Membrane traffic in polarized epithelial cells.

Epithelial cells contain apical and basolateral surfaces with distinct compositions. Sorting of certain proteins to the basolateral surface involves the epithelial-specific mu 1b clathrin adaptor subunit. Recent results have shown that targeting to the basolateral surface utilizes the exocyst, whereas traffic to the apical surface uses syntaxin 3. Endocytosis at the apical surface is regulated by ARF6. Transcytosis of IgA is regulated by the p62Yes tyrosine kinase.

Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly.

Cellular polarization involves the generation of asymmetry along an intracellular axis. In a multicellular tissue, the asymmetry of individual cells must conform to the overlying architecture of the tissue. However, the mechanisms that couple cellular polarization to tissue morphogenesis are poorly understood. Here, we report that orientation of apical polarity in developing Madin-Darby canine kidney (MDCK) epithelial cysts requires the small GTPase Rac1 and the basement membrane component laminin. Dominant-negative Rac1 alters the supramolecular assembly of endogenous MDCK laminin and causes a striking inversion of apical polarity. Exogenous laminin is recruited to the surface of these cysts and rescues apical polarity. These findings implicate Rac1-mediated laminin assembly in apical pole orientation. By linking apical orientation to generation of the basement membrane, epithelial cells ensure the coordination of polarity with tissue architecture.

Apical targeting in polarized epithelial cells: There's more afloat than rafts.

Most metazoan cells are 'polarized'. A crucial aspect of this polarization is that the plasma membrane is divided into two or more domains with different protein and lipid compositions or example, the apical and basolateral domains of epithelial cells or the axonal and somatodendritic domains of neurons. This polarity is established and maintained by highly specific vesicular membrane transport in the biosynthetic, endocytic and transcytotic pathways. Two important concepts, the 'SNARE' and the 'raft' hypotheses, have been developed that together promise at least a partial understanding of the underlying general mechanisms that ensure the necessary specificity of these pathways.

An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby canine kidney cells.

The polymeric immunoglobulin receptor is expressed in a variety of polarized epithelial cells. Newly made receptor travels first to the basolateral surface. The receptor is then endocytosed, transported across the cell in vesicles, and exocytosed at the apical surface. We have now deleted the membrane spanning and cytoplasmic portions of the receptor by site-directed mutagenesis, thus converting the receptor to a secretory protein. When expressed in polarized Madin-Darby canine kidney (MDCK) cells the truncated protein is secreted at both surfaces, with a ratio of apical-to-basal secretion of 3.4. In contrast, when the exogenous secretory protein chicken lysozyme is expressed in these cells, it is released at both sides with a ratio of apical-to-basal secretion of 0.43. (Koder-Koch, C., R. Bravo, S. Fuller, D. Cutler, and H. Garoff, 1985, J. Cell Biol., 43:297-306). Lysozyme is thought to lack a signal that targets it to one surface or the other, and so its secretion may represent a default, bulk flow pathway to both surfaces. When compared with lysozyme, the truncated polymeric immunoglobulin receptor is preferentially secreted apically by a factor of 3.4:0.43 or 7.8. We suggest that the lumenal portion of the polymeric immunoglobulin receptor contains a signal that targets it to the apical surface.

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes.

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non-transcytosing molecules.

Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells.

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.

Functional expression of the polymeric immunoglobulin receptor from cloned cDNA in fibroblasts.

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.

Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells.

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.

Dynamics of beta-catenin interactions with APC protein regulate epithelial tubulogenesis.

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, beta-catenin, with several molecular partners have been shown to be important for cell signaling and cell-cell adhesion. To examine if beta-catenin has a role in tubulogenesis, we tested the effect of expressing NH2-terminal deleted beta-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH2-terminal deleted beta-catenins inhibited formation of these cell extensions. Both DeltaN90 beta-catenin, which binds to alpha-catenin, and DeltaN131 beta-catenin, which does not bind to alpha-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of beta-catenin distinct from its role in cadherin-mediated cell-cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH2-terminal deleted beta-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH2-terminal deleted beta-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for beta-catenin in cell migration and indicate that dynamic beta-catenin-APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide.

NH2-terminal deletion of beta-catenin results in stable colocalization of mutant beta-catenin with adenomatous polyposis coli protein and altered MDCK cell adhesion.

beta-Catenin is essential for the function of cadherins, a family of Ca2+-dependent cell-cell adhesion molecules, by linking them to (alpha)-catenin and the actin cytoskeleton. beta-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant beta-catenins in MDCK epithelial cells to gain insights into the regulation of beta-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length beta-catenin, beta-catenin mutant proteins with NH2-terminal deletions before (deltaN90) or after (deltaN131, deltaN151) the alpha-catenin binding site, or a mutant beta-catenin with a COOH-terminal deletion (delta C) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All beta-catenin mutant proteins form complexes and colocalize with E-cadherin at cell-cell contacts; deltaN90, but neither deltaN131 nor deltaN151, bind alpha-catenin. However, beta-catenin mutant proteins containing NH2-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal-deleted beta-catenin poorly colocalize with APC protein. NH2-terminal deletions result in increased stability of beta-catenin bound to APC protein and E-cadherin, compared with full-length beta-catenin. At low density, MDCK cells expressing NH2-terminal-deleted beta-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH2 terminus, but not the COOH terminus of beta-catenin, regulates the dynamics of beta-catenin binding to APC protein and E-cadherin. Changes in beta-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of beta-catenin binding to alpha-catenin. These results demonstrate that regulation of beta-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.

Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells.

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells.

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207-1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle-like structures as well as from secretory granules.

Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis.

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5-trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.

The SNARE machinery is involved in apical plasma membrane trafficking in MDCK cells.

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide-sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against alpha-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide-sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and alpha-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.

Redundant and distinct functions for dynamin-1 and dynamin-2 isoforms.

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.

Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis.

The endosomal compartment of polarized epithelial cells is a major crossroads for membrane traffic. Proteins entering this compartment from the cell surface are sorted for transport to one of several destinations: recycling to the original cell surface, targeting to lysosomes for degradation, or transcytosis to the opposite surface. The polymeric immunoglobulin receptor (pIgR), which is normally transcytosed from the basolateral to the apical surface, was used as a model to dissect the signals that mediate this sorting event. When exogenous receptor was expressed in Madin-Darby Canine Kidney (MDCK) cells, it was shown that phosphorylation of pIgR at the serine residue at position 664 is required for efficient transcytosis. Replacement of this serine with alanine generated a receptor that is transcytosed only slowly, and appears to be recycled. Conversely, substitution with aspartic acid (which mimics the negative charge of the phosphate group) results in rapid transcytosis. It was concluded that phosphorylation is the signal that directs the pIgR from the endosome into the transcytotic pathway.

Alternate splicing of rabbit polymeric immunoglobulin receptor.

Rabbits have a minimum of two polymeric immunoglobulin receptor primary translation products. A cDNA clone of the smaller product lacked two of the five receptor domains. These two domains were on a single exon. As there was one receptor gene, we suggest that this exon can be spliced in or out.

Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway.

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.

Dimerization of the polymeric immunoglobulin receptor controls its transcytotic trafficking.

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-zeta chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:zeta chimeric receptor expressed in Jurkat cells initiates a zeta-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR's transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.