. Overexpression of rab5 increased endocytic transport and stimulated early endosome fusion in vitro, whereas inhibition of rab5 led to the opposite effects. Recently, it was proposed that rab5 recruits EEA1 (one of its many effectors) to sites of endosome fusion along with NSF and syntaxin 13 that together drive formation of a large multimeric complex which then coordinates fusion pore assembly (368). This general role of rab5 in endocytosis strongly suggests an important role in the early steps of the transcytotic pathway in most polarized cells. Consistent with this is the presence of rab5 on early apical and basolateral endosomes in hepatocytes, hepatic WIF-B cells, in mouse kidney epithelia and MDCK cells (67, 272, 583). Furthermore, when overexpressed in MDCK cells, increased rates of fluid-phase internalization from the apical and basolateral PM were observed, suggesting a role for rab5a in endocytosis from both domains (67). In endothelial cells, rab5 is also localized to the PM and early endosomes, suggesting a role in endocytosis. However, it is not found associated with caveolae, suggesting it is not required for endothelial transcytosis (509).

Rab17 has been strongly implicated in regulating transcytosis in a number of different epithelial cells, first because of its restricted expression pattern and subcellular location, and more recently from functional studies. In kidney, rab17 expression was induced only upon mesenchymal differentiation to polarized cells (337). In intestinal tissue sections, it was detected only in polarized cells and not in surrounding, nonpolarized cells. In enterocytes, rab17 was detected in the basolateral domain, whereas in kidney cells it was found both at the basolateral PM and in apical tubules underlying the brush border (337). In MDCK and polarized Eph 4 cells, it was detected in subapically located vesicular structures (247, 631), and in hepatocytes, rab17 copurified with transcytotic vesicles (210). Functionally, the overexpression of wild-type rab17 in MDCK cells impaired the basolateral to apical transcytosis of dIgA (247). Conversely, in Eph 4 cells expressing GTPase-deficient mutants, the basolateral to apical transcytosis of Tf-R and a chimeric receptor was enhanced as was apical recycling of the chimeric receptor (631). Rab17 has also been copurified with a population of transcytotic vesicles from rat liver, suggesting it is an important regulator of hepatic basolateral to apical transcytosis, too. Surprisingly, both rab1 and -2 also copurified with the vesicles, implicating them as additional regulators of transcytosis (265). The next steps will be pinpointing the site of function in the transcytotic pathway, examining whether the role of rab17 is universal among epithelial cells and to identify the cellular effectors that rab17 activity regulates.

Rabs 11 and 25 have also been identified as important regulators of basolateral to apical transcytosis from functional studies performed in gastric parietal and MDCK cells (82, 134, 610). Both of these rabs have been localized to the apical recycling endosome in MDCK cells (82), and when the respective GTP-binding mutants were overexpressed, basolateral to apical transcytosing IgA accumulated in these structures (610). Interestingly, the activated form of rab25 inhibited transport more than the rab11 mutant. Also interesting is the observation that unlike for nonpolarized cells, rab11 is not required for Tf recycling (610). The puzzle is why these rabs apparently regulate the same transport steps at the apical PM. Do the differential responses suggest separable roles in transport? Also, why is rab11 required for Tf recycling in nonpolarized cells, but not polarized cells? Although rab25 expression is enriched in epithelial tissues, it is surprisingly absent in liver (193), pointing out yet another important difference in apical PM targeting in polarized hepatocytes.

The recent identification of two rab11 effectors, rip11 and myosin Vb, has further confirmed a role for this rab in regulating late steps in basolateral to apical transcytosis (304, 458). In both cases, dominant negative mutations significantly impaired IgA apical PM delivery, and corresponding accumulations of IgA in the apical recycling endosome were observed in MDCK cells. Like rab11, myosin Vb was found to regulate Tf recycling only in nonpolarized cells (304) and rip11 mutants did not impair basolateral transferrin recycling (458). Are different rab11 effectors required for transferrin recycling in nonpolarized cells? How are these two effectors both regulated by rab11 at the same transport step? Careful dissection of the molecular events required for vesicle budding from the apical recycling endosome, transport to the apical PM, and subsequent docking and fusion are required to specifically identify how these molecules function.

The expression patterns of rabs13 and 3B are also highly dependent on the polarized state of a cell (611, 632). In nonpolarized cells, they were found in cytoplasmic vesicles, whereas in polarized cells, they were recruited to tight junctions. Rab13 has so far been detected at tight junctions of Caco-2 cells, mouse intestinal cells, kidney, and liver (632) while rab3B has been observed in tight junctions of colonic epithelia, kidney, and liver (611). In both cases, their localizations were dependent on the integrity of the tight junction. When junctions were disassembled by Ca2+ withdrawal, staining of rabs13 and 3B at the cell surface was lost, suggesting these rabs function in vesicle delivery to the tight junction and, in particular, regulate vesicular delivery of junctional components. At present whether any transcytotic vesicles are also specifically delivered to the cell surface at sites of cell-cell contact, and by extension, under the control of these rab isoforms, is not yet known. Immunoadsorption and examination of the vesicles with which these rab isoforms are associating will provide important clues to their function.

Interestingly, in a recent report (595) overexpressed, myc-tagged rab3B was localized not to tight junctions in MDCK cells, but to apically located structures that also contained unoccupied pIgA-R. At present, there are no explanations for this different staining pattern. Nonetheless, in the presence of dIgA, rab3B dissociated from pIgA-R and the GTP-bound mutant rab3B impaired dIgA-activated transcytosis to approximately control (-dIgA) levels. Together these results suggest that rab3B is a negative regulator of ligand-stimulated transcytosis in MDCK cells. It will be interesting to see if rab3B also regulates other forms of ligand-activated transcytosis in other cell types and whether the sites of delivery are at or near the tight junction.

Because of the congestion of different rab isoforms in the apical and subapical region of the polarized epithelial cell, it is important to identify the specific intermediates participating in basolateral-to-apical transcytosis. How many different subapical compartments exist, and of those, which receive transcytosing molecules? How do the rabs distribute among them? Furthermore, much of the morphological and functional analysis on the different rab isoforms has been performed in transfected cells overexpressing either wild-type or mutated proteins. Although these studies provided insight into rab function, careful enumeration and examination of the endogenous rab isoforms in a single cell type is required to clearly understand their role in vivo. Nonetheless, the concentration of rab proteins in the subapical regions of epithelial cells is striking and may point to the complexity of intracellular compartments and membrane transport events at this PM domain. It remains to be determined whether other putative transport machinery molecules or rab effectors are also concentrated at the apical regions of cells.

6. The exocyst

Over a dozen genes have been identified in yeast that are required for TGN to PM transport (275, 408, 409), and of these, more than two-thirds of the gene products form a multimeric complex referred to as the exocyst. The exocyst subunits were localized to the yeast PM, but only to sites of rapid cell growth at small bud tips (573). Interestingly, this expression pattern differs from the yeast t-SNARE molecules, which are evenly distributed at the PM. From these data, it was suggested that the exocyst mediates vesicle delivery to restricted regions of the cell surface such that it additionally discriminates (beyond SNARE function) whether and/or where a vesicle docks (573).

Mammalian homologs to the exocyst subunits are ubiquitously expressed and also form a multimeric complex that is mainly peripherally associated with the PM (239, 282, 574). The mammalian counterparts of the yeast Sec6, Sec8, and Sec10 exocyst have been localized to tight junctions in MDCK cells, and their discrete staining patterns were dependent on intact junctional complex formation (200, 324). Anti-rSec6 antibodies blocked transport of LDL-R from the TGN to the basolateral PM in permeabilized cells, whereas direct transport of an apical PM antigen, p75, was not changed (200). Conversely, overexpression of mammalian Sec10 enhanced PM transport of E-cadherin and basolateral secretion, whereas apical delivery of the integral PM protein gp135 was unchanged (324). Surprisingly, Sec10 overexpression also enhanced apical secretion. Taken together, these results implicate the exocyst as an important regulator of vesicle targeting to both PM domains, but delivery to the apical domain may be restricted to vesicles carrying secreted cargo. The placement of the complex further implicates the tight junction as an important site for vesicle delivery to either domain. Whether transcytotic vesicles are specifically recruited to these or other sites on the PM inhabited by the exocyst has not been rigorously examined, but remains an interesting possibility.

7. Annexins

Annexins are a large family of proteins grouped together due to shared amino acid sequence similarity and their biochemical properties (103, 387, 563). One distinguishing feature of most annexins is their ability to aggregate membrane vesicles in the presence of Ca2+ in vitro (103). This activity led to the idea that annexins initiate membrane-membrane contact that results in fusion. Of the numerous annexin isoforms, a few have been identified as playing roles in polarized membrane targeting, but many of those were implicated based only on their subcellular location (see Table 5). However, more direct evidence has been demonstrated for annexins XIIIa and XIIIb, the former being an intestinal-specific isoform and the latter, a kidney-specific isoform (150, 618). In SLO-permeabilized MDCK cells, addition of either of these annexin isoforms enhanced direct apical PM delivery of HA (150, 309). Only the addition of recombinant XIIIa inhibited basolateral delivery of VSV-G. Accordingly, the addition of anti-annexin XIIIb antibodies specifically blocked transport of TGN-derived vesicles to the apical cell surface while transport to the basolateral surface was not changed (299). Unfortunately, the roles of annexin XIIIa and XIIIb in transcytosis were not tested in these studies. Some evidence for a role for annexin II in transcytosis comes from studies performed in isolated hepatocyte couplets (620). Upon induction of transcytosis by the addition of bile salts, annexin II immunofluorescence increased dramatically and was redistributed sequentially from beneath the basolateral PM to perinuclear structures and finally to the apical pole (620). Whether the annexin was simply a passenger on these transcytotic intermediates or was mediating transport activity is not known.

8. Dynamin

Dynamins are a family of high molecular mass (100 kDa), peripheral membrane-associated GTPases that function in the early stages of endocytosis (502, 586). Examination of dynamin function in transfected or microinjected mammalian cells has indicated its participation in clathrin-mediated endocytosis (106, 568) and suggested a role for dynamin in mediating fission of caveolae from the PM (223, 416). A role for dynamin in transcytosis has best been established in endothelial cells. Not only was dynamin found associated with caveolae in these cells, but it was also identified as a required factor for the release of caveolae from endothelial PM preparations in vitro (416). Caveolae-mediated internalization of cholera toxin B was also impaired in cultured endothelial cells expressing dynamin dominant negative constructs (416). Although these results imply that dynamin is a general regulator of caveolae-mediated internalization in endothelial cells, the effects of the dominant negative dynamins on transcytosing proteins were not analyzed.

Examination of dynamin isoforms in other polarized mammalian epithelial cells has been limited to cultured MDCK cells. Dynamin-2 was found at both PM domains and corresponding defects in IgA and Tf internalization from both domains were observed in cells expressing dominant negative dynamin mutants (7). Interestingly, dynamin-1 (the brain-specific isoform) localized only to the apical PM, and when the dominant negative dynamin-1 was expressed, internalization was inhibited only from that domain (7). Whether this is a physiologically relevant finding is not yet clear but may point to similarities in PM dynamics at the apical domain in epithelial cells and at the synapse in neurons. Nonetheless, many transcytosing receptors (including pIgA-R) are internalized via clathrin-coated vesicles, a process requiring dynamin activity. The prediction is that dynamin is required for the internalization of transcytosing molecules in polarized epithelial cells. Does dynamin regulate internalization of all transcytosing molecules, through caveolae, clathrin-coated, or noncoated endocytic vesicles, and at both PM domains?

B. Cytoskeleton

1. Microtubules and microtubule-based motors

In addition to the asymmetric distribution of PM proteins, the polarity of epithelial cells is also reflected in the organization of the cytoskeleton. In nonpolarized cells, microtubules emanate from a juxtanuclear microtubule organizing center (MTOC). In polarized cells, there is accumulating evidence that microtubules are instead (or additionally?) organized from sites at or near the apical PM such that the emanating microtubules are oriented with their minus ends at the apical PM and their plus ends attached to or near the basolateral PM (22, 131, 374, 475). Such an arrangement also dictates the placement of organelles within the interior of the epithelial cell (410, 434). In particular, in many epithelial cells, the compartments of the transcytotic pathway are linearly situated along the parallel microtubules. However, this arrangement is not universal to all polarized cells. For example, the microtubules in endothelial cells are arranged parallel, perpendicular, and obliquely to the long axis and in some cases even form criss-crossed helical arrays (53, 481).

In general, disruption of microtubules does not inhibit internalization of molecules (either soluble of membrane-associated) into early endosomal compartments, but does impair their movement to other compartments. This is true for molecules destined for transcytosis and internalized from the basolateral side in epithelial cells where the transcellular path is long, such as in colchicineor nocodazole-treated MDCK cells, isolated hepatocytes or Caco-2 cells (56, 110, 137, 190, 222, 246, 346, 362). Interestingly, addition of colchicine impaired transcytotic delivery of albumin in endothelial cells in situ despite the comparatively short distance between the apical and basolateral surfaces (13). However, addition of microtubule disrupting agents does not impair transcytosis of molecules internalized from the apical surface in MDCK or Caco-2 cells (246, 362). The reasons for this differential dependence on microtubules to the two domains are not yet understood. One possibility is that basolaterally destined vesicles have a shorter path to the lateral surface and thus a higher probability of encountering their target by simple diffusion. Alternatively, the machinery associated with basolaterally destined vesicles may promote more efficient and specific binding interactions with their target.

Microtubules are probably not a direct requirement for transcytosis; they likely facilitate delivery by providing the tracks upon which vesicles are translocated (43). Thus, when microtubules are disrupted, the kinetics of delivery are slowed, i.e., it takes a vesicle longer to encounter its appropriate target membrane by diffusion than when tracked along microtubules. However, this passive role for microtubules does not account for the mistargeting observed for some transcytosing molecules upon microtubule disruption. For example, the apical PM proteins, aminopeptidase N, DPP IV, and alkaline phosphatase, lost their polarized expression patterns in Caco-2 cells treated with nocodazole or colchicine (56, 137, 190). One explanation for the mistargeting is that different vesicle populations require the activities of distinct assemblies of docking and fusion molecules that differ in their binding specificities. Those vesicles with more promiscuous docking capabilities are able to associate with the improper domain upon microtubule disruption (e.g., apically targeted vesicles) resulting in apparent missorting. Alternatively, the mistargeting may reflect normal basolateral delivery of these PM proteins and the subsequent need for microtubules to facilitate transport to the apical PM domain.

The microtubule-based motor molecule that has received the most attention as a possible regulator of transcytosis is cytoplasmic dynein. In vitro analysis indicated that this megadalton, multisubunit molecule translocates vesicles in an ATP-dependent manner toward microtubule minus ends and that its activity is enhanced by another megadalton, multisubunit molecule, dynactin (279, 373). Since the microtubule minus ends are anchored in the apical PM in many epithelial cells, it is thought that dynein mediates delivery of vesicles from the basolateral to the apical cell surface. Although this is an attractive proposal, the evidence confirming it is only circumstantial and largely stems from the effects of microtubule disruption on transcytosis. Additional evidence comes from the observation that transport in this direction is NEM sensitive. Although NEM sensitivity is a litmus test for the involvement of NSF in transport, this alkylating agent is also a potent inhibitor of dynein ATP-ase activity at similar concentrations (93).

Despite the absence of direct evidence that dynein regulates transcytotic vesicle delivery, a recent study examining the trafficking of transfected rhodopsin in MDCK cells implicates this motor in direct apical vesicle targeting. The 14-kDa endogenous dynein light chain Tctex-1 directly binds transfected rhodopsin in MDCK cells. When RP3, a non-rhodopsin-binding Tctex-1 homolog, was overexpressed in MDCK cells, it displaced the endogenous Tctex-1 in the dynein complex and disrupted apical delivery of rhodopsin (567). Interestingly, the apical distributions of HA and gp135 were not changed in these cells, nor were any basolateral antigens, suggesting that distinct light chains might regulate vesicle translocation. In permeabilized MDCK cells, when cytoplasmic dynein activity was abolished by either ultraviolet/vanadate photocleavage or immunodepletion from cytosolic extracts, direct apical delivery was impaired (298). On the other hand, immunodepletion of kinesin, a plus-end directed microtubule motor, inhibited transport to both the basolateral (to the plus ends as expected) and the apical (to the minus ends which was unexpected) domains. One possible explanation for this last result is that kinesin may be required to translocate vesicles through the microtubule meshwork that is postulated to exist between the Golgi and apical PM in MDCK cells (22). Finally, a surprising finding is that apical-organized microtubules were not required for transport from the TGN to either cell surface domain in MDCK cells (199). This implies that microtubules of both polarities, and by extension, both motor proteins facilitate transport to either PM domain, a report that contradicts numerous reports. Such continued confusion highlights the need for more information from multiple systems before we can confidently assign specific and/or generalizable roles for microtubules and their motors in transcytosis.

2. Actin and actin-based motors

The actin cytoskeleton also has a unique organization in many polarized cells. In general, actin microfilaments extend to the basolateral PM and form attachments through interactions with proteins of zonulae adherens, tight junctions, and focal adhesions. At the apical surface, actin is found as the core filament of microvilli and also as a dense subcortical web (58, 146, 366). At the basolateral domain, the actin-associated proteins fodrin and ankyrin form a scaffold that restricts the movements of certain integral PM proteins, including the Na+-K+-ATPase, thereby stabilizing the basolateral population (208, 366, 386). At the apical surface, actin was shown to be an important factor in maintaining the apical distribution of gp135, a membrane glycoprotein in MDCK cells, even in cells not in contact with their neighbors (419).

In general, actin is thought to be an important regulator of endocytosis from the apical, but not basolateral, PM (16). In MDCK, Caco-2, and pancreatic acinar cells, addition of the actin disrupting agent cytochalasin D impaired internalization only from the apical domain (197, 261, 528). Clathrin-coated pits accumulated at the apical PM in each cell type, suggesting a block in clathrin-mediated internalization. In treated Caco-2 cells, apical internalization of folate was also decreased, implying that caveolae-mediated internalization was altered, and in MDCK cells, apical uptake of Lucifer yellow, which is internalized via noncoated vesicles, was decreased (197, 261). Thus actin may be important in many modes of apical internalization. However, there are exceptions to this generalization, as it was observed that the receptor-mediated internalization of ligand from the basolateral domain of hepatocytes was impaired by cytochalasin B treatment (280). Furthermore, treatment of Caco-2 cells with latrunculin B, another actin-disrupting agent, misdirected basolaterally internalized Tf-R and LDL-R into the apically directed transcytotic pathway (136). Limited evidence from studies in MDCK cells suggests that actin is also involved in facilitating steps in postendocytic transcytotic trafficking. In the presence of cytochalasin D, the delivery of transcytosing dIgA from the basolateral early endosome to the apical recycling endosome was impaired 45% (346). Interestingly, dIgA transcytosis was completely blocked when both cytochalasin D and nocodazole were added, suggesting that microfilaments and microtubules work in concert to facilitate transport to the apical cell surface (16, 346).

In support of the evidence indicating a role for actin in regulating membrane dynamics, recent studies have also implicated specific actin-based motors as important players in polarized vesicle trafficking. In particular, many classes of myosin motors have been localized to the subcortical actin network in many different epithelial cell types. The single-headed, short-tailed myosin I isoform has been localized to the apical brush border of intestinal (440) and kidney cells (94). They have also been found in association with zymogen granules at the apical aspect of pancreatic acinar cells (452) and in hepatocytes (25, 95). Also present at the intestinal brush border, although much less abundant, are the related myosin isoforms V and VI (218). Myosin VI has also been placed at the apical brush border of the proximal tubule cell line LLC-PK1 (215), whereas myosin Va has been also localized to subapical structures in polarized hepatic WIF-B cells (321) and myosin Vb at the apical recycling endosome in MDCK cells.

Functional studies on two different myosin isoforms have also placed them as potential regulators of transcytosis. Brush-border myosin 1 (BBM1) plays a role in postendocytic traffic at the basolateral pole, whereas myosin Vb is functioning at the apical pole (136, 304, 315). RhoA and rac1, two small GTPases that regulate actin cytoskeletal dynamics, also have also been implicated as regulators of polarized membrane transport with rhoA functioning at the basolateral surface and rac1 at the apical PM (270, 315). In particular, basolateral-to-apical transcytosing IgA accumulated in basolateral early endosomes in MDCK cells expressing inactivated forms of rhoA, whereas it accumulated in apical early endosomes in cells expressing dominant negative forms of myosin Vb or a constitutively activated form of rac1 (270, 304, 315). The mechanisms by which these motors, GTPases, and actin regulate membrane transport in polarized epithelial cells are not yet known but are the subject of a recent review (16).

C. Lipids and Transcytosis

The identification of proteins as important regulators of membrane transport is widely accepted and nearly indisputable. In the last several years, it has become more accepted that lipids also play significant roles in membrane transport. In particular, phosphoinositides, PC, cholesterol, and glycosphingolipids have been shown to be important players. Here we will focus on the roles of phosphoinositide 3-phosphate [PI(3)P], cholesterol, and glycosphingolipids in polarized membrane transport. Other lipids and their modifying enzymes have been the subject of many recent reviews (99, 242, 353).

1. PI(3)P

In the past several years, the role PI(3)P lipids play in regulating membrane transport has received considerable attention. This interest arose from early studies examining the effects of the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin and LY294002 on membrane trafficking (98, 524, 584). To date, many transport pathways, including transcytosis, have been shown to be wortmannin and/or LY294002 sensitive. In MDCK cells, wortmannin treatment impaired basolateral to apical dIgA transcytosis (78, 212). Likewise, both wortmannin and LY294002 disrupted transcytosis of pIgA-R and three newly synthesized resident apical PM proteins to the WIF-B apical domain (582) while wortmannin perfusion in isolated rat livers decreased the biliary release of basolaterally internalized HRP (156). Both agents were also observed to impair transcytosis in both directions of ricin in FRT cells and of neonatal FcR in IMCD cells (212, 370). Although the transcytosing proteins in treated WIF-B cells eventually reached their final destination (the apical PM), they transiently accumulated in basolateral early endosomes, indicating a block early in the transcytotic pathway (583).

Mammalian cells encode at least three different classes of PI 3-kinase isoforms (23, 162). Class I includes the p85/p110 heterodimeric kinases which consist of an 110-kDa catalytic subunit associated with a regulatory 85-kDa subunit. The other member is PI 3-kinase-{gamma}, whose catalytic activity is regulated by the {beta}{gamma}-subunits of heterotrimeric G proteins (80, 162, 591a). Class II PI 3-kinases include higher molecular weight kinases that contain C2 domains and class III kinases share the highest sequence similarity with the sole isoform identified in yeast, Vps34p (224, 604). The last kinase is also under the control of a regulatory subunit, p150 (in mammalian cells) or Vps15p (in yeast) (225, 427). All mammalian PI 3-kinase isoforms are sensitive to wortmannin and LY294002 (class II kinases at higher concentrations), which has led to ambiguity in distinguishing the roles that specific PI 3-kinases play in membrane transport. However, more recent studies that examined the effects of microinjection of specific inhibitory reagents on membrane transport have helped to begin assigning specific roles to specific kinase (529).

Using these inhibitory reagents in polarized hepatic cells, we found that specific inhibition of the class III PI 3-kinase Vps34p led to the formation of prelysosomal vacuoles containing endocytosed resident apical PM proteins and to the transient accumulation of transcytosing apical proteins in basolateral early endosomes (583). These results indicate that the lipid product of Vps34p, PI(3)P, regulates the two endocytic pathways differentially, at an early endosomal stage from the basolateral surface and from prelysosomes to lysosomes from the apical surface. The current model of PI(3)P's role in endocytosis invokes recruitment of Vps34p/p150 by activated rab5 to the sites of endosome-endosome fusion and local production of PI(3)P (98, 559, 625). The PI(3)P-binding protein early endosomal antigen 1 (EEA1) is recruited to these sites where PI(3)P and rab5 binding stabilize its membrane association. The stabilized EEA1 molecules then form oligomers that coordinate the formation of a large vesicle docking site also containing NSF and syntaxin13, all of which drive endosome fusion. Thus, in nonpolarized cells, when PI(3)P lipids were depleted by wortmannin, EEA1 association with early endosomes was lost and the subsequent events were affected. Likewise, we found that EEA1 dissociated from basolateral early endosomes in treated WIF-B cells; concomitantly, we observed delayed basolateral to apical transcytosis. All of these results are consistent with the current model. In contrast, the block we observed at a late endocytic step in the apical pathway is not consistent with the existing model (583). Disruption of EEA1 function in the fusion of early endosomes arising from the apical surface should have blocked an earlier step in the pathway. However, a subpopulation of EEA1 near the apical surface remained membrane-bound under PI(3)P-depleting conditions, suggesting that the protein remained active and allowed progression of endocytosed proteins along the apical route thereby revealing a block downstream.

Injection of p110{alpha} inhibitory antibodies into WIF-B cells was also found to impair basolateral to apical transcytosis as did anti-Vps34p injection (583). However, increased basolateral surface staining of the transcytosing markers was observed with no corresponding intracellular accumulations, suggesting that Vps34p and p110{alpha} act at separate steps of the pathway, with p110{alpha} possibly acting at internalization. Together, these results lead to a number of questions. Are the apical and basolateral pathways in other polarized epithelial cells also differentially regulated by PI(3P)? Are the functions of p110{alpha} and Vps34p in basolateral-to-apical transcytosis conserved among other epithelial cells? What PI(3)P-binding proteins are required for membrane transport at the distinct steps?

2. Cholesterol and glycosphingolipids

Glycosphingolipids and cholesterol are enriched in cell surface membranes in all eukaryotic cells. In polarized epithelial cells, the apical surface is even further enriched for these lipid species (158, 281, 295, 597). One possible function of cholesterol and glycosphingolipids is to impart structural rigidity and decreased permeability to the apical domain, which in turn protects the cell against the harsh external environment it faces (e.g., the detergent-like bile or high acidity) (295). The differences in these environments would therefore dictate the lipid compositions required for appropriate protection and function in different epithelial cell types. The intrinsic properties of glycosphingolipids and cholesterol promote their assembly into specialized membrane domains called "rafts" (63, 214). Within these cell surface domains are selected proteins that are recruited based on their biophysical properties. In particular, GPI-anchored proteins that are predominantly expressed at the apical surfaces of epithelial cells localize to rafts.

These observations in combination with studies performed mainly in MDCK cells have led to the "raft" hypothesis for protein sorting. According to this hypothesis, rafts form in the biosynthetic pathway where they recruit apically destined proteins (especially GPI-linked proteins); the rafts with their recruited cargo are then transported directly to the apical domain in vesicles. There is considerable experimental evidence to support this hypothesis. For example, GPI anchors have been shown to be sufficient to target proteins to the apical domain (62, 325). Furthermore, rafts have been isolated based on their insolubility in nonionic detergents (especially Triton X-100) at 4°C, GPI-anchored proteins copurify with them (297), and cholesterol-depleting drugs and sphingolipid synthesis inhibitors disrupt delivery of apical PM residents (252, 297). Recent work has also suggested that caveolins and caveolae-like vesicles are important for PM delivery of GPI-anchored proteins. In caveolin-1 or caveolin-3 knock-out mice, GPI-anchored proteins were retained at the TGN in mouse embryo fibroblasts or muscle tissue, respectively (551).

However, there are many observations that are inconsistent with the raft hypothesis of sorting (612). In FRT cells, both glycosphingolipids and GPI-anchored proteins are sorted to the basolateral domain, whereas in certain MDCK strains, they are evenly distributed between the two domains. However, in both cases, other apical PM proteins are sorted properly to the apical surface. In hepatocytes, GPI-anchored proteins, such as 5'-nucleotidase, are first transported to the basolateral domain before apical delivery (499). Furthermore, many nonapical proteins have also been detected in purified raft fractions.

Despite the debate, the question still remains. Do rafts sort apically destined proteins in the transcytotic pathway? If so, are rafts present at the basolateral domain or in other transcytotic intermediates? Two approaches have been taken to begin answering these questions. First is examining whether transcytosing proteins are present in detergent-insoluble fractions and second is whether they are internalized from the cell surface via caveolae, specialized raft domains. So far, the first approach has yielded conflicting results from studies performed in enterocytes as well as FRT and MDCK cells (210, 493). In polarized enterocytes from mouse intestinal explants, a significant proportion of basolateral to apical transcytosing IgA (secreted from neighboring mucosal plasma cells) was found in detergent-insoluble rafts (210). Because IgA is internalized and delivered to the apical surface via the pIgA-R, the data imply that the receptor must also be raft associated. Accordingly, ~50% of the pIgA-R was recovered in detergent-insoluble fractions (210). This result contradicts that reported for pIgA-R in MDCK and FRT cells; pIgA-R was not found in Triton X-100-insoluble fractions at any point during its life cycle (493). The reasons for these opposing observations are likely not due to differences in raft preparation, since the methods used were very similar. Instead, the differences may be related to the intrinsic differences between cell types, between in vitro versus in vivo systems, or between endogenously (in enterocytes) or exogenously (in FRT and MDCK cells) expressed molecules. None of these possibilities has yet been well explored.

The second approach to determine whether rafts sort apically destined proteins in the transcytotic pathway has focused on examining interactions of transcytosing proteins with caveolae. Because not all rafts are associated with caveolin, caveolae have more recently been classified as specialized rafts. Much of what we know about caveolae in polarized epithelial cells comes from studies in endothelial cells where these structures are highly abundant (see sect. IV). Otherwise, caveolae have been examined in only a limited number of polarized cells. Despite the enrichment of cholesterol and glycosphingolipids at the apical domain, caveolae have only been observed at the basolateral domain of MDCK cells and in kidney epithelial cells in situ (57, 435, 498). Like endothelial cells, MDCK caveolae formation is dependent on cholesterol levels (205, 267, 511). In both cases, no morphologically definable caveolae were observed in cells treated with cholesterol-depleting drugs such as filipin or cyclodextrin. In endothelial cells, the cholesterol-dependent loss of caveolae corresponded to decreases in albumin transcytosis both in vitro and in vivo (267, 511). When the drugs were withdrawn and cholesterol synthesis stimulated by the addition of mevalonate in MDCK cells, or the addition of 10–20% serum in endothelial cells, the caveolae reformed (205, 511). FRT and Caco-2 cells express little to no detectable caveolin, and no caveolae have been observed (322, 603). However, when caveolin-1 was overexpressed in these cells, caveolae were observed in both domains in FRT cells and only at the basolateral domain of Caco-2 cells (322, 603). Interestingly, the formation of caveolae in FRT cells did not promote apical sorting of GPI-anchored proteins or their sorting into rafts (322), suggesting that rafts are not responsible for apical targeting in these cells. Alternatively, other components may be lacking in FRT cells that are necessary for sorting into rafts and/or subsequent apical delivery.

Our challenge is to begin carefully examining the detergent solubility properties of multiple endogenous apical PM protein types (e.g., GPI-linked, single transmembrane, polytopic) during their entire life cycles within one cell type to determine the role of rafts in transcytosis. The detergents used should not be limited to Triton X-100, as it has been recently reported that in PC12 cells, subpopulations of rafts exist with different solubility properties (482). The effects of both cholesterol and glycosphingolipid disrupters on transcytosis should be directly tested. Also, examination of purified organellar intermediates for raft components and protein insolubility properties may help clarify at which step(s) rafts are required. Such a strategy has shown that recycling endosomes (immunoisolated with anti-Tf-R antibodies) are enriched for raft components including glycosphingolipids, cholesterol, caveolin, and another raft-associated protein, flotillin (167). Such careful and consistent experimentation will more clearly determine the role of rafts in apical vesicle targeting. Recent evidence also suggests that the PM-associated t-SNAREs are organized in cholesterol-dependent surface domains in nonpolarized cells (87, 303). Are these domains required for vesicle delivery to the apical PM?

D. Perturbations of Transcytosis

The use of perturbants has long been a way to begin dissecting the mechanisms and molecules involved in regulating complex cellular processes. Chemical perturbants are commonly used and have provided insight into understanding vesicle transport in mammalian cells. Of particular note are alkylating agents (e.g., NEM), drugs that specifically disrupt the cytoskeletal systems of the cell and specific lipid kinase inhibitors which were discussed in section V, AC. In this section, we focus on the perturbation of acute regulation of vesicle transport by agents that alter the functions of heterotrimeric G proteins, intracellular calcium (Cai) homeostasis, protein kinase A (PKA), or protein kinase C (PKC) activity (Table 6).


1. Heterotrimeric G proteins and PKA

Accumulating evidence supports a role for heterotrimeric G proteins in regulating vesicle transport in both the endocytic and exocytic pathways (4, 41, 42, 220, 412, 445, 561). Examination of the effects of various agents on epithelial cells, especially MDCK cells, have indicated that transcytosis is also regulated by G proteins. The first clues came from treating cells with nonselective G protein activators, guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) and AlF. In both cases, transcytosis to the apical, but not basolateral, domain was slightly enhanced (42). Addition of specific G protein ADP-ribosylating toxins (cholera or pertussis toxins) further indicated that transport to the apical domain (from both the TGN and via transcytosis) is regulated by the Gs{alpha}-subunit (31, 42, 211). Mastoparan, (a Gi-activating peptide) had no effect on pIgA-R apical delivery while anti-Gs {alpha}-antibodies were slightly inhibitory, confirming a role for Gs in transcytosis to the apical domain (42). In intact cells, the overexpression of the wild-type or constitutively active Gs{alpha}-subunit led to a modest increase in transcytosis (211). Interestingly, the addition of either the Gs{alpha}- or {beta}{gamma}-subunits to an in vitro system led to a small increase in the formation of transcytotic vesicles (42). However, it is not known whether the {beta}{gamma}-subunits were acting to inhibit another G protein that negatively regulates transcytosis or in concert with the {alpha}-subunit.

One well-studied result of Gs activation is the activation of adenylyl cyclase and the subsequent increased production of cAMP. PKA is then activated by the increased cAMP levels, which puts in motion numerous (but not well-defined) cellular processes. To determine whether this cascade of events is involved in vesicle transport, another handful of chemical perturbants has been useful. In particular, forskolin (a direct activator of adenylyl cyclase) has been shown to enhance transcytosis to the apical, but not basolateral, domain in MDCK cells (211). This effect was also seen by the addition of exogenous cAMP to both MDCK cells and intact rat hepatocytes (211, 217). A PKA inhibitor, H-89, produced the opposite effect, consistent with a role for PKA in regulating transcytosis (211). Some evidence suggests that G protein/PKA regulates transcytosis from apical endosomal compartments to the apical PM (31). Interestingly, this is also the site(s) at which at least nine small-molecular-weight GTP-binding proteins have been proposed to function, further exposing the complexity of vesicle transport events in this region of a polarized epithelial cell.

Trimeric G proteins are also important regulators of endothelial transcytosis, but in this case, Gi has been examined most extensively. In polarized endothelial cells in vitro, addition of pertussis toxin (a Gi inhibitor) or expression of a dominant negative Gi{alpha} peptide inhibited apical to basolateral albumin transcytosis mediated by activated gp60 (379). Previously, treatment of endothelial cells with tyrosine kinase inhibitors suggested that a src-mediated signaling pathway regulated transcytosis (Ref. 576 and discussion in sect. IVA3); thus Minshall and colleagues examined whether Gi and src signaling were coupled. Consistent with this hypothesis, overexpression of dominant negative src prevented the association of Gi{alpha} with caveolin (caveolae?) in caveolin-1-overexpressing cells. Furthermore, dominant negative src also inhibited albumin transcytosis. From these results, the authors suggested that gp60 activation recruits Gi to caveolae that in turn sets off a src-mediated signaling cascade that activates transcytosis (379). This situation is somewhat analogous to the activation of pIgA-R transcytosis by ligand binding where another tyrosine kinase, p62yes, is involved (341). Thus tyrosine phosphorylation may be a common mechanism for regulating activated transcytotic pathways.

2. Calcium, calmodulin, and PKC

The use of another set of pharmacological agents has indicated that vesicle trafficking is also acutely regulated by changes in Cai levels. Thapsigargin, a selective inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases, enhanced transcytosis to the apical domain in MDCK cells, whereas BAPTA, a Ca2+-chelating compound, inhibited it (79). Neither agent altered transcytosis in the opposite direction. The mechanism whereby Cai fluxes are manifested in changes in vesicle transport is not known, but likely includes alterations in Ca2+-dependent enzyme activities. This has been substantiated by studies examining the effects of the calmodulin antagonists W-7, W-13, and trifluoperazine in MDCK cells (18, 244, 329). All three agents significantly impaired basolateral to apical transcytosis of dIgA while transcytosis in the same direction of the fluid-phase marker ricin was enhanced. In both cases, endocytosis from the basolateral surface was unchanged, suggesting that the agents were acting later in the pathway. Consistent with this is the finding that dIgA accumulated in large endosomal structures located in the apical region of cells treated with W-13 (18). Interestingly, apical to basolateral transcytosis of ricin was not altered by these agents, yet an increase in its endocytosis from the apical domain was observed (329). However, megalin-mediated transcytosis of thyroglobulin in the same direction was inhibited in thyroid cells (FRTL-5) treated with W-7 and trifluoperazine (349). Thus CaM regulation is important for many steps in transcytosis. The challenge is to pinpoint the specific calmodulin-dependent enzymes that are functioning at these transport steps to understand the differential effects of these agents.

PKC has also received attention as a possible regulator of transcytosis based both on the effects of changing Cai levels and on the use of phorbol esters, potent PKC activators. When one such phorbol ester, phorbol 12-myristate 13-acetate (PMA), was applied to MDCK cells, both apical recycling and basolateral to apical transcytosis of dIgA and transferrin were enhanced, suggesting PKC was acting at an apical recycling compartment (79). PMA treatment also led to the membrane recruitment of {alpha} and {epsilon} PKC isoforms (79). Interestingly, dIgA binding to its receptor (conditions that activate its own transcytosis) also activated PKC-{epsilon}, which led to increased levels of inositol 1,4,5-trisphosphate and Cai, the latter of which stimulated transcytosis (79, 548). The rise in Cai is likely mediated via inositol 1,4,5-trisphosphate-sensitive intracellular stores; thus ligand binding initiates a signal that is propagated across the cell independent of the ligand-receptor complex itself (340). Paradoxically, treatment of MDCK cells with H-7, a specific PKC inhibitor, did not inhibit dIgA or ricin transcytosis in MDCK cells (18, 329). At present, there is no good explanation for these disparate results.

3. Possible mechanisms

In all the studies cited above, it is important to point out that in many cases, the inhibitory and stimulatory effects of transfection or addition of pharmacological agents on PM targeting were small. Does the size of the response in vitro reflect loss of normal regulation that would be observed in vivo or does it represent fine-tuning that may be critical for proper organ function? If it is the latter, what appears to be a minimal change in vitro may have a large impact on the overall homeostatic balance of the organism. Thus physiological studies are needed. Specifically, our challenge is to identify the molecules in membrane transport that are regulated by phosphorylation or calmodulin/Ca2+ binding. Few if any direct links have yet been established, but there are some examples of where the modifications of molecules implicated in transcytosis correlate with their proposed functions.

Many of the SNARE molecules are phosphorylated in vitro by purified kinases, and the modification alters their binding properties. In particular, {alpha}-SNAP is a substrate for PKA and when phosphorylated, its ability to bind the core docking and fusion complex was decreased 10-fold (230). In vitro, syntaxin 4 was shown to be phosphorylated by PKA, casein kinase II (CKII), and PKC, and this phosphorylation disrupted its binding to SNAP23 or SNAP25 (92, 159, 474). When syntaxin 4 was used as bait in a yeast two-hybrid screen, a novel SNARE kinase (SNAK) was identified, but surprisingly, SNAP-23 was overwhelmingly its preferred substrate in vitro and in vivo (73). SNAK-phosphorylated SNAP-23 was not associated with the ternary complex, whereas phosphorylation of syntaxin 1 by CKII enhanced t-SNARE's association with SNAP-25 (154). Interestingly, by using phosphospecific antibodies to stain neurons, it was found that the phosphorylated form of syntaxin 1 was localized to discrete regions along the axonal PM that did not colocalize with synaptic vesicles (154). Another SNARE hypothesis molecule, Munc18–1, is a substrate for PKC and cyclin-dependent kinase 5, and phosphorylation in this case inhibited binding to syntaxin 1 (123a, 164, 527).

Several rabs and rab effector proteins have also been shown to be phosphorylated. In vitro, GDI phosphorylation is mediated by PKA (555). In vivo, the phosphorylated GDI associated to the cytosolic form of rab5, while the unphosphorylated GDI was bound to the membrane-associated rab5. These data suggest that the cycling of rab proteins between donor and acceptor membranes is also a regulated process. The rab effector protein rabphilin 3A is phosphorylated by PKA in vitro (411). Another rab effector, rab8ip, is a serine/threonine protein kinase itself (GC kinase) that is activated by the stress response in lymphocytes (466). The phosphorylation state of rip11, a rab11 effector, may regulate its membrane binding properties. In polarized MDCK cells, conditions that decrease rip11 phosphorylation (e.g., staurosporine treatment) enhanced its binding to membranes (458). Caveolin, the major structural protein of caveolae, has been shown to be tyrosine phosphorylated in endothelial cells under conditions where transcytosis was stimulated (191, 318, 358). Both kinesin and cytoplasmic dynein are phosphorylated in vitro and in vivo, and this modification has been correlated with their ability to transport vesicles (including transcytotic vesicles?) along microtubules (320, 486, 494). The light chain of the actin based myosin I motor proteins is calmodulin, which is thought to regulate motor activity (449). Annexin binding to membrane lipids, and by extension, ability to promote intermembrane associations, is dependent on Ca2+ (103).

E. Transcytosis Versus Direct PM Delivery

Transcytosis is only one pathway that molecules take to a specific PM domain. Both newly synthesized PM proteins and secreted molecules can also be delivered directly from the TGN to either PM domain. How different are the mechanisms regulating vesicle transport along these pathways? As expected, transport directly from the TGN to either the apical or basolateral domains is regulated differently. In particular, differences in the involvement of SNARE molecules were observed in permeabilized MDCK cells (17, 253, 332). Addition of anti-NSF antibodies, NEM, mutant NSF, rab-GDI, or tetanus and botulinum F neurotoxins all inhibited basolateral targeting of VSV-G protein, whereas targeting from the TGN to the apical domain of HA was not changed (17, 253). This implies that basolateral targeting requires NSF, rab proteins, and VAMP 2. Conversely, syntaxin 3 overexpression or anti-syntaxin 3 antibodies inhibited only apical delivery (300, 332). Addition of {alpha}-SNAP antibodies and treatment of cells with botulinum E inhibited transport to both domains (17, 253), whereas TI-VAMP antibodies inhibited apical transport (the effects of theses antibodies on basolateral targeting were not examined) (300). Taken together, targeting to both domains requires SNARE molecules, but in different combinations. Surprisingly, apical targeting is NSF independent, suggesting the involvement of an as yet unidentified homolog. Microtubule and actin filament disruption also has differential effects on direct delivery to either domain in MDCK cells; apical delivery is inhibited, whereas, in most cases, basolateral is not (56, 137, 190, 362, 420, 598). In addition, PKA, PKC, and calmodulin-mediated mechanisms appear to acutely regulate delivery mainly to the apical domain (79, 329, 447).

How different are the targeting mechanisms regulating transcytosis? This question has so far been best addressed in studies performed in MDCK cells. Unlike TGN to apical delivery of HA-containing vesicles, pIgA-R-mediated transcytosis appears to require NSF activity (17, 253). Furthermore, syntaxin 3 (required for direct apical targeting) is not involved in mediating transcytosis of IgA (332). SNAP-23, on the other hand, is involved in both basolateral to apical transcytosis, and direct apical and basolateral targeting (332). Also, both direct and transcytotic delivery to the basolateral domain does not require microtubules (see sect. VB). Based on the effects of pertussis toxin on MDCK cells, transport from the TGN to the basolateral PM was found to involve the Gi{alpha} subunit of heterotrimeric G proteins, whereas transcytosis to this domain was not (31, 211, 446). From these results (and others) it is clear that the mechanisms cells use to regulate cell surface delivery are complex and are dependent on factors that are not yet understood.

Another interesting twist to apical PM sorting has come from studies looking at the raft-associated protein referred to as MAL. This 17-kDa tetra-spanning TMD protein, first identified in myelin and lymphocytes (hence MAL), is also expressed in many epithelial cell types where it is concentrated at the TGN (287). With the use of an antisense approach, it was shown that MDCK cells lacking MAL showed decreased specific apical delivery of a single TMD apical protein surrogate, the influenza HA the ectopic expression of human MAL rescued the defect (459). Thus MAL has been implicated as an important player in apical sorting. Interestingly, liver does not express this isoform of MAL, a finding consistent with the absence of a direct apical delivery mechanism for the single-TMD class of apical PM proteins in hepatocytes. Recently, another MAL family member has been identified, MAL2, that is enriched in hepatic cells (120). In HepG2 cells treated with antisense MAL2 oligonucleotides, transcytosis of pIgA was impaired from early endosomes to a juxta-apical compartment. Thus different MAL isoforms may be responsible for specialized domain-specific protein sorting. This is further suggested by the finding that yet another MAL family member, BENE, is expressed in endothelial cells where it is associated with caveolae (119).

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