Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Apical macropinocytosis in polarized MDCK cells: regulation by N-ethylmaleimide-sensitive proteins

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.     

EFA6 regulates endosomal trafficking and affects early endosomes in polarized MDCK cells

The small-GTPase family of ADP ribosylation factors (ARFs) recruit coat proteins to promote vesicle budding. ARFs are activated by an association with sec7-containing exchange factors which load them with GTP. In epithelial cells, the small GTPase ARF6 operates within the endocytic system and has been shown to associate with ARNO to promote apical endocytosis and early to late endosomal trafficking. EFA6 has been shown to stimulate tight-junction formation and maintenance. Here, we show that in polarized epithelial MDCK cells, EFA6 is localized to early endosomes, causes their dramatic enlargement, and promotes basolateral targeting of IgA, which is normally targeted to the apical PM. These results suggest that the physiological function of ARF6 within the endocytic system is regulated by the exchange factor it associates with.     

Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO(4)) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a cellular compartment in close proximity but not overlapping with the basolateral surface. Surface biotinylation studies indicate that hephaestin in the peri-basolateral location is accessible to the extra-cellular environment. These results support the hypothesis that hephaestin is involved in iron mobilization of iron from the intestine to circulation.     

Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells.

Lysosomal acid phosphatase (LAP) is synthesized as a type I membrane glycoprotein and targeted to lysosomes via the plasma membrane. Its cytoplasmic tail harbours a tyrosine-containing signal for rapid internalization. Expression in Madine-Darby canine kidney cells results in direct sorting to the basolateral cell surface, rapid endocytosis and delivery to lysosomes. In contrast, a deletion mutant lacking the cytoplasmic tail is delivered to the apical plasma membrane where it accumulates before it is slowly internalized. A chimeric protein, in which the cytoplasmic tail of LAP is fused to the extracytoplasmic and transmembrane domain of the apically sorted haemagglutinin, is sorted to the basolateral plasma membrane. A series of truncation and substitution mutants in the cytoplasmic tail was constructed and comparison of their polarized sorting and internalization revealed that the determinants for basolateral sorting and rapid internalization reside in the same segment of the cytoplasmic tail. The cytoplasmic factors decoding these signals, however, tolerate distinct mutations indicating that different receptors are involved in sorting at the trans-Golgi network and at the plasma membrane.     

Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medical recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.     

Apical to basolateral surface area ratio and polarity of MDCK cells grown on different supports.

We have established conditions under which Madin-Darby canine kidney cells develop a well-polarized monolayer on polycarbonate filters and on transparent filters. These filters have biochemical and mechanical advantages over the nitrocellulose filters which have been widely used. Transepithelial resistance was established 10 h after plating and stabilized after 24 h. The distribution of protein antigens was followed by surface immunofluorescence and quantitated by a surface immunoassay that we developed. Uvomorulin was localized to the lateral membrane, with low amounts detectable on the basal membrane. The 58-kDa antigen was distributed over the entire basolateral domain, including cell processes extending into the filter pores. This distribution was confirmed by immunogold labeling of frozen sections. The 114-kDa antigen was found to be present at similar surface densities on both the apical and the basolateral domain. The support used for growth had profound effects on the cell morphology. A morphometric analysis of the plasma membrane of both strains of the cell line showed an increase in the number and size of the microvilli, and a smoother basal membrane as compared to published data on nitrocellulose filters. The apical to basolateral surface area ratio was therefore modified.     

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.     

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin

E-cadherin plays an essential role in cell polarity and cell-cell adhesion; however, the pathway for delivery of E-cadherin to the basolateral membrane of epithelial cells has not been fully characterized. We first traced the post-Golgi, exocytic transport of GFP-tagged E-cadherin (Ecad-GFP) in unpolarized cells. In live cells, Ecad-GFP was found to exit the Golgi complex in pleiomorphic tubulovesicular carriers, which, instead of moving directly to the cell surface, most frequently fused with an intermediate compartment, subsequently identified as a Rab11-positive recycling endosome. In MDCK cells, basolateral targeting of E-cadherin relies on a dileucine motif. Both E-cadherin and a targeting mutant, DeltaS1-E-cadherin, colocalized with Rab11 and fused with the recycling endosome before diverging to basolateral or apical membranes, respectively. In polarized and unpolarized cells, coexpression of Rab11 mutants disrupted the cell surface delivery of E-cadherin and caused its mistargeting to the apical membrane, whereas apical DeltaS1-E-cadherin was unaffected. We thus demonstrate a novel pathway for Rab11 dependent, dileucine-mediated, mu1B-independent sorting and basolateral trafficking, exemplified by E-cadherin. The recycling endosome is identified as an intermediate compartment for the post-Golgi trafficking and exocytosis of E-cadherin, with a potentially important role in establishing and maintaining cadherin-based adhesion.     

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.     

A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells.

An important function of the low affinity IgG Fc receptor FcRII-B2 (FcR) on macrophages is the internalization of soluble antigen-antibody complexes for lysosomal degradation. Most endocytic receptors possess tyrosine-containing cytoplasmic determinants required for endocytosis. In many proteins, signals which overlap with the endocytosis determinant and share the same critical tyrosine residue also mediate basolateral sorting in the trans-Golgi network of epithelial cells. Despite the presence of two tyrosine residues in the FcR cytosolic domain, neither one is absolutely required for coated pit localization or basolateral targeting. Nevertheless, a short domain of 13 residues containing one of the non-critical tyrosine residues mediates endocytosis and basolateral delivery. Alanine scan mutagenesis of this region now revealed a critical role of a leucine-leucine motif in both events. These findings suggest that endocytosis and basolateral sorting can be mediated by both tyrosine- and di-leucine-based signals and confirm the close relationship between the two determinants already observed for 'classical' tyrosine-dependent motifs.     

Specific efflux of glutathione from the basolateral membrane domain in polarized MDCK cells during ricin-induced apoptosis.

Although the depletion of reduced glutathione (GSH) has been observed in a variety of apoptotic systems, little is known about the mechanism of GSH depletion. In this study we used polarized MDCK cells to study the GSH flux during ricin-induced apoptosis. Here we report that the specific accumulation of GSH occurred in the basolateral medium during ricin treatment with similar kinetics to in apoptotic changes such as an increase in caspase-3 like activity and DNA fragmentation, while there was no significant increase in the GSH level in apical medium. These results suggest that GSH efflux occurred through a GSH-specific channel or transporter located in the basolateral membrane domain of polarized MDCK cells undergoing apoptosis. Treatment with other protein toxins such as modeccin, Pseudomonas toxin, and diphtheria toxin, which can induce apoptotic cell death, also resulted in selective GSH efflux from the basolateral side. Thus, GSH efflux through a specific transporter may be a common step of apoptosis induced by these toxins, while these toxins have different intoxication mechanisms leading to protein synthesis inhibition. Pretreatment of cells with Z-Asp-CH(2)-DCB, a caspase family inhibitor, inhibited ricin-induced basolateral GSH efflux as well as DNA fragmentation, suggesting that the activation of caspases, i.e. those that are inhibited by Z-Asp-CH(2)-DCB, is implicated in the opening of the GSH transporter.     

Apical and non-polarized secretion of serpins from MDCK cells

Corticosteroid binding globulin, a member of the serpin family, was previously shown to be secreted mainly apically from MDCK cells in an N-glycan independent manner [Larsen et al. (1999) FEBS Lett. 451, 19-22]. Apart from N-glycosylation, serpins are not known to carry any other posttranslational modifications, suggesting the presence of a proteinaceous apical sorting signal. In the present study we have expressed four other members of the serpin family: alpha1-antitrypsin, C1 inhibitor, plasminogen activator inhibitor-1 and antithrombin in MDCK cells. Tight monolayers of transfected cells were grown on filters and the amounts of recombinantly expressed serpins in the apical and the basolateral media were determined. alpha1-Antitrypsin and C1 inhibitor were found mainly in the apical medium whereas plasminogen activator inhibitor-1 and antithrombin were found in roughly equal amounts in the apical and basolateral media. Control experiments showed that all four serpins are transported along the exocytotic pathway in an uncomplicated way that does not involve transcytosis or differences in stability on the two sides of the cells. We conclude that some members of the serpin family including corticosteroid binding globulin, alpha1-antitrypsin and C1 inhibitor are secreted mainly apically from MDCK cells whereas plasminogen activator inhibitor-1 and antithrombin are secreted in a non-polarized manner.     

Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells

In MDCK cells, basolateral sorting of most membrane proteins has been shown to depend on distinct cytoplasmic domain determinants. These signals can be divided into those which are related to signals for localization at clathrin-coated pits and those which are unrelated. The LDL receptor bears two tyrosine-containing signals, one of each class, that can independently target receptors from the Golgi complex and from endosomes to the basolateral plasma membrane. We have now investigated the other structural features required for the activity of both determinants. We find that both depend, at least in part, on clusters of 1-3 acidic amino acids located on the COOH-terminal side of each tyrosine. While single residues adjacent to each tyrosine were also found to be critical, the two signals differed in that only the coated pit-unrelated signal could tolerate a phenylalanine in place of its tyrosine residue. We also found that the structural requirements for basolateral targeting of the "coated pit-related" signal were distinct from those required for rapid endocytosis. Apart from sharing a common tyrosine residue, no feature of the NPXY motif for coated pit localization was required for basolateral targeting. We also investigated basolateral targeting of the mouse macrophage Fc receptor (FcRII-B2) which contains a tyrosine-independent coated pit localization signal. Basolateral transport and endocytosis were found to depend on a common dileucine-type motif. Thus, basolateral targeting determinants, like coated pit domains, can contain either tyrosine- or di-leucine-containing signals. The amino acids in the vicinity of these motifs determine whether they function as determinants for endocytosis, basolateral targeting, or both.