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W J Nelson

Publications and source records attributed to W J Nelson.

At least 109 records · Page 6Linked to original sources
PubMed1990

Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity.

Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.

Animals↗
PubMed1990

Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin-Darby canine kidney epithelial cells.

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.

Animals↗
PubMed1990

Steps in the morphogenesis of a polarized epithelium. I. Uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts.

The development of cell polarity in Madin-Darby canine kidney (MDCK) cells has been analyzed under conditions in which cells are induced to form multicellular epithelial cysts in stages that mimic the ontogeny of epithelial tissues and organs in vivo. The morphogenesis of MDCK cysts in suspension culture or in a collagen gel proceeds in distinct stages involving the initial aggregation of cells followed by development of a closed monolayer of polarized epithelial cells that surrounds a central lumen. The polarity of cells was determined at each stage by analyzing the distributions of marker proteins of the apical (gp135) and basal-lateral (Na+,K(+)-ATPase) domains of the plasma membrane, the tight junction (ZO-1) and proteins involved in cell-cell (uvomorulin) and cell-substratum contact (type IV collagen). We show that cells have a distinctive and opposite polarity in cysts formed in suspension culture compared to those formed in collagen gels. In suspension culture, the basal-lateral membrane faces the central lumen and the apical membrane faces the outside, whereas in collagen gel, the basal-lateral membrane faces the outside collagen and the apical membrane faces the central lumen. Detailed analysis of the distributions of marker proteins during the morphogenesis of these three-dimensional structures indicates that: (1) cell-cell contact is sufficient to trigger the segregation of marker proteins of the apical and basal-lateral membrane domains to distinct regions of the membrane; (2) cell-cell contact induces association of the tight junction protein ZO-1 with the contact zone between cells; (3) localization of the tight junction protein ZO-1 to the apex of the lateral membrane and the establishment of the epithelial axis, however, requires the formation of a basal lamina and cell-substratum contact; (4) in suspension culture, MDCK cysts secrete and establish a basal lamina in the central lumen. These results show that cell-cell and cell-substratum contact have distinct roles in the morphogenesis of polarized epithelia. We suggest that the mechanisms involved in triggering cell polarity may be common to different polarized epithelia in vivo.

Animals↗
PubMed1990

Steps in the morphogenesis of a polarized epithelium. II. Disassembly and assembly of plasma membrane domains during reversal of epithelial cell polarity in multicellular epithelial (MDCK) cysts.

A fundamental aspect in the morphogenesis of a polarized epithelium is the formation of structurally and functionally distinct apical and basal-lateral domains of the plasma membrane. The formation of these membrane domains involves the accumulation of domain-specific proteins and removal of incorrectly localized proteins. The mechanisms involved in these processes are not well understood. We have approached this problem by detailed analysis of the distribution and fate of proteins specific for different membrane domains during reversal of epithelial polarity. In the preceding paper we showed that MDCK cells form multicellular cysts comprising a closed monolayer of polarized cells. The orientation of cell polarity depends upon whether cysts are formed in suspension culture or in a collagen gel. Here, we show that, when fully developed cysts formed in suspension culture are placed in a collagen gel, polarity is rapidly reversed without cell dissociation. We show that during the process of polarity reversal, plasma membrane domains are disassembled by uptake of proteins into cytoplasmic vesicles, followed by protein degradation that probably occurs in lysosomes. The disassembly and assembly of the apical and the basal-lateral membrane domains occur in a sequential order with different kinetics. Our results provide further insights into the establishment of protein specificity of plasma membrane domains in polarized cells.

Animals↗
PubMed1990

Involvement of the membrane-cytoskeleton in development of epithelial cell polarity.

The generation of cell surface polarity in transporting epithelial cells occurs in three distinct stages that involve cell-cell recognition and adhesion, cell surface remodelling to form biochemically and functionally distinct cell surface domains, and development of vectorial function. A widely used model system to study mechanisms involved in these stages is the Madin-Darby canine kidney (MDCK) cell line. Under appropriate growth conditions, MDCK cells develop in similar stages into polarized, multicellular epithelial structures. Analysis of membrane-cytoskeletal proteins ankyrin and fodrin during development of MDCK cell surface polarity shows that they gradually assemble into an insoluble protein complex on the basal-lateral membrane domain upon cell-cell adhesion, concomitantly with the redistribution of Na+,K(+)-ATPase, a marker protein of the basal-lateral membrane. Biochemical analysis shows that ankyrin, fodrin occur in a complex with Na+,K(+)-ATPase and the cell adhesion molecule uvomorulin in MDCK cells. A model is presented in which assembly of membrane-cytoskeletal complexes at sites of uvomorulin-induced cell-cell contact causes a remodelling of the cell surface distribution of specific membrane proteins which, in turn, contributes to the generation of epithelial cell surface polarity.

Animals↗
PubMed1989

Morphogenesis of the polarized epithelial cell phenotype.

Polarized epithelial cells play fundamental roles in the ontogeny and function of a variety of tissues and organs in mammals. The morphogenesis of a sheet of polarized epithelial cells (the trophectoderm) is the first overt sign of cellular differentiation in early embryonic development. In the adult, polarized epithelial cells line all body cavities and occur in tissues that carry out specialized vectorial transport functions of absorption and secretion. The generation of this phenotype is a multistage process requiring extracellular cues and the reorganization of proteins in the cytoplasm and on the plasma membrane; once established, the phenotype is maintained by the segregation and retention of specific proteins and lipids in distinct apical and basal-lateral plasma membrane domains.

Animals↗
PubMed1989

A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity.

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.

Animals↗
PubMed1989

Regulation of desmosome assembly in epithelial cells: kinetics of synthesis, transport, and stabilization of desmoglein I, a major protein of the membrane core domain.

Desmosomes are composed of two morphologically and biochemically distinct domains, a cytoplasmic plaque and membrane core. We have initiated a study of the synthesis and assembly of these domains in Madin-Darby canine kidney (MDCK) epithelial cells to understand the mechanisms involved in the formation of desmosomes. Previously, we reported the kinetics of assembly of two components of the cytoplasmic plaque domain, Desmoplakin I/II (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685 and 106:687-699. We have now extended this analysis to include a major glycoprotein component of the membrane core domain, Desmoglein I (DGI; Mr = 150,000). Using metabolic labeling and inhibitors of glycoprotein processing and intracellular transport, we show that DGI biosynthesis is a sequential process with defined stages. In the absence of cell-cell contact, DGI enters a Triton X-100 soluble pool and is core glycosylated. The soluble DGI is then transported to the Golgi complex where it is first complex glycosylated and then titrated into an insoluble pool. The insoluble pool of DGI is subsequently transported to the plasma membrane and is degraded rapidly (t1/2 less than 4 h). Although this biosynthetic pathway occurs independently of cell-cell contact, induction of cell-cell contact results in dramatic increases in the efficiency and rate of titration of DGI from the soluble to the insoluble pool, and its transport to the plasma membrane where DGI becomes metabolically stable (t1/2 greater than 24 h). Taken together with our previous study of DPI/II, we conclude that newly synthesized components of the cytoplasmic plaque and membrane core domains are processed and assembled with different kinetics indicating that, at least initially, each domain is assembled separately in the cell. However, upon induction of cell-cell contact there is a rapid titration of both components into an insoluble and metabolically stable pool at the plasma membrane that is concurrent with desmosome assembly.

Animals↗
PubMed1988

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

Animals↗
PubMed1988

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.

Animals↗
PubMed1988

Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect.

Madin-Darby canine kidney (MDCK) cells are highly differentiated and have retained the morphogenetic properties necessary to form polarized, multicellular epithelial structures (cysts) in vitro that resemble epithelial tissues in vivo. We introduced the c-src gene into MDCK cells to elevate the level of the plasma membrane-associated cellular tyrosine kinase, pp60c-src, to levels two- to ninefold higher than that expressed in parent MDCK cells. Our results revealed a highly discriminatory biological action of pp60c-src on the morphogenetic properties of MDCK cells. Elevated expression of pp60c-src conferred on MDCK cells the ability to undergo dramatic changes of cell shape that includes the formation of long cell processes (100 to 200 microns), never observed in control MDCK cells. The morphogenesis of multicellular epithelial cysts was altered by elevated levels of pp60c-src and led to predictable distortions of their three-dimensional architecture. However, these cells established morphologically normal cell polarity, formed adhesive epithelial cell-cell contacts indistinguishable from those of control MDCK cells, and exhibited neither focus-forming ability or anchorage-independent growth potential. Finally, we showed that MDCK cells expressing elevated levels of pp60c-src exhibit increased phosphorylation of a more limited number of phosphotyrosine-containing proteins than MDCK cells expressing pp60v-src. We suggest that a natural function of pp60c-src is to regulate the morphogenetic properties which determine the shape of differentiated cells and multicellular structures.

Animals↗
PubMed1988

Workers' compensation: coverage, benefits, and costs, 1985.

The 84.3 million workers protected by workers' compensation laws in 1985 represented 87 percent of all wage and salary workers in that year. Both the amount of benefits paid to workers and the cost of the program to employers rose substantially from 1984 to 1985. Benefit payments totaled $22.5 billion-14.1 percent higher than in 1984 and the largest annual increase since 1978-79. About two-thirds of the payments in 1985 were money payments ($15.1 billion) and the remainder ($7.4 billion) went for medical care for disabled workers. Private insurance companies made nearly three-fifths of these payments and State funds and self-insured employers each paid about one-fifth of the total benefit amount in 1985. For the first time since 1978, the annual growth in employer costs exceeded the growth in workers' benefits, resulting in a slight decrease in the loss ratio for 1984-85. Employer costs were up nearly 17 percent from the previous year, reaching an estimated $29.3 billion. Covered payrolls increased by 7 percent in that same period. Total benefit payments as a percent of payroll also increased noticeably in 1985.

Costs and Cost Analysis↗