RESUMEN
We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca(2+) is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca(2+) is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca(2+) starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca(2+) is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62-65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca(2+) starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca(2+) removal. Phosphoaminoacid analysis showed that the 62-65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability.
Asunto(s)
Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Animales , Western Blotting , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular , Quelantes/farmacología , Ácido Egtácico/farmacología , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/ultraestructura , Expresión Génica , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Ocludina , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/fisiologíaRESUMEN
This investigation utilized immunocytochemical and fluorescent protocols to analyze the roles of cellular proliferation and apoptosis in the regulation of differentiation and senescence of the rat small intestinal mucosa. Specifically, the study localized apoptotic zones of the villus through the use of enzymatic tags; established the transition point between cell growth and differentiation, i.e. the point of no return where crypt cells differentiate into absorptive cells with barrier functions; and the role that plasmalemmal, cytoskeletal, junctional and extracellular matrix (ECM) elements may play in the regulation of differentiation and migration of epithelial cells from crypt to villus. Apoptosis was relegated to the villus tip forming a prominent 'apoptotic cuff' of cells. Close scrutiny of these cuffs reveals the presence of apoptotic cells adjacent to non-apoptotic (healthy) cells. Mid-villus epithelial cells were non-apoptotic and all cells in the crypt-villus unit expressed Bcl-2 activity. Intestinal lactase expression was prominent in post-mitotic cells along the villus, while cells in the crypt and base were negative for lactase activity. In contrast, all the cells of the crypt-villus unit were intensely reactive for F-actin. Close scrutiny of isolated cells and frozen sections indicates specific localization of actin in the microvillus region, apical cytoplasm, basolateral and lateral plasmalemma which was in close proximity to fibronectin in the basement lamina. Occludin positive junctional networks were prominent at villus tips, where senescent and apoptotic cells were also most prominent, suggesting that tight junctional integrity was essential to barrier, digestive and absorptive functions in all regions of the mucosa.
Asunto(s)
Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Actinas/análisis , Animales , Apoptosis , Diferenciación Celular , Femenino , Intestino Delgado/citología , Intestino Delgado/fisiología , RatasRESUMEN
This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca2+ removal and was characterized by a marked drop of TER. The reintroduction of Ca2+ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca2+ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Uniones Intercelulares/efectos de los fármacos , Proteínas de la Membrana/química , Fragmentos de Péptidos/farmacología , Animales , Tampones (Química) , Calcio/farmacología , Línea Celular , Polaridad Celular , Pollos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ocludina , Fosfoproteínas/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
In secretory cells, microtubule- (Mt-) based motor enzymes are thought to support transport of secretory vesicles to the cell surface for subsequent release. At present, the role of Mts and kinesin in secretory vesicle transport in exocrine epithelial cells has not been defined. Furthermore, it is unclear whether an agonist-induced secretory event modifies kinesin function and distribution, thus altering vesicle transport. To this end, we utilized isolated rat pancreatic acini and cultured rat pancreatic acinar cells to examine the role of Mts and kinesin in regulated secretion. Exposure of cells to cytoskeletal antagonistic drugs demonstrated that the observed movements of apically clustered zymogen granules (ZGs) are supported by Mts, but not actin. Morphological studies of Mt organization in polarized acini show that Mt plus ends extend outward from the apical membrane toward the cell center. Immunofluorescence microscopy in both cell models revealed a clear association of kinesin with apical ZGs, while quantitative immunoblot analysis of pancreatic subcellular fractions confirmed kinesin enrichment on ZG membranes. In addition, microinjection of kinesin antibodies into cultured acinar cells inhibited ZG movements. Indirect immunofluorescence staining of isolated cells and quantitative Western blotting of isolated ZGs revealed that kinesin association with granule membranes increased up to 3-fold in response to a secretory stimulus. Autoradiographic studies of 32P-labeled acini showed up to a 6-fold increase in kinesin heavy chain (KHC) phosphorylation during stimulated secretion. These studies provide the first direct evidence that Mts and kinesin support ZG movements and that physiological agonists induce a marked phosphorylation of KHC while increasing the association of kinesin with ZG membranes. These changes during agonist stimulation suggest that the participation of kinesin in zymogen secretion is regulated.
Asunto(s)
Cinesinas/metabolismo , Páncreas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Gránulos Citoplasmáticos , Precursores Enzimáticos/metabolismo , Microtúbulos/fisiología , Datos de Secuencia Molecular , Páncreas/citología , Fosforilación , Ratas , Ratas Sprague-Dawley , Secretina/farmacología , Sincalida/farmacologíaRESUMEN
Reversible protein phosphorylation has been implicated in the regulation of organelle transport by cytoplasmic dynein. Motor function may be modulated directly by the phosphorylation of dynein or through the phosphorylation of an accessory factor. Dynactin binds to cytoplasmic dynein and is a required activator for dynein-driven vesicular motility. In metabolic labeling studies we have determined that the p150Glued subunit of dynactin is a phosphoprotein. Treatment of Rat2 cells with okadaic acid or with activators of protein kinase A or protein kinase C caused a marked increase in the incorporation of 32P into p150Glued; the increased phosphorylation correlated with activated vesicular transport. Phosphoamino-acid analysis of p150Glued isolated from cells treated with okadaic acid or with activators of either protein kinase A or protein kinase C indicated exclusive labeling of phosphoserine. These results suggest that the phosphorylation of dynactin may serve to regulate intracellular transport catalyzed by cytoplasmic dynein.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Complejo Dinactina , Dineínas/química , Dineínas/metabolismo , Activación Enzimática/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/metabolismo , Modelos Biológicos , Ácido Ocadaico/farmacología , Orgánulos/metabolismo , Fosforilación , Conformación Proteica , Proteína Quinasa C/metabolismo , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Hamster islet tumor (HIT) cells retain much of the capacity of normal beta cells to act as glucose sensors. When stimulated with glucose or glucose plus forskolin, HIT cells release much more insulin than unstimulated cells. Ultrastructural analysis reveals that the secretory product of these cells is stored in membrane-bound granules that associate with microtubules under certain circumstances. Immunofluorescence studies using insulin antibody confirm the presence of insulin in granular structures in these cells. The microtubule inhibitor Nocodazole reduces the number of polymerized microtubules and inhibits the sustained phase of insulin secretion in HIT cells. Thus, the structural integrity of microtubules is important for the sustained phase of the insulin secretion to occur. The microtubule stabilizing drug taxol does not decrease insulin secretion. Since taxol blocks microtubule depolymerization, microtubule polymerization-depolymerization alone does not appear to be responsible for insulin granule transport. The increased use of these drugs in cancer research and therapy makes it important to understand their effects on insulin secretion.
Asunto(s)
Adenoma de Células de los Islotes Pancreáticos/metabolismo , Adenoma de Células de los Islotes Pancreáticos/patología , Glucosa/farmacología , Insulina/metabolismo , Nocodazol/farmacología , Paclitaxel/farmacología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Adenoma de Células de los Islotes Pancreáticos/ultraestructura , Animales , Colforsina/farmacología , Cricetinae , Técnica del Anticuerpo Fluorescente , Secreción de Insulina , Microscopía Electrónica , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Neoplasias Pancreáticas/ultraestructura , Células Tumorales CultivadasRESUMEN
The roles that complex carbohydrates play in gamete recognition and fertilization are well-documented. This study was designed to identify and map sites of N-linked oligosaccharides in boar and bull sperm. Plant lectins, conjugated with FITC, RITC and gold particles, were used as probes for specific sugar moieties in sperm membranes. Results showed strong labeling of sperm heads by most lectins. However, lectins with specificities for mannosyl, glucosyl, and sialic acid were predominantly distributed over the sperm acrosomal region. Lectins specific for fucosyl residues predominantly stained the postacrosomal regions of both boar and bull sperm. Lectin gold studies revealed that mannosyl and galactosyl-linked residues occupied the acrosomal membrane and plasma membrane in the postacrosomal region, but not the nucleus or nuclear membrane. The cytoplasmic droplet, middle piece and flagellum of boar and bull sperm were moderately stained by lectins. The microquantitative analysis confirmed the fluorescence data. The presence of fucosyl residues predominantly in the postacrosomal regions lends credence to the suggestion that glycoproteins rich in fucosyl-like residues may be a universal carbohydrate moiety involved in recognition processes in mammalian fertilization.