RESUMEN
The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.
Asunto(s)
Uniones Intercelulares/análisis , Glomérulos Renales/análisis , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Animales , Anticuerpos , Endotelio Vascular/análisis , Células Epiteliales , Técnica del Anticuerpo Fluorescente , Immunoblotting , Glomérulos Renales/citología , Glomérulos Renales/crecimiento & desarrollo , Túbulos Renales Distales/análisis , Masculino , Ratas , Ratas Endogámicas , Proteína de la Zonula Occludens-1Asunto(s)
Uniones Intercelulares/análisis , Proteínas de la Membrana , Secuencia de Aminoácidos , Animales , Clonación Molecular , Conexinas , ADN/genética , Regulación de la Expresión Génica , Inmunohistoquímica , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and octyl glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoring a gap junction-like structure.
Asunto(s)
Uniones Intercelulares/ultraestructura , Hígado/ultraestructura , Proteínas de la Membrana/análisis , Animales , Colesterol , Conexinas , Detergentes , Digitonina , Técnica de Fractura por Congelación , Glucósidos , Uniones Intercelulares/análisis , Liposomas , Microscopía Electrónica , Fosfatidilcolinas , Ratas , SolubilidadRESUMEN
Cell-cell adhesion is controlled by many molecules found on the cell surface. In addition to the constituents of well-defined junctional structures, there are the molecules that are thought to play a role in the initial interactions of cells and that appear at precise times during development. These include the cadherins and cell adhesion molecules (CAMs). Representatives of these families of adhesion molecules have been isolated from most of the major tissues. The notable exception is the vascular endothelium. Here we report the identification of a cell surface molecule designated "endoCAM" (endothelial Cell Adhesion Molecule), which may function as an endothelial cell-cell adhesion molecule. EndoCAM is a 130-kD glycoprotein expressed on the surface of endothelial cells both in culture and in situ. It is localized to the borders of contiguous endothelial cells. It is also present on platelets and white blood cells. Antibodies against endoCAM prevent the initial formation of endothelial cell-cell contacts. Despite similarities in size and intercellular location, endoCAM does not appear to be a member of the cadherin family of adhesion receptors. The serologic and protease susceptibility characteristics of endoCAM are different from those of the known cadherins, including an endogenous endothelial cadherin. Although the precise biologic function of endoCAM has not been determined, it appears to be one of the molecules responsible for regulating endothelial cell-cell adhesion processes and may be involved in platelet and white blood cell interactions with the endothelium.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/aislamiento & purificación , Moléculas de Adhesión Celular/aislamiento & purificación , Adhesión Celular , Animales , Anticuerpos Monoclonales , Antígenos de Diferenciación Mielomonocítica/análisis , Plaquetas/análisis , Calcio/farmacología , Bovinos , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/farmacología , Células Cultivadas , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Sueros Inmunes , Uniones Intercelulares/análisis , Uniones Intercelulares/ultraestructura , Peso Molecular , Péptido Hidrolasas , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Inhibidores de Proteasas , TripsinaRESUMEN
Antibodies were raised in rabbits to highly purified preparations of bovine brain clathrin. The serum stained by immunofluorescence rat liver sections at tight junctions in a pattern that was identical to that previously reported (B. R. Stevenson et al.: J. Cell Biol. 103, 755-766 (1986] in which a monoclonal antibody specific to a 220 kDa (ZO-1) liver tight junction component was used. The serum also stained regions of the cell surface corresponding to the positions of intercellular junctions in confluent MDCK and HepG-2 cell cultures. Analysis of brain clathrin preparations resolved by polyacrylamide gel electrophoresis by immunoblotting with the serum indicated reaction with clathrin heavy and light chains as well as towards a 220 kDa polypeptide that was a minor component. Affinity purification of the serum provided antibodies directed mainly to clathrin light chains and these antibodies, as well as an independent antiserum to clathrin heavy chains, immunofluorescently stained liver tissue and cells in a manner typical of coated membranes/vesicles. These results suggested, by difference, that antibodies to a 220 kDa polypeptide, a minor constituent in brain clathrin preparations, were responsible for staining intercellular tight junctions in epithelia. The 220 kDa polypeptide present in brain clathrin preparations was demonstrated to be immunologically distinct from liver myosin heavy chain as well as erythrocyte and brain ankyrin. Comparison by two-dimensional mapping of the 220 kDa in brain clathrin with the clathrin heavy chain (180 kDa) polypeptide showed they were different proteins, but the 220 kDa polypeptide present in rat liver tight junctions was highly similar to the 220 kDa present in bovine brain clathrin preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Clatrina/análisis , Uniones Intercelulares/análisis , Péptidos/análisis , Animales , Ancirinas , Proteínas Sanguíneas/análisis , Química Encefálica , Bovinos , Membrana Celular/ultraestructura , Clatrina/aislamiento & purificación , Eritrocitos/análisis , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Hígado/análisis , Hígado/ultraestructura , Proteínas de la Membrana/análisis , Microscopía Electrónica , Peso Molecular , Miosinas/análisis , RatasRESUMEN
In the previous study, we succeeded in isolating the cell-to-cell adherens junctions from rat liver (Tsukita, S., and S. Tsukita. 1989. J. Cell Biol. 108:31-41.). In this study, we have obtained mAbs specific to the 400-kD protein, which was identified as one of the major constituents of the undercoat of isolated adherens junctions. Immune blot analyses showed that this protein occurs in various types of tissues. Immunofluorescence microscopy and immune electron microscopy have revealed that this protein is distributed not only at the undercoat of adherens junctions but also along actin bundles associated with the junction in nonmuscle cells: stress fibers in cultured fibroblasts and circumferential bundles in epithelial cells. The partially purified protein molecule looks like a slender rod approximately 400 nm in length. By virtue of its molecular shape, we have named this protein 'tenuin' (from Latin 'tenuis', thin or slender).
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Citoesqueleto/ultraestructura , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/aislamiento & purificación , Citoesqueleto de Actina/análisis , Animales , Anticuerpos Monoclonales , Complejo Antígeno-Anticuerpo , Electroforesis Discontinua , Técnica del Anticuerpo Fluorescente , Uniones Intercelulares/análisis , Hígado/ultraestructura , Microscopía Electrónica , Peso Molecular , Conformación Proteica , Ratas , Estrés MecánicoAsunto(s)
Moléculas de Adhesión Celular/análisis , Comunicación Celular , Uniones Intercelulares/análisis , Proteínas de la Membrana/análisis , Animales , Moléculas de Adhesión Celular/metabolismo , Conexinas , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/metabolismoRESUMEN
The relative localization of ZO-1 and cingulin, the only two known components of the tight junction, was compared in Madin-Darby canine kidney (MDCK) cells, chicken small intestine, rat kidney distal convoluted tubule, and a hepatoma cell line. Immunoblot analysis demonstrated that cingulin and ZO-1 are immunologically unrelated and that, in the colon, cingulin is a single polypeptide with a molecular mass of 140 kDa. Immunofluorescent localization of cingulin and ZO-1 in confluent monolayers of MDCK cells showed identical staining patterns. However, subconfluent MDCK cells showed distinct localizations of the two proteins. Both cingulin and ZO-1 were found at the plasma membrane only at areas of cell-cell contact, but cingulin was diffusely distributed within the cytoplasm, whereas ZO-1 showed a more clustered internal arrangement. Cingulin and ZO-1 were identically localized at the plasma membrane of hepatoma tissue culture (HTC) cells at sites of cell-cell contact. In chicken intestine examined at the ultrastructural level, immunogold particles associated with cingulin were found approximately three times farther from the junctional membrane than those affiliated with ZO-1.
Asunto(s)
Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Animales , Anticuerpos Monoclonales , Línea Celular , Pollos , Duodeno/ultraestructura , Técnica del Anticuerpo Fluorescente , Uniones Intercelulares/análisis , Riñón , Túbulos Renales Distales/ultraestructura , Neoplasias Hepáticas Experimentales , Proteínas de la Membrana/inmunología , Microscopía Electrónica , Microvellosidades/ultraestructura , Músculo Liso/ultraestructura , Fosfoproteínas/inmunología , Ratas , Proteína de la Zonula Occludens-1RESUMEN
In this study we have examined a protein associated with bile canaliculi of mouse and rat hepatocytes that is detected by monoclonal antibody BG9.1. The protein is seen by indirect-immunofluorescence microscopy as 2 discrete parallel lines at the lateral borders of adjacent hepatocytes. This pattern is present during development in the day 13 fetal mouse liver. Electron microscopy with immuno-gold labeling indicated that the protein is associated with the cytoplasmic surface of junctional complexes located on either side of bile canaliculi. BG9.1 reacts with a protein of 192,000 apparent molecular weight on immunoblots of plasma membrane isolated from mouse and rat hepatocytes. It has been reported that unlike most cellular components, tight junctions are not soluble in sodium deoxycholate. Extraction of isolated hepatic plasma membrane sheets with deoxycholate and other reagents did not eliminate the pattern seen by indirect-immunofluorescence microscopy and enhanced the intensity of reactions on immunoblots. BG9.1 also binds to the junctional-complex region in other epithelial cell types. These results indicate that BG9.1 detects a deoxycholate-insoluble protein associated with junctional complexes and suggests that the protein is a component of tight junctions.
Asunto(s)
Uniones Intercelulares/análisis , Hígado/ultraestructura , Proteínas/análisis , Animales , Anticuerpos Monoclonales , Membrana Celular/análisis , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Microscopía Electrónica , Peso Molecular , Ratas , Ratas EndogámicasRESUMEN
SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100 mM) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.
Asunto(s)
Disulfuros/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Miocardio/ultraestructura , Compuestos de Sulfhidrilo/metabolismo , Animales , Conexinas , Etilmaleimida/farmacología , Uniones Intercelulares/análisis , Uniones Intercelulares/ultraestructura , Yodoacetamida/farmacología , Proteínas de la Membrana/análisis , Microscopía Electrónica , Miocardio/metabolismo , RatasRESUMEN
In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-polyacrylamide gels, an 83K (K = 10(3) Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, suggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin-membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.
Asunto(s)
Proteínas de Microfilamentos , Células de Sertoli/ultraestructura , Actinas/análisis , Actinas/metabolismo , Actinas/fisiología , Animales , Western Blotting , Adhesión Celular/fisiología , Fraccionamiento Celular/métodos , Proteínas del Citoesqueleto/análisis , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Uniones Intercelulares/análisis , Uniones Intercelulares/ultraestructura , Masculino , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/metabolismo , Microscopía Electrónica , Ratas , Ratas Endogámicas , Células de Sertoli/análisis , Células de Sertoli/citología , VinculinaRESUMEN
To explain of the mechanism of the enhanced nasal epithelial permeability to HRP in patients with nasal allergy, the inferior turbinate mucosa was removed from 6 normal adults and 7 adults with nasal allergy. Difference of the fine structure of the intercellular junction was compared between normal mucosa and mucosa of nasal allergy by electron microscope. Staining pattern of four kinds of HRP-conjugated lectin (HRP-WGA, PNA, UEA-I and RCA-I) was also studied by electron microscope. There was no significant difference in the intercellular space of the mucosa between the normal mucosa and mucosa of nasal allergy. In the epithelial cell membrane, pattern of HRP-lectin staining was almost similar in both groups. In normal nasal epithelium, the intercellular junction consisted of junctional complex; adherent junction, desmosome and gap junction. The intercellular space was approximately 150-250 A in width. The tight junction was located beneath the luminal surface of the epithelium, and belt-like continuation connecting the adjacent cells. It was concluded that enhanced permeability to HRP in nasal allergy was not morphologic changes of the intercellular junction and component and distribution of the glycoconjugates in epithelial cellular membrane, but this may be based on functional changes.
Asunto(s)
Uniones Intercelulares/ultraestructura , Lectinas , Mucosa Nasal/ultraestructura , Rinitis Alérgica Perenne/patología , Rinitis Alérgica Estacional/patología , Glicoconjugados/análisis , Histocitoquímica , Humanos , Uniones Intercelulares/análisis , Microscopía Electrónica , Mucosa Nasal/análisis , Rinitis Alérgica Perenne/metabolismo , Rinitis Alérgica Estacional/metabolismoRESUMEN
A procedure has been developed to isolate gap junction-enriched subcellular fractions from Drosophila. Crude membranes from larval homogenates were extracted with 1% N-lauroyl sarcosine in 6 M urea and the gap junctions were collected by centrifugation. The major proteins were separated by SDS PAGE and purified by electro-elution. Electron microscopy revealed structurally pleiomorphic gap junctions in the fractions which included (1) conventional, 16-18 nm-wide septalaminar, (2) collapsed, 13-15 nm-wide pentalaminar, (3) split, and (4) aggregated forms. The fractions contained five major proteins with apparent molecular weights of 18, 26, 36, 52 and 54 kD. Evidence based on (1) the degradation and aggregation behavior of the major proteins following electro-elution and reelectrophoresis, (2) immunological cross-reactivities by affinity-purified antibodies against the major proteins on immunoblots, and (3) immunofluorescent staining of presumptive gap junctions in Drosophila imaginal discs at the light-microscopic level and immunogold staining of purified gap junctions at the electron-microscopic level suggests that the major proteins are interrelated and of gap-junction origin.
Asunto(s)
Drosophila melanogaster/anatomía & histología , Uniones Intercelulares/análisis , Proteínas del Tejido Nervioso/aislamiento & purificación , Animales , Uniones Intercelulares/ultraestructura , Larva , Microscopía Electrónica , Peso Molecular , Proteínas del Tejido Nervioso/inmunología , Fracciones Subcelulares/análisis , Fracciones Subcelulares/ultraestructuraRESUMEN
Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.
Asunto(s)
Uniones Intercelulares/análisis , Hígado/análisis , Proteínas de la Membrana/análisis , Animales , Células Cultivadas , Conexinas , AMP Cíclico/metabolismo , Técnica del Anticuerpo Fluorescente , Semivida , Hepatectomía , Inmunohistoquímica , Uniones Intercelulares/ultraestructura , Hígado/embriología , Hígado/metabolismo , Hígado/ultraestructura , Regeneración Hepática , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Peso Molecular , Fosforilación , RatasRESUMEN
The postnatal development of the Sertoli cell barrier, tubular lumen, fluid flow, and cytoskeletal elements in Sertoli and myoid cells was investigated in the Sprague-Dawley rat. With the aid of hypertonic fixatives, a barrier to the rapid entry of fluid was noted in the majority of tubules on the 15th and 16th postnatal (p.n.) days and was completely formed in all tubules prior to p.n. day 18. The actin forming the ectoplasmic specialization (ES), a cytoskeletal complex related to the occluding junctions composing the barrier, began its development during the period of initial barrier formation (16 p.n. day) and progressively attained its adult prominence. The ES developed its characteristic adult pattern and adult fluorescent intensity at about p.n. day 22. Some seminiferous tubules showed very small lumina as early as p.n. day 10. All tubules were not open until p.n. day 30. The size (diameter) of the lumen increased slowly from p.n. day 10 until p.n. day 30 when it started to increase rapidly until about p.n. day 50. Fluid flow in seminiferous tubules was detected as early as p.n. day 20 and increased in amount thereafter. Myoid cell actin filament bundles, running in parallel, were present at p.n. day 10. Actin formed a meshwork pattern characteristic of the adult on, or slightly prior to, p.n. day 22. These data indicate that there is a temporal relationship between the development of the actin cytoskeleton within the Sertoli cell and initial formation of the Sertoli cell barrier. Similarly, there is a temporal relationship between the development of the actin cytoskeleton of myoid cells and tubular fluid flow. The rapid increase in tubular lumen diameter, however, does not correlate with the initial development of Sertoli and myoid cytoskeletal elements.
Asunto(s)
Barrera Hematotesticular , Citoesqueleto/ultraestructura , Músculos/citología , Semen/metabolismo , Túbulos Seminíferos/citología , Células de Sertoli/citología , Testículo/citología , Citoesqueleto de Actina/análisis , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/análisis , Actinas/metabolismo , Animales , Comunicación Celular , Citoesqueleto/metabolismo , Citoesqueleto/fisiología , Uniones Intercelulares/análisis , Uniones Intercelulares/fisiología , Uniones Intercelulares/ultraestructura , Masculino , Músculos/análisis , Músculos/fisiología , Ratas , Ratas Endogámicas , Túbulos Seminíferos/embriología , Túbulos Seminíferos/fisiología , Células de Sertoli/embriología , Células de Sertoli/fisiologíaRESUMEN
Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.
Asunto(s)
Uniones Intercelulares/análisis , Proteínas de la Membrana/análisis , Miocardio/ultraestructura , Animales , Northern Blotting , Western Blotting , Tejido Conectivo/ultraestructura , Conexinas , Femenino , Técnica del Anticuerpo Fluorescente , Células de la Granulosa/ultraestructura , Inmunohistoquímica , Hígado/ultraestructura , Microscopía Electrónica , Músculo Liso/ultraestructura , Ratas , Distribución TisularRESUMEN
A new isolation procedure for cell-to-cell adherens junctions has been developed using rat liver. From the bile canaliculi-enriched fraction obtained by homogenization of the liver and sucrose gradient centrifugation, the fraction rich in adherens junction was recovered by detergent treatment followed by sucrose gradient centrifugation. Light and electron microscopy revealed that this final fraction was mainly composed of the belt-like adherens junctions with their associated short actin filaments. Biochemical and immunological analyses have shown that vinculin is highly enriched in this fraction. Considering that vinculin is known to be localized in the cell-to-cell adherens junctions, we can conclude that we have succeeded in isolating the cell-to-cell adherens junctions. Furthermore, the constituents of the undercoat (dense layer underlying the membrane) of adherens junctions were selectively extracted from the fraction rich in junctions. Upon SDS electrophoresis of this extract, 10 polypeptides including vinculin, alpha-actinin, and actin were dominant. The results obtained are discussed with special reference to the molecular organization of the undercoats of cell-to-cell adherens junctions.
Asunto(s)
Canalículos Biliares/ultraestructura , Conductos Biliares Intrahepáticos/ultraestructura , Proteínas del Citoesqueleto , Uniones Intercelulares/ultraestructura , Hígado/ultraestructura , Actinina/análisis , Actinas/análisis , Animales , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Desmoplaquinas , Desmosomas , Uniones Intercelulares/análisis , Queratinas/análisis , Glicoproteínas de Membrana/análisis , Microscopía Electrónica , Proteínas Musculares/análisis , Ratas , VinculinaRESUMEN
In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.
Asunto(s)
Proteínas del Citoesqueleto/análisis , Citoesqueleto/ultraestructura , Epitelio Pigmentado Ocular/ultraestructura , Animales , Bovinos , Pollos , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/análisis , Citoesqueleto/fisiología , Técnica del Anticuerpo Fluorescente , Cobayas , Humanos , Uniones Intercelulares/análisis , Uniones Intercelulares/ultraestructura , Proteínas de Filamentos Intermediarios/análisis , Filamentos Intermedios/ultraestructura , Microscopía Electrónica , Epitelio Pigmentado Ocular/análisis , Epitelio Pigmentado Ocular/fisiología , Rana ridibunda , Ratas , Especificidad de la Especie , Xenopus laevisRESUMEN
MIP and MP70 are putative gap junction components in the plasma membranes of the mammalian lens fibre cells. We show now that MP70 can be solubilized separately from MIP in mild detergent solutions, and that this treatment results in the dissociation of the fibre gap junctions. Solubilized MP70 was isolated as 16.9 S particles by velocity gradient centrifugation and in the electron microscope had the appearance of short double-membrane structures consistent with connexon-pairs. These observations open a new experimental avenue in which to characterize separately the two putative lens gap junction proteins structurally and functionally.