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 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.

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.

Brefeldin A (BFA) inhibits basolateral membrane (BLM) delivery and dimerization of transcobalamin II receptor in human intestinal epithelial Caco-2 cells. BFA effects on BLM cholesterol content.

Brefeldin A (BFA) treatment of Caco-2 cells (5 microg/ml for 12 h) reduced by 90% the cholesterol, but not the phospholipid (PL), levels of the basolateral membrane (BLM), thus altering its PL/cholesterol molar ratio from 2.6 to 22.0, and decreasing its steady state fluorescent anisotropy (rs) from 0.27 to 0.15. BFA treatment for 12 h also resulted in complete loss of transcobalamin II receptor (TC II-R) activity/protein levels in the BLM and the disappearance of trans-Golgi network (TGN) morphology as revealed by confocal immunofluorescence microscopy using antibody to TGN 38. However, BFA treatment had no effect on either total cellular cholesterol, TC II-R activity, or PL levels. When cells treated with BFA for 12 h were exposed to BFA-free medium for 0-24 h, all of the effects were reversed, including reappearance of normal TGN morphology. TC II-R delivered to the BLM during this period was progressively sialylated and changed its physical state from a monomer (8 h) to a dimer (12 h), coinciding with increased delivery (11-53 pmol) of cholesterol to the BLM and an increase in the BLM rs from 0.15 to 0.21. These results indicate that cholesterol, but not PL, delivery to the BLM of Caco-2 cells is BFA-sensitive, and cholesterol, by influencing the higher order of the BLM, is essential for TC II-R dimerization.

A conserved domain is present in different families of vesicular fusion proteins: a new superfamily.

We have analyzed conserved domains in t-SNAREs [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors in the target membrane], proteins that are believed to be involved in the fusion of transport vesicles with their target membrane. By using a sensitive computer method, the generalized profile method, we were able to identify a new homology domain that is common in the two protein families previously identified to act as t-SNAREs, the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) families, which therefore constitute a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain was also identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode, and mammalian proteins were identified that belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins.

Overexpression of full- or partial-length MAP4 stabilizes microtubules and alters cell growth.

To investigate the in vivo functions of MAP4, a microtubule-associated protein expressed almost ubiquitously in vertebrate cells, we prepared stably transfected clonal mouse Ltk- cell lines expressing full-length MAP4 (L-MAP4 cells) or its MT-binding domain (L-MTB cells). Although transfectants showed no dramatic defect in morphology, organellar distribution, or level of MT polymer, as compared to naive Ltk- cells or L-MOCK cells (transfected with vector alone), MTs in L-MAP4 and L-MTB cells showed greater stability than those in control cells, as monitored by the level of post-translationally detyrosinated alpha-tubulin and by a quantitative nocodazole-resistance assay. In vivo, the MT-binding domain of MAP4 stabilized MTs less potently than full-length MAP4, in contrast to the equivalent efficacy demonstrated in studies of in vitro MT polymerization (Aizawa et al. (1991), J. Biol. Chem. 266, 9841-9846), L-MAP4 and L-MTB cells grew significantly more slowly than control cells; this growth inhibition was not due to mitotic arrest or cell death. L-MAP4 and L-MTB cells also exhibited greater tolerance to the MT-depolymerizing agent, nocodazole, but not to the MT-polymerizing agent, Taxol. Our results demonstrate that MAP4 and its MT-binding domain are capable of MT stabilization in vivo, and that increasing the intracellular level of MAP4 affects cell growth parameters.

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.

Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells.

Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3, and 4 were found to be endogenously present in Madin-Darby canine kidney cells. The localization of syntaxins 1A, 1B, 2, 3, and 4 in stably transfected Madin-Darby canine kidney cell lines was studied with confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures, with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, whereas the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity.

Calmodulin binds to the basolateral targeting signal of the polymeric immunoglobulin receptor.

We have identified a major calmodulin (CaM)-binding protein in rat liver endosomes using 125I-CaM overlays from two-dimensional protein blots. Immunostaining of blots demonstrates that this protein is the polymeric immunoglobulin receptor (pIgR). We further investigated the interaction between pIgR and CaM using Madin-Darby canine kidney cells stably expressing cloned wild-type and mutant pIgR. We found that detergent-solubilized pIgR binds to CaM-agarose in a Ca(2+)-dependent fashion, and binding is inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide), suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is the most prominent 35S-labeled CaM-binding protein in the detergent phase of Triton X-114-solubilized, metabolically labeled pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically cross-linked to both solubilized and membrane-associated pIgR, suggesting that binding can occur while the pIgR is in intact membranes. The CaM binding site is located in the membrane-proximal 17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR constitutes an autonomous basolateral targeting signal. However, binding of CaM to various pIgR mutants suggests that CaM binding is not necessary for basolateral targeting. We suggest that CaM may be involved in regulation of pIgR transcytosis and/or signaling by pIgR.

Differential expression of alternatively spliced forms of MAP4: a repertoire of structurally different microtubule-binding domains.

We previously reported that the microtubule (MT)-binding domain of microtubule-associated protein 4 (MAP4) contains three 18-amino acid imperfect repeats that are homologous to the repeats found in the MT-binding domains of the neuronal MAPs, MAP2 and tau [Chapin, S. J., & Bulinski, J. C. (1991) J. Cell Sci. 98, 27-36]. Here we report the isolation of clones encoding additional isoforms of MAP4, which differ in the number of repeated elements contained within their MT-binding domains. In addition to clones encoding three repeats, we isolated clones encoding a four-repeat isoform, whose MT-binding domain bears a striking similarity to the four-repeat isoform of tau. Other MAP4 clones that we isolated encode five repeats. The additional repeat in the five-repeat isoform of MAP4 is quite unusual in its amino acid sequence; this unusual repeat was also found by Aizawa et al. [Aizawa, H., et al. (1990) J. Biol. Chem. 265, 13849-13855] among the repeats encoded by bovine MAP4 clones possessing four repeats. In humans, MAP4 was recently shown to be encoded by a single-copy gene [West, R. R., et al. (1991) J. Biol. Chem. 266, 21886-21896]; we demonstrated that the human MAP4 gene is located on human chromosome 3p21. Expression of multiple MAP4 isoforms from this gene, which appears to result from alternative RNA splicing, was investigated by RNase protection analysis of mammalian cell lines and rat tissues. The five-repeat isoform was the only form detectable in most cell lines, and it was the most abundant isoform expressed in rat lung, liver, kidney, spleen, and testis. However, in rat brain, heart, and skeletal muscle, although the five-repeat isoform was expressed at all developmental stages examined, the tau-like four-repeat isoform was also expressed, and its expression increased during development. The three-repeat isoform was expressed in heart and, to a lesser extent, in brain, skeletal muscle, and lung. Our results demonstrate that several different MAP4 isoforms are expressed in the rat in different tissues and at various developmental stages. Furthermore, our data suggest that differential expression of MAP4 isoforms possessing distinct MT-binding domains may be involved in the changes in MT dynamics or function that are known to accompany differentiation.

Cellular microtubules heterogeneous in their content of microtubule-associated protein 4 (MAP4).

Previous immunolocalization studies using many primate cultured cell lines demonstrated that a microtubule-associated protein of M(r) approximately 210,000 which is now called MAP4, is present along the length of microtubules in interphase and mitotic cells [Bulinski and Borisy (1980) J. Cell Biol. 87:802-808; DeBrabander et al. (1981) J. Cell Biol. 91:438-455]. Since MAP4 has been implicated as a microtubule stabilizer, we asked whether all classes of microtubules possess an equal complement of MAP4. We have reexamined the cellular distribution of MAP4, using both conventional double-label immunofluorescence and an antibody blocking technique [Schulze and Kirschner (1987) J. Cell Biol. 104:277-288] to highlight microtubules lacking, or depleted in, MAP4. These techniques have revealed that thin processes extending from monkey kidney cells (TC-7), and those made by human neuroblastoma cells (IMR-32) in response to retinoic acid, are often deficient in MAP4 immunoreactivity. Since both types of cellular processes contain stable microtubules, which are enriched in detyrosinated (Glu) tubulin, we tested the ability of MAP4 to bind to microtubules made from pure Glu and pure tyrosinated (Tyr) tubulin in vitro. MAP4 bound to both types of microtubules, and the similar saturation level of MAP4 binding to Glu and Tyr microtubules suggested that differential binding to these forms of tubulin does not contribute directly to a mechanism for segregation of MAP4 on microtubules in vivo. In TC-7 cells, we also observed MAP4-depletion on single microtubules, distal regions of broad cytoplasmic extensions, and midbodies of dividing cells. MAP4 depletion may reflect recent, rapid growth of microtubules to which MAP4 has not yet bound, or the presence of other MAPs that may compete with MAP4 for binding sites on the MT. We suggest that different levels of MAP4 on microtubules may directly modulate microtubule dynamics within single cells, as well as other microtubule functions such as those involving microtubule motor activity.

Non-neuronal 210 x 10(3) Mr microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domains of neuronal MAP2 and tau.

A polyclonal antiserum raised against a HeLa cell microtubule-associated protein of Mr 210,000 (210 kD MAP or MAP4), an abundant non-neuronal MAP, was used to isolate cDNA clones encoding MAP4 from a human fetal brain lambda gt11 cDNA expression library. The largest of these clones, pMAP4.245, contains an insert of 4.1 kb and encodes a 245 kD beta-galactosidase fusion protein. Evidence that pMAP4.245 encodes MAP4 sequences includes immunoabsorption of MAP4 antibodies with the pMAP4.245 fusion protein, as well as identity of protein sequences obtained from HeLa 210 kD MAP4 with amino acid sequences encoded by pMAP4.245. The MAP4.245 cDNA hybridizes to several large (approximately 6-9 kb) transcripts on Northern blots of HeLa cell RNA. DNA sequencing of overlapping MAP4 cDNA clones revealed a long open reading frame containing a C-terminal region with three imperfect 18-amino acid repeats; this region is homologous to a motif present in the microtubule (MT)-binding domain of two prominent neuronal MAPs, MAP2 and tau. The pMAP4.245 sequence also encoded a series of unrelated repeats, located in the MAP's projection domain, N-terminal to the MT-binding domain. MAP4.245 fusion proteins bound to MTs in vitro, while fusion proteins that contained only the projection domain repeats failed to bind specifically to MTs. Thus, the major human non-neuronal MAP resembles two neuronal MAPs in its MT-binding domain, while most of the molecule has sequences, and presumably functions, distinct from those of the neuronal MAPs.