Biomedical subjects

W J Nelson

Publications and source records attributed to W J Nelson.

At least 73 records · Page 4Linked to original sources

PubMed1995

Differential expression of Na(+)-K(+)-ATPase, ankyrin, fodrin, and E-cadherin along the kidney nephron.

Ionic homeostasis in vertebrates is maintained by epithelial cells that line kidney nephrons. Transport of ions and solutes is coupled to Na+ reabsorption from the ultrafiltrate and requires specific subcellular distribution and activity of Na(+)-K(+)-ATPase along the nephron. Studies using cell culture models of renal epithelia indicate that the subcellular distribution of Na(+)-K(+)-ATPase is regulated by interactions with the submembrane cytoskeleton and E-cadherin-mediated adherens junctions. We have now examined the relevance of these in vitro observations to the subcellular organization of these proteins in different nephron segments of the adult mouse kidney using immunofluorescence microscopy. Our results demonstrate that segmental and subcellular distributions of Na(+)-K(+)-ATPase and the membrane-cytoskeletal proteins, ankyrin and fodrin, vary in parallel along the nephron and do not parallel variations in expression of the tight junction protein ZO-1 or E-cadherin. These data indicate that a mechanism for restricting Na(+)-K(+)-ATPase subcellular distributions through interactions with the membrane cytoskeleton is likely to be relevant in vivo.

Animals↗

PubMed1995

Differential expression of cell-cell and cell-substratum adhesion proteins along the kidney nephron.

Structural and functional differences among epithelial cells of kidney nephrons may be regulated by variations in cell-to-cell (cell-cell) and cell-to-substratum (cell-substratum) junctions. Using immunofluorescence microscopy, we demonstrate that the cadherin-associated proteins alpha- and beta-catenin are localized to basolateral membranes of cells in all nephron segments, whereas plakoglobin, a protein associated with both classical and desmosomal cadherins, is localized to noninterdigitated lateral membranes in the distal half of the nephron where it colocalizes with desmoplakin and cytokeratin K8. Plakoglobin is also present in capillary endothelial cells where staining for the other catenins and desmosomal proteins is not observed. Immunofluorescence for laminin A and alpha 6-integrin, proteins that mediate cell-substratum contacts, reveal no correlations with the other staining patterns observed. These data indicate that plakoglobin and beta-catenin subserve distinct functions in cell-cell adhesion and suggest that E-cadherin-mediated contacts generate a basal level of cell-cell adhesion, whereas desmosomal junctions provide additional strength to cell-cell contacts in the distal nephron.

Animals↗

PubMed1994

Feedback regulation of cell-substratum adhesion by integrin-mediated intracellular Ca2+ signaling.

Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, would healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion.

Amino Acid Sequence↗

PubMed1994

Organization and function of the cytoskeleton in polarized epithelial cells: a component of the protein sorting machinery.

Development and maintenance of cell-surface polarity in epithelial cells requires specialized localization of proteins to functionally and structurally distinct plasma membrane domains. The organization of these domains is dependent upon targeted delivery of transport vesicles between different membrane compartments, and upon protein sorting in the membranes of the Golgi complex and cell surface. Increasing evidence has been gathered in recent years that cytoskeletal components facilitate these processes.

Actin Cytoskeleton↗

PubMed1994

Beta-catenin: a common target for the regulation of cell adhesion by Wnt-1 and Src signaling pathways.

Beta-catenin is a cytosolic protein originally identified through its association with the cadherin class of cell-adhesion proteins. However, recent studies have demonstrated that there are cadherin-independent pools of beta-catenin and that beta-catenin binds at least one other protein, the product of the tumor-suppressor gene APC. Furthermore, beta-catenin is the target of two signal transduction pathways mediated by the proto-oncogenes src and wnt-1. This raises the possibility that beta-catenin plays a pivotal role in balancing cellular responses to both adhesive and proliferative signals.

Animals↗

PubMed1994

Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin.

Wnt-1 homologs have been identified in invertebrates and vertebrates and play important roles in cellular differentiation and organization. In Drosophila, the products of the segment polarity genes wingless (the Wnt-1 homolog) and armadillo participate in a signal transduction pathway important for cellular boundary formation in embryonic development, but functional interactions between the proteins are unknown. We have examined Wnt-1 function in mammalian cells in which armadillo (beta-catenin and plakoglobin) is known to bind to and regulate cadherin cell adhesion proteins. We show that Wnt-1 expression results in the accumulation of beta-catenin and plakoglobin. In addition, binding of beta-catenin to the cell adhesion protein, cadherin, is stabilized, resulting in a concomitant increase in the strength of calcium-dependent cell-cell adhesion. Thus, a consequence of the functional interaction between Wnt-1 and armadillo family members is the strengthening of cell-cell adhesion, which may lead to the specification of cellular boundaries.

Amino Acid Sequence↗

PubMed1994

Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.

Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells.

The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta-catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100-insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.

Animals↗

PubMed1994

Golgi spectrin: identification of an erythroid beta-spectrin homolog associated with the Golgi complex.

Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

Animals↗

PubMed1994

Determinants of apical membrane formation and distribution in multicellular epithelial MDCK cysts.

Madin-Darby canine kidney epithelial cells form three-dimensional cysts in spinner culture with a defined cell surface polarity. Transfer of cysts from spinner culture to a collagen gel matrix results in rapid loss of apical membrane proteins from the outside surface of the cyst, degradation of extracellular matrix (ECM) from the cyst lumen, and de novo formation of the apical membrane at the luminal surface. Degradation of endogenous ECM was inhibited with 1,10-phenanthroline, an inhibitor of metalloproteinases, resulting in cysts in which cells are surrounded by either cell-cell or cell-substratum contacts. The consequence of the lack of a free cell surface on the formation of a new apical membrane domain in these cysts was analyzed. Changes in cell surface polarity were followed with antibodies to marker proteins of the apical or basolateral membranes. In the absence of a free cell surface, the apical membrane formed de novo by accumulation and fusion of presorted vesicles containing apical membrane proteins; the coalescence of these vesicles results in the formation of a central lumen. These results provide novel insights into the generation of membrane domains and formation of a lumen in complex, three-dimensional epithelial structures in development.

Animals↗

PubMed1994

Disability trends in the United States: a national and regional perspective.

Between 1978 and 1993, the number of persons receiving disability benefits under either the Supplemental Security Income program or the Old-Age, Survivors, and Disability Insurance program increased more than 43 percent--from 4.7 million to 6.7 million. In 1993, 4.08 percent of the U.S. resident population aged 18-64 were receiving a disability benefit under one or both of the programs, compared with 3.37 percent in 1978. This ratio had declined to 2.93 percent in 1983. The article examines the change in growth since 1975 in each of the two disability programs and provides an overview of key legislative changes occurring during the period. The emphasis is on regional and State changes during the 1987-92 period, identifying those areas where growth has been the most dramatic.

Adolescent↗

PubMed1994

Cell polarity in human renal cystic disease.

BACKGROUND: In polycystic kidney disease (PKD), altered cellular polarity with mislocation of Na/K-ATPase, and net fluid secretion may have a role in cyst development and progression. EXPERIMENTAL DESIGN: Cell polarity was assessed in surgically excised human normal, autosomal dominant PKD, and acquired PKD occurring in end stage renal disease on long-term dialysis kidneys quick frozen (< 5 minutes) or fixed to minimize ischemic changes. RESULTS: Findings were similar in autosomal dominant PKD and acquired PKD kidneys. By ultrastructure, in cysts, cells were polarized, however, their basement membranes were greatly thickened and reticulated. By immunohistology, in cell-lining cysts, Na/K-ATPase, fodrin, and ankyrin were localized primarily to basolateral cell membranes and uvomorulin was localized to lateral cell membranes. In about 25% of the cells, however, Na/K-ATPase was localized to the apical as well as the basolateral membranes. Both in autosomal dominant PKD and normal kidney cell monolayers in vitro, cationic ferritin was normally absorbed by apical endocytosis, and transferred to apical vacuoles and phagolysosomes. CONCLUSIONS: These findings indicate intact structural and functional polarity in cell-lining cysts; however, in about 25% of the cells, Na/K-ATPase, fodrin, and ankyrin are localized to apical and lateral cell membranes, probably due to cell dedifferentiation. The notable changes in the basement membranes of cysts suggest a key role for the extracellular matrix in the pathogenesis of PKD.

Adult↗

PubMed1993

Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion.

Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.

Animals↗

PubMed1993

Regulation of cell surface polarity in renal epithelia.

In the kidney, polarized epithelial cells play critical roles in ion, fluid and solute reabsorption from the ultrafiltrate to the blood supply. Detailed analysis of protein distributions has revealed that ion channels, transporters and pumps are restricted to distinct domains of the plasma membrane that face either the ultrafiltrate (apical membrane) or the blood supply (basal-lateral membrane). The importance of the development and maintenance of the polarized distributions of these proteins in renal epithelia for normal cell function is demonstrated by the fact that several disease states are characterized by abnormal distributions of proteins; for example in polycystic kidney disease, Na+/K(+)-ATPase has been detected in the apical and lateral membranes, compared with normal cells where Na+/K(+)-ATPase is localized in the basal-lateral membrane domain. Recent studies indicate that the development of restricted distributions of proteins at the cell surface of Madin Darby canine kidney epithelial cells is determined by direct sorting of proteins in the trans Golgi network into vesicles that are delivered vectorially to either the apical or basal-lateral membrane. Upon arrival at the plasma membrane, some proteins, such as Na+/K(+)-ATPase, may be selectively retained by binding to the membrane cytoskeleton.

Animals↗

PubMed1993

Spatial and temporal dissection of immediate and early events following cadherin-mediated epithelial cell adhesion.

Cell-cell adhesion is at the top of a molecular cascade of protein interactions that leads to the remodeling of epithelial cell structure and function. The earliest events that initiate this cascade are poorly understood. Using high resolution differential interference contrast microscopy and retrospective immunohistochemistry, we observed that cell-cell contact in MDCK epithelial cells consists of distinct stages that correlate with specific changes in the interaction of E-cadherin with the cytoskeleton. We show that formation of a stable contact is preceded by numerous, transient contacts. During this time and immediately following formation of a stable contact, there are no detectable changes in the distribution, relative amount, or Triton X-100 insolubility of E-cadherin at the contact. After a lag period of approximately 10 min, there is a rapid acquisition of Triton X-100 insolubility of E-cadherin localized to the stable contact. Significantly, the total amount of E-cadherin at the contact remains unchanged during this time. The increase in the Triton X-100 insoluble pool of E-cadherin does not correlate with changes in the distribution of actin or fodrin, suggesting that the acquisition of the Triton X-100 insolubility is due to changes in E-cadherin itself, or closely associated proteins such as the catenins. The 10 minute lag period, and subsequent prompt and localized nature of E-cadherin reorganization indicate a form of signaling is occurring.

Actins↗

PubMed1993

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia.

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Defining E-cadherin-associated protein complexes in epithelial cells: plakoglobin, beta- and gamma-catenin are distinct components.

Ca(2+)-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include alpha-, beta-, and gamma-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either beta- or gamma-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and beta-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of gamma-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and beta-catenin co-immunoprecipitate with E-cadherin. Laser-scanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with gamma-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for < 50% of the gamma-catenin signal. Two-dimensional gel electrophoresis resolved the gamma-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI approximately 6.1). The other protein comigrated with gamma-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of approximately 4.25; we refer to this protein as gamma-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains alpha-, beta-, and gamma-catenin. Although plakoglobin shares sequence similarity with beta-catenin, and comigrates with gamma-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The association of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.

Amino Acid Sequence↗

PubMed1993

Epithelial cell adhesion and development of cell surface polarity: possible mechanisms for modulation of cadherin function, organization and distribution.

Epithelial cell adhesion is principally regulated by calcium-dependent cell adhesion proteins, termed cadherins. Recent studies indicate that cadherin function is modulated by a class of proteins, termed catenins, that bind to the cytoplasmic domain of cadherin. Here we review the evidence that catenins regulate cadherin function in cell-cell adhesion, and discuss their role in initiating cell surface polarity in epithelial cells.

Amino Acid Sequence↗