Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.     

N-glycans are not a universal signal for apical sorting of secretory proteins.

In MDCK cells, N-glycans have been shown to determine the sorting of secretory proteins and membrane proteins to the apical domain in the absence of a dominant basolateral targeting signal. We have examined the sorting of endogenous proteins in ECV304 cells in the presence and absence of tunicamycin, an inhibitor of N-linked glycosylation. A prominent apically secreted protein of 71 kDa was not N-glycosylated and continued to be secreted apically in the presence of tunicamycin. In contrast, other endogenous proteins that were N-glycosylated were secreted preferentially into the basolateral medium or without polarity. When rat growth hormone was expressed in MDCK and ECV304 cells, we observed 65 and 94% of the secretion to the basolateral medium, respectively. Introduction of a single N-glycan caused 83% of the growth hormone to be secreted at the apical surface in MDCK cells but had no significant effect on the polarity of secretion of growth hormone in ECV304 cells. These results indicate that not all cell lines recognise N-glycans as a signal for apical sorting and raises the possibility of using ECV304 cells as a model system for analysis of apical sorting molecules.     

Guanosine nucleotides modulate the inhibitory effect of brefeldin A on protein secretion

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the endoplasmic reticulum (ER), leaving no definable Golgi-apparatus, and blocks transport of proteins from the ER to distal secretory compartments of the cell. Using pulse-chase experiments the present study shows that BFA (1 microgram/ml) inhibits basal and CCK-stimulated protein secretion in isolated pancreatic acinar cells by 65 +/- 6% and 84 +/- 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 micrograms/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (1 mM). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP-binding protein. Investigating the effect of BFA on small molecular weight GTP-binding proteins we observed that [alpha-32P]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP-binding proteins may be the molecular target of Brefeldin A in pancreatic acinar cells.     

A Secretory Golgi Bypass Route to the Apical Surface Domain of Epithelial MDCK Cells

Proteins leave the endoplasmic reticulum (ER) for the plasma membrane via the classical secretory pathway, but routes bypassing the Golgi apparatus have also been observed. Apical and basolateral protein secretion in epithelial Madin-Darby canine kidney (MDCK) cells display differential sensitivity to Brefeldin A (BFA), where low concentrations retard apical transport, while basolateral transport still proceeds through intact Golgi cisternae . We now describe that BFA-mediated retardation of glycoprotein and proteoglycan transport through the Golgi apparatus induces surface transport of molecules lacking Golgi modifications, possessing those acquired in the ER. Low concentrations of BFA induces apical Golgi bypass, while higher concentrations were required to induce basolateral Golgi bypass. Addition of the KDEL ER-retrieval sequence to model protein cores allowed observation of apical Golgi bypass in untreated MDCK cells. Basolateral Golgi bypass was only observed after the addition of BFA or upon cholesterol depletion. Thus, in MDCK cells, an apical Golgi bypass route can transport cargo from pre-Golgi organelles in untreated cells, while the basolateral bypass route is inducible.     

A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells

Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.     

The Viral Spike Protein Is Not Involved in the Polarized Sorting of Coronaviruses in Epithelial Cells

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK_MHVR cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK_MHVR cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020–2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.     

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.     

Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells.

The effects of brefeldin A (BFA) on transferrin (Tf) transcellular transport, Tf receptor (TfR) distribution, and TfR-mediated endocytosis in filter-grown Madin-Darby canine kidney (MDCK) cells were studied. BFA (1.6 micrograms/ml) markedly enhanced the transcytosis of 125I-labeled Tf (125I-Tf) in both apical-to-basal and basal-to-apical directions; yet, BFA did not enhance the transcytosis of either native horseradish peroxidase (HRP) or membrane-bound HRP-poly(L-lysine) conjugates. Furthermore, this enhanced transcytosis of 125I-Tf was abolished either by competition with excess unlabeled Tf or by incubation at temperatures less than or equal to 25 degrees C. In addition, BFA treatment to MDCK cells: (a) increased 125I-Tf specific binding to the apical membrane and decreased 125I-Tf specific binding to the basal membrane; (b) decreased TfR recycling at the basolateral membrane; (c) altered the apical/basolateral distribution of TfRs in favor of the apical side; and (d) markedly increased 59Fe extraction, but not transcytosis, from apically endocytosed 59Fe-loaded Tf. These effects are consistent with a model in which BFA alters the traffic pattern of internalized Tf by decreasing basolateral TfR recycling, while diverting the nonrecycled fraction to the apical side of the cell. Our results indicate that, unlike the reported inhibition of polymeric IgA transcytosis (Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627), BFA can enhance the transcytosis of Tf in MDCK cells. Thus, by altering the intracellular traffic of ligand-receptor complexes, BFA can elicit either a decrease or an increase in transcytosis depending on the nature of the intracellular receptor processing.     

Selective anchoring in the specific plasma membrane domain: a role in epithelial cell polarity

We have studied the role of restrictions to lateral mobility in the segregation of proteins to apical and basolateral domains of MDCK epithelial cells. Radioimmunoassay and semiquantitative video analysis of immunofluorescence on frozen sections showed that one apical and three basolateral glycoproteins, defined by monoclonal antibodies and binding of beta-2-microglobulin, were incompletely extracted with 0.5% Triton X-100 in a buffer that preserves the cortical cytoskeleton (Fey, E. G., K. M. Wan, and S. Penman. 1984. J. Cell Biol. 98:1973-1984; Nelson, W. T. and P. J. Veshnock. 1986. J. Cell Biol. 103:1751-1766). The marker proteins were preferentially extracted from the "incorrect" domain (i.e., the apical domain for a basolateral marker), indicating that the cytoskeletal anchoring was most effective on the "correct" domain. The two basolateral markers were unpolarized and almost completely extractable in cells prevented from establishing cell-cell contacts by incubation in low Ca++ medium, while an apical marker was only extracted from the basal surface under the same conditions. Procedures were developed to apply fluorescent probes to either the apical or the basolateral surface of live cells grown on native collagen gels. Fluorescence recovery after photobleaching of predominantly basolateral antigens showed a large percent of cells (28- 52%) with no recoverable fluorescence on the basal domain but normal fluorescence recovery on the apical surface of most cells (92-100%). Diffusion coefficients in cells with normal fluorescence recovery were in the order of 1.1 x 10(-9) cm2/s in the apical domain and 0.6-0.9 x 10(-9) cm2/s in the basal surface, but the difference was not significant. The data from both techniques indicate (a) the existence of mobile and immobile protein fractions in both plasma membrane domains, and (b) that linkage to a domain specific submembrane cytoskeleton plays an important role in the maintenance of epithelial cell surface polarity.     

Brefeldin A inhibits protein synthesis in cultured cells.

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1 microgram/ml. Inhibition was both time-dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA-resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA-sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.     

The influence of pH on the vesicular traffic to the surface of the polarized epithelial cell, MDCK

The effect of pH on the secretion of the gp 80 glycoprotein complex and lysozyme from MDCK cells was examined by treatment of the cells with either NH4Cl, chloroquine or monensin. In untreated cells gp 80 is sorted with approximately 75% efficiency into the apical pathway. Lysozyme is secreted in a nonpolar fashion at both cell surfaces. Treatment of the cells with the drugs had nearly identical effects on the transport kinetics and on the ratio of the proteins released at the two plasma membrane domains. At increasing drug concentrations, the transport of both proteins to the apical and the basolateral cell surface was equally retarded. Furthermore, we observed a dose-dependent decrease in the amount of gp 80 and lysozyme released at the basolateral cell surface, which was accompanied by a nearly equivalent increase in the secretion of the two proteins at the apical plasma membrane domain. A twofold rise in the apical to basolateral ratio was already found at drug concentrations which only marginally affected the kinetics of transport. These results show that an increase in intravesicular pH not only redirects secretory proteins sorted into the basolateral pathway (Caplan et al. Nature, 329, 632 (1987] but also secretory proteins devoid of sorting information for that pathway, presumably by modulating the vesicular traffic to the basolateral cell surface.     

Access to nectin favors herpes simplex virus infection at the apical surface of polarized human epithelial cells

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.     

Low Rho activity in hepatocytes prevents apical from basolateral cargo separation during trans‐Golgi network to surface transport

Hepatocytes, the main epithelial cells of the liver, organize their polarized membrane domains differently from ductal epithelia. They also differ in their biosynthetic delivery of single‐membrane‐spanning and glycophosphatidylinositol‐anchored proteins to the apical domain. While ductal epithelia target apical proteins to varying degrees from the trans‐Golgi network (TGN) to the apical surface directly, hepatocytes target them first to the basolateral domain, from where they undergo basolateral‐to‐apical transcytosis. How TGN‐to‐surface transport differs in both scenarios is unknown. Here, we report that the basolateral detour of a hepatocyte apical protein is due, in part, to low RhoA activity at the TGN, which prevents its segregation from basolateral transport carriers. Activating Rho in hepatocytic cells, which switches their polarity from hepatocytic to ductal, also led to apical‐basolateral cargo segregation at the TGN as is typical for ductal cells, affirming a central role for Rho‐signaling in different aspects of the hepatocytic polarity phenotype. Nevertheless, Rho‐induced cargo segregation was not sufficient to target the apical protein directly; thus, failure to recruit apical targeting machinery also contributes to its indirect itinerary.     

Vesicular stomatitis virus infects and matures only through the basolateral surface of the polarized epithelial cell line,MDCK

We have used Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters to study the polarity of virus infection and maturation. The cells form epithelia-like monolayers, which display high (>1000 Ω cm²) electrical resistance and a cuboidal morphology. Vesicular stomatitis virus (VSV) was found to infect the monolayer at least 100 times more efficiently when applied through the filter to the basolateral surface than when applied to the apical surface. The avian influenza, fowl plague virus (FPV), infected the monolayer through either the apical or basolateral surface. The polarity of virus budding was evaluated by harvesting virus from the two sides of the monolayer. More than 99% of released influenza hemagglutinin titre was found on the apical side of the filter, while more than 98% of budded VSV was found on the basal side. This polarity of budding was retained through 10 hr of viral infection, as was the polarity of surface expression of viral envelope proteins revealed by immunofluorescence. The strong preference of VSV for basolateral maturation is paralleled by an equally strong preference for infection through the basolateral membrane of this polar epithelial cell.     

Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells

We have studied the effects of brefeldin A (BFA) on endocytosis and intracellular traffic in polarized MDCK cells by using the galactose-binding protein toxin ricin as a membrane marker and HRP as a marker of fluid phase transport. We found that BFA treatment rapidly increased apical endocytosis of both ricin and HRP, whereas basolateral endocytosis was unaffected, as was endocytosis of HRP in the poorly polarized carcinoma cell lines HEp-2 and T47D. Tubular endosomes were induced by BFA both apically and basolaterally in some MDCK cells, comparable with those seen in HEp-2 and T47D cells. In addition, in MDCK cells, BFA induced formation of small (< 300 nm) vesicles, labeled both after apical and basolateral uptake of HRP, as well as some very large (> 700 nm) vacuoles, which were only labeled when HRP was present in the apical medium. In contrast, neither in MDCK nor in HEp-2 or T47D cells, did BFA have any effect on lysosomal morphology. Moreover, transcytosis in the basolateral-apical direction was stimulated both for HRP and ricin. Other vesicular transport routes were less affected or unaffected by BFA treatment. Two closely related structural analogues of BFA (B16 and B21), unable to produce the changes in Golgi and endosomal morphology seen after BFA treatment in a number of different cell lines, were also unable to mimic the effects of BFA on MDCK cells.     

Isolation and characterization of mutant CHO cell lines with compartment-specific resistance to brefeldin A

22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle- specific coats.     

Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome

We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule- requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).     

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.     

Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2)

The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco-2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface.