RESUMEN
Previous results from our laboratory have indicated a requirement for CK intermediate filaments (IF) for the organization of the apical domain in polarized epithelial cells in culture. The results seemed to be challenged by the phenotype of cytokeratin (CK) 8-deficient mice, which comprises only colorectal hyperplasia, female sterility and a weaker hepatocyte integrity. In this work localization with anti-CK antibodies indicated that many Ck8-/- epithelia still form IF in CK8-deficient mice, perhaps because of the expression of the promiscuous CK7. In the small intestine, only villus enterocytes lacked IFs. These cells appeared to lose syntaxin 3, and three apical membrane proteins (alkaline phosphatase, sucrase isomaltase and cystic fibrosis transmembrane conductance regulator) as they progressed along the villus. At the distal third of the villi, gamma-tubulin was found scattered within the cytoplasm of enterocytes, in contrast to its normal sub-apical localization, and the microtubules were disorganized. These results could not be attributed to increased numbers of apoptotic or necrotic cells. The only other cell type we found without IFs in CK8 null mice, the hepatocyte, displayed increased basolateral levels of one apical marker (HA4), indicating a correlation between the lack of intermediate filaments and an apical domain phenotype. These data suggest a novel function for intermediate filaments organizing the apical pole of simple polarized epithelial cells.
Asunto(s)
Filamentos Intermedios/metabolismo , Queratinas/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Femenino , Yeyuno/citología , Yeyuno/metabolismo , Queratinas/genética , Queratinas/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Datos de Secuencia Molecular , FenotipoRESUMEN
The striking similarities between microvillus inclusions (MIs) in enterocytes in microvillus inclusion disease (MID) and vacuolar apical compartment in tissue culture epithelial cells, led us to analyze endoscopic biopsies of duodenal mucosa of a patient after the samples were used for diagnostic procedures. Samples from another patient with an unrelated disease were used as controls. The MID enterocytes showed a decrease in the thickness of the apical F-actin layer, and normal microtubules. The immunofluorescence analysis of the distribution of five apical membrane markers (sucrase isomaltase, alkaline phosphatase, NHE-3 Na+/H+ exchanger, cGMP-dependent protein kinase, and cystic fibrosis trans-membrane conductance regulator), showed low levels of these proteins in their standard localization at the apical membrane as compared with normal duodenal epithelium processed in parallel. Instead, four of these markers were found in a diffuse distribution in the apical cytoplasm, below the terminal web (as indicated by co-localization with F-actin and cytokeratin 19), and in MIs as well. The basolateral protein Na(+)-K+ATPase, in contrast, was normally localized. These results support the hypothesis that MID may represent the first genetic defect affecting apical membrane traffic, possibly in a late step of apical exocytosis.
Asunto(s)
Actinas/análisis , Enfermedades Duodenales/patología , Enterocitos/ultraestructura , Cuerpos de Inclusión/ultraestructura , Proteínas de la Membrana/análisis , Microvellosidades/ultraestructura , Membrana Celular/química , Membrana Celular/ultraestructura , Polaridad Celular , Células Cultivadas , Preescolar , Enfermedades Duodenales/genética , Enfermedades Duodenales/metabolismo , Femenino , Humanos , Cuerpos de Inclusión/química , Queratinas/análisis , Microvellosidades/química , Transporte de Proteínas , Vesículas Secretoras/química , Vesículas Secretoras/ultraestructuraRESUMEN
We have previously shown that a thin ( approximately 1 microm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of gamma-tubulin-containing structures (potential microtubule-organizing centers) in CACO-2 cells and demonstrated perfect colocalization of centrosomes and nearly 50% of noncentrosomal gamma-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide-mediated downregulation of cytokeratin 19, using two different antisense sequences, was more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitated in multi-protein fragments of the cytoskeleton with gamma-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol did not affect coimmunoprecipitation. These results from three independent techniques, indicate that insoluble gamma-tubulin-containing structures are attached to apical intermediate filaments.
Asunto(s)
Polaridad Celular , Centrosoma/metabolismo , Células Epiteliales/citología , Filamentos Intermedios/metabolismo , Queratinas/metabolismo , Tubulina (Proteína)/metabolismo , Acrilamida/farmacología , Actinas/metabolismo , Células CACO-2 , Centrosoma/efectos de los fármacos , Centrosoma/ultraestructura , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Regulación hacia Abajo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/ultraestructura , Queratinas/genética , Microscopía Electrónica , Peso Molecular , Nocodazol/farmacología , Oligonucleótidos Antisentido/genética , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina , Unión Proteica/efectos de los fármacos , SolubilidadRESUMEN
In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40-70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical-basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37 degrees C or in n-octyl-beta-D-glycoside at 4 degrees C (representative of GPI-anchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+-K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.
Asunto(s)
Polaridad Celular/fisiología , Citoesqueleto/fisiología , Queratinas/fisiología , Actinas/análisis , Animales , Células CACO-2 , Línea Celular , Regulación hacia Abajo , Células Epiteliales , Humanos , Intestino Delgado/química , Queratinas/análisis , Proteínas de la Membrana/análisis , Microvellosidades , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , ARN Mensajero , Ratas , Estómago/química , Tubulina (Proteína)/análisisRESUMEN
Because the mechanisms that govern mitosis are a key to the understanding of cell growth, the proteins associated with chromosomes specifically during this phase have received thorough attention. In the present work we report an Mr58000 protein in MDCK epithelial cells, recognized by a monoclonal antibody (LFM-1) that decorates chromosomes during M-phase. Cell fractionation methods followed by immunoblotting and immunofluorescence showed that this protein is associated with the nuclear fraction. Biochemical extraction procedures on isolated metaphase chromosomes from nocodazole-synchronized cells indicated that the Mr58000 protein behaves as a chromosomal scaffold protein, that is, it remains in the pellets after high salt (2M NaCl) or 3'-5' diiodosalicylic acid treatments, even in DNAse pre-digested samples. In addition, confocal microscopy of those chromosomes revealed the LFM-1 epitopes distributed on the external surface and the axis of chromatids. Parallel analysis of interphase nuclei revealed LFM-1 epitopes inside G1-, but excluded from G2-phase nuclei. These results were independently confirmed on nuclei sorted by flow cytometry and in cell populations synchronized by release of G1-/S-phase hydroxyurea arrest. The Mr58000 and a minor Mr38000 protein (which was enriched only in mitotic chromosomes of synchronized cells) were analyzed by Edman degradation. They shared the sequence at the amino-terminal end but failed to show total homology with known proteins. These results suggest that LFM-1 antigens fit some of the predictions of the licensing factor model, and may have a role in cell cycle dependent events.
Asunto(s)
Ciclo Celular , Núcleo Celular/química , Proteínas Nucleares/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Antígenos Nucleares , Línea Celular , Cromátides/química , Perros , Células Epiteliales , Epítopos/análisis , Interfase , Yodobenzoatos , Mitosis , Datos de Secuencia Molecular , Peso Molecular , Nocodazol , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Salicilatos , Cloruro de SodioRESUMEN
Madin-Darby canine kidney and other epithelial cell lines (e.g. Caco-2, MCF-10A and MCF-7) develop intracellular vacuoles composed of apical membrane displaying microvilli (VACs) when impaired from forming normal cell-to-cell contacts. In a previous publication, we showed that VACs are rapidly exocytosed upon treatment with 8-Br-3',5'-cyclic adenosine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, and that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present work, we tested the hypothesis that cAMP may be a positive modulator of the 'constitutive' exocytic pathway. To mimic conditions in cells with incomplete intercellular contacts, the intracellular levels of cAMP were decreased by means of two independent approaches: (i) pores were induced in the plasma membrane with the polypeptidic antibiotic subtilin, thus allowing small molecules (including cAMP) to permeate and move out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellular levels of cAMP were measured and, in porated cells, equilibrated to simulate the corresponding physiological intracellular concentrations. The decrease in cAMP within the physiological range resulted in a decreased rate of transport of an apical marker of the constitutive pathway (influenza virus hemagglutinin) from the trans-Golgi network to the apical plasma membrane. Likewise, the delivery of a number of cellular apical proteins to the plasma membrane was retarded at low cAMP concentrations. The inhibitors of adenylate cyclase failed to block basolateral delivery of vesicular stomatitis virus G protein. This differential modulatory effect may represent a differentiation-dependent control of the insertion of apical membrane in epithelial cells.
Asunto(s)
Proteínas Bacterianas , AMP Cíclico/fisiología , Exocitosis , Glicoproteínas de Membrana , Proteínas de la Membrana/metabolismo , Péptidos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenosina/análogos & derivados , Adenosina/farmacología , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Antibacterianos/farmacología , Bacteriocinas , Transporte Biológico , Línea Celular , Polaridad Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Perros , Epitelio , Exocitosis/efectos de los fármacos , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/metabolismo , Riñón , Microvellosidades/metabolismo , Tionucleótidos/farmacología , Vacuolas/metabolismo , Vidarabina/farmacología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Ascites 13762 rat mammary adenocarcinoma cells contain an abundant heterodimeric cell surface glycoprotein complex. It is composed of a transmembrane subunit and a sialomucin subunit and is the product of a single gene. The transmembrane subunit has two EGF-like domains and activates the proto-oncogene receptor kinase p185neu. Southern blot comparisons of the ascites tumor and rat liver demonstrated the presence of the gene encoding the complex in normal tissues and showed an amplification of about fivefold in the ascites tumor. Polymerase chain reaction assays showed the presence of mRNA for the complex in rat brain and lung, but not in liver, pancreas, placenta, intestine, kidney, ovary and uterus. Northern blot analyses showed that the 9 kb transcript for the complex is expressed at an approximately 500-fold higher level in the ascites cells than in rat brain. Immunocytochemical studies using antiserum directed against the transmembrane subunit showed its presence in bronchial epithelium, brain ependymal and neurons of four day old animals and in the endoderm and neuronal cells of embryos. Similar immunocytochemical studies showed the presence of the transmembrane subunit in some human breast tumors. These results suggest that the gene encoding this complex is regulated in a tissue-specific manner, is overexpressed in some tumors and may play a role in tumor progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor ErbB-2/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Northern Blotting , Southern Blotting , Encéfalo/metabolismo , Embrión de Mamíferos/metabolismo , Activación Enzimática , Receptores ErbB/metabolismo , Humanos , Inmunohistoquímica , Pulmón/metabolismo , Mucina 4 , Reacción en Cadena de la Polimerasa , Proto-Oncogenes Mas , Ratas , Células Tumorales CultivadasRESUMEN
Although many pieces of evidence support the notion of a role for the cytoskeleton in epithelial polarization, no cytoskeletal component has been found to be specifically apical, except for some actin-binding proteins. Here we report the apical distribution of a 53 kDa cytokeratin. Furthermore, this cytokeratin co-purified with biotinylated apical plasma membrane proteins in high density complexes. Differential biotinylation of the basolateral domain showed that the 53 kDa protein is mainly attached to the apical membrane, although a companion 58 kDa protein attaches to both apical and basolateral membrane proteins. Immunoprecipitation experiments indicated that a number of apical components are directly or indirectly linked to the 53 kDa protein. These results indicate the existence of a terminal web-like structure in non-brush-border cells, which attaches to the apical domain and may play a role in apical polarization, especially during the acquisition of polarity from non-polarized cellular stages.
Asunto(s)
Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Epitelio/metabolismo , Epitelio/ultraestructura , Animales , Línea Celular , Membrana Celular/metabolismo , Polaridad Celular , Perros , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Queratinas/química , Queratinas/aislamiento & purificación , Queratinas/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Peso MolecularRESUMEN
The MAT-C1 subline of the 13762 rat mammary adenocarcinoma has highly stable, branched microvilli and immobile cell surface receptors. A membrane- and microfilament-associated 58-kDa protein (p58) in the MAT-C1 microvilli has been implicated in the stabilization of the microvilli and microfilament-membrane interactions. This protein is associated with a high M(r) glycoprotein complex containing the (proto)oncogene p185neu and other signal transduction components in a putative microfilament-associated signal transduction particle. Amino acid sequences were obtained from two trypsin peptides of p58. Screening a MAT-C1 cDNA library with a degenerate oligonucleotide derived from the larger peptide and polymerase chain reaction amplification of cDNA ends permitted the isolation of overlapping cDNAs encoding the 427-amino acid open reading frame of p58. In vitro transcription and translation using a full-length cDNA gave a protein of approximately 55 kDa, which reacts with anti-p58 antiserum and reconstitutes into a complex with actin and glycoproteins from the membrane-microfilament interaction site. When COS-7 cells were transfected with the full-length cDNA, p58 was localized in a punctate distribution. In addition, the transfected cells exhibited fewer microfilament cables than untransfected neighboring cells. The amino acid sequence showed a surprising similarity to mammalian retroviral Gag proteins and included regions corresponding to p15, p12 and the N-terminal 80% of p30. Comparisons of p58 and the corresponding regions of the Gag proteins for Moloney murine leukemia virus indicated that about 60% of their amino acid residues were identical. These studies suggest that p58 is the product of an endogenous retroviral gene whose expression as a cellular protein alters the properties of the tumor cell to provide a selective advantage for tumor growth in the animal.
Asunto(s)
Productos del Gen gag/química , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario , ADN Viral , Productos del Gen gag/genética , Proteínas de la Membrana/química , Proteínas de Microfilamentos/química , Microvellosidades/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Ratas , Retroviridae/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Células Tumorales CultivadasRESUMEN
We have previously shown that an integral plasma membrane glycoprotein (AP2) is highly polarized to the apical domain in confluent Madin-Darby canine kidney (MDCK) epithelial cells. However, when the monolayers are prevented from forming intercellular contacts, approximately 60% of the AP2 cellular content is stored in the intracellular vacuolar apical compartment (VAC). In the current work we found that AP2 was present in the non-tumorigenic human mammary epithelial cell line MCF-10A, in the breast carcinoma cell lines MCF-7 and T47D, and in breast ductal carcinomas in vivo. By radioimmunoassay, an intracellular compartment of AP2 was identified in the mammary cell lines in culture. In MCF-10A, this compartment behaved as in MDCK cells; namely it was observed only when the cells cannot form cell-cell contacts. However, in the carcinoma cell lines MCF-7 and T47D, a significant AP2 intracellular compartment was observed also under conditions permissive for the formation of intercellular contacts. These results were confirmed by immunofluorescence and immunoelectron microscopy experiments that showed VACs in MCF-7 and T47D, even in cells with extensive intercellular contacts. In MCF-7 cells, the addition of serum caused a partial decrease of the AP2 intracellular compartment. The exocytosis of VACs occurred towards the center of multi-cellular groups, forming intercellular lumens, similar to those transiently observed in MDCK cells and to structures described by others during embryo development. Altogether, these results suggest that VAC exocytosis is controlled by cell-cell contact signalling, which may be defective in carcinoma cells.
Asunto(s)
Neoplasias de la Mama/ultraestructura , Compartimento Celular/fisiología , Exocitosis/fisiología , Vacuolas/ultraestructura , Animales , Biomarcadores de Tumor/análisis , Fenómenos Fisiológicos Sanguíneos , Neoplasias de la Mama/fisiopatología , Comunicación Celular/fisiología , División Celular/fisiología , Supervivencia Celular/fisiología , Medio de Cultivo Libre de Suero , Perros , Células Epiteliales , Epitelio/química , Humanos , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/biosíntesis , Células Tumorales CultivadasRESUMEN
Vacuolar apical compartment (VAC) is a transient organelle originally observed in Madin-Darby canine kidney (MDCK) epithelial cells impaired from forming cell-cell contacts. VACs are large vacuoles which contain microvilli and apical plasma membrane markers (among others, a 184-kDa plasma membrane protein, AP2), but exclude basolateral membrane markers. Upon reestablishment of cell-cell contacts, VACs are rapidly (within 1 h) exocytosed toward intercellular spaces, after which the apical plasma membrane drifts toward its final destination (Vega-Salas, Salas, and Rodriguez-Boulan. 1988. J. Cell Biol. 107, 1717-1728). In this work, we studied the role of cAMP as a mediator for the exocytosis of VACs. We shifted confluent cells from low to normal calcium medium (thus reestablishing cell-cell contacts and causing VAC exocytosis), a shift which resulted in a significant rise of cellular levels of both total intracellular and protein-bound cAMP. The 8-Br analog of cAMP (8-Br-cAMP) (5-50 microM) caused externalization of the intracellular compartment of AP2 as measured by radioimmunoassay. A similar effect was observed with 3-isobutyl-1-methylxanthine. 8-Br-cAMP also caused the appearance of AP2-positive VAC images in nonpermeabilized cells, namely, VACs that become accessible to extracellular antibodies upon fusion with the plasma membrane. Lanthanum, which abolishes the peak of intracellular free calcium during a calcium switch, failed to block the exocytosis. On the other hand, 12-O-tetradecanoylphorbol-13-acetate induced only a modest exocytic response. Finally, 8-Br-cAMP induced VAC exocytosis in sparse MDCK cells grown in normal calcium medium. These data indicate that cAMP is a mediator between the extracellular signal provided by cell-cell contacts and VAC exocytosis.
Asunto(s)
AMP Cíclico/fisiología , Exocitosis/efectos de los fármacos , Sistemas de Mensajero Secundario , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Complejo 2 de Proteína Adaptadora , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Adhesión Celular/fisiología , Línea Celular/efectos de los fármacos , Biosíntesis de Proteínas , Vacuolas/efectos de los fármacosRESUMEN
Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.
Asunto(s)
Adhesión Celular , Polaridad Celular , Células Epiteliales , Matriz Extracelular/metabolismo , Laminina/metabolismo , Receptores de Laminina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Cromatografía de Afinidad , Perros , Vidrio , Riñón , Datos de Secuencia Molecular , Receptores de Laminina/inmunologíaAsunto(s)
Antígenos de Superficie/análisis , Epitelio/inmunología , Animales , Biotina , Fraccionamiento Celular , Transformación Celular Viral , Células Cultivadas , Expresión Génica , Genes Virales , Humanos , Procesamiento de Imagen Asistido por Computador , Transfección , Grabación en Video , Proteínas Virales/análisisAsunto(s)
Epitelio/fisiología , Animales , Transporte Biológico , Fenómenos Biomecánicos , Comunicación Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales , Epitelio/metabolismo , Glicoproteínas/metabolismo , Homeostasis , Proteínas del Envoltorio Viral/metabolismoRESUMEN
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.
Asunto(s)
Membrana Celular/ultraestructura , Citoesqueleto/fisiología , Epitelio/ultraestructura , Proteínas de la Membrana/fisiología , Anticuerpos Monoclonales/inmunología , Compartimento Celular , Línea Celular , Membrana Celular/fisiología , Microscopía Fluorescente , Polietilenglicoles , SolubilidadRESUMEN
The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.
Asunto(s)
Comunicación Celular , Membrana Celular/fisiología , Exocitosis , Orgánulos/fisiología , Animales , Calcio/fisiología , Células Cultivadas , Epitelio/fisiología , Técnica del Anticuerpo Fluorescente , Cinética , Fusión de Membrana , Modelos Biológicos , Orgánulos/metabolismo , Radioinmunoensayo , Vacuolas/metabolismo , Vacuolas/fisiologíaRESUMEN
Experimental conditions that abolish or reduce to a minimum intercellular contacts between Madin-Darby canine kidney epithelial cells result in the appearance of an intracellular storage compartment for apical membrane proteins. Subconfluent culture, incubation in 1-5 microM Ca++, or inclusion of dissociated cells within agarose or collagen gels all caused the intracellular accumulation of a 184-kD apical membrane protein within large (0.5-5 micron) vacuoles, rich in microvilli. Influenza virus hemagglutinin, an apically targeted viral glycoprotein, is concentrated within these structures but the basolateral glycoprotein G of vesicular stomatitis virus and a cellular basolateral 63-kD membrane protein of Madin-Darby canine kidney cells were excluded. This novel epithelial organelle (VAC), which we designate the vacuolar apical compartment, may play an as yet unrecognized role in the biogenesis of the apical plasma membrane during the differentiation of normal epithelia.
Asunto(s)
Comunicación Celular , Membrana Celular/metabolismo , Proteínas de la Membrana/biosíntesis , Animales , Línea Celular , Perros , Técnica del Anticuerpo Fluorescente , Homeostasis , Riñón , Cinética , Microscopía Electrónica , Peso Molecular , Radioinmunoensayo , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
The time course of development of polarity of an apical (184-kD) and a basolateral (63-kD) plasma membrane protein of Madin-Darby canine kidney cells was followed using semiquantitative immunofluorescence on semithin (approximately 0.5-micron) frozen sections and monoclonal antibody probes. The 184-kD protein became highly polarized to the apical pole within the initial 24 h both in normal medium and in 1-5 microM Ca2+, which results in well-spread, dome-shaped cells, lacking tight junctions and other lateral membrane interactions. In contrast, the basolateral 63-kD membrane protein developed full polarity only after incubation in normal Ca2+ concentrations for greater than 72 h, a time much longer than that required for the formation of tight junctions (approximately 18 h) and failed to polarize in 1-5 microM Ca2+. These results demonstrate that intradomain restriction mechanisms independent of tight junctions, such as self-aggregation or specific interactions with the submembrane cytoskeleton, participate in the regionalization of at least some epithelial plasma membrane proteins. The full operation of these mechanisms depends on the presence of normal cell-cell interactions in the case of the basolateral 63-kD antigen but not in the case of the apical 184-kD protein.
Asunto(s)
Comunicación Celular , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/análisis , Animales , Anticuerpos Monoclonales , Línea Celular , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Perros , Células Epiteliales , Epitelio/fisiología , Uniones Intercelulares/fisiología , Riñón , Microscopía Electrónica , Peso MolecularRESUMEN
Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12-15 hr displayed high-affinity binding sites for 125I-lathyritic (soluble) collagen (120,000/cell, KD = 30 nM) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2 = 6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a two-step model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.
Asunto(s)
Proteínas Portadoras/fisiología , Receptores Inmunológicos/fisiología , Animales , Adhesión Celular , Línea Celular , Colágeno , Células Epiteliales , Fibronectinas , Cinética , Laminina , Receptores de ColágenoRESUMEN
We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization.