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Biomedical subjects

K M Yamada

Publications and source records attributed to K M Yamada.

At least 19 recordsLinked to original sources
PubMed1992

Distinct regions of human fibronectin are essential for fibril assembly in an in vivo developing system.

In early vertebrate development, the proper assembly of fibronectin into fibrils is crucial for embryonic cells to adhere and to migrate on the extracellular matrix. The molecular mechanisms by which such a process occurs in vivo are poorly understood. In the amphibian embryo Pleurodeles waltl fibronectin fibrils appear first at the blastula stage. They form a fibrillar matrix on the basal surface of animal cells facing the blastocoel. Using competition and perturbation experiments with purified proteolytic fragments and domain-specific monoclonal antibodies, we demonstrate that at least three fibronectin sites are essential for assembly of fibronectin fibrils in the blastula of Pleurodeles waltl. Two sites, the RGDS sequence and the synergistic domain in the 10th type III repeat, are both involved in receptor recognition. A third site that spans the 9th type I and 1st type III homology sequences is also likely to participate in fibronectin-fibronectin interactions.

Amino Acid Sequence↗
PubMed1992

Regulation of fibronectin receptor distribution.

To determine the role of each intracellular domain of the fibronectin receptor in receptor distribution, chimeric receptors were constructed containing the human interleukin-2 receptor (gp55 subunit) as the extracellular and transmembrane domains, in combination with either the alpha 5 or beta 1 intracellular domain of the fibronectin receptor as the cytoplasmic domain. These chimeric receptors were transiently expressed in normal fibroblasts, and their localization on the cell surface was determined by immunofluorescence using antibodies to the human interleukin-2 receptor. The alpha 5 chimera was expressed diffusely on the plasma membrane. The beta 1 chimera, however, colocalized with the endogenous fibronectin receptor at focal contacts of cells spread on fibronectin. On cells spread in the presence of serum, the beta 1 chimera colocalized both with the fibronectin receptor at sites of extracellular fibronectin fibrils and with the vitronectin receptor at focal contacts. The beta 1 intracellular domain alone, therefore, contains sufficient information to target the chimeric receptor to regions of the cell where ligand-occupied integrin receptors are concentrated. The finding that the beta 1 chimeric protein behaves like a ligand-occupied receptor, even though the beta 1 chimera cannot itself bind extracellular ligand, suggests an intracellular difference between occupied and unoccupied receptors, and predicts that the distribution of integrin receptors can be regulated by ligand occupancy. We tested this prediction by providing a soluble cell-binding fragment of fibronectin to cells spread on laminin. Under conditions preventing further ligand adsorption to the substrate, this treatment nevertheless resulted in the relocation of diffuse fibronectin receptors to focal contacts. Similarly, a redistribution of diffuse vitronectin receptors to focal contacts occurred on cells spread on laminin after the addition of the small soluble peptide GRGDS. We conclude that the propensity for receptor redistribution to focal contacts driven by the beta 1 cytoplasmic domain alone is suppressed in heterodimeric unoccupied fibronectin receptors, and that ligand occupancy can release this constraint. This redistribution of integrin receptors after the binding of a soluble substrate molecule may provide a direct means of assembling adhesion sites.

Amino Acid Sequence↗
PubMed1992

Altered glycosylation and cell surface expression of beta 1 integrin receptors during keratinocyte activation.

We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered beta 1 integrin subunit glycosylation accompanied by an increase in cell surface beta 1 integrin receptors. Activated keratinocytes contained two forms of the beta 1 integrin subunit, approximately 93 kDa and approximately 113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Pre-activated keratinocytes contained beta 1 integrin subunits ranging from approximately 97 to 110 kDa. These beta 1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of beta 1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller beta 1 subunits found in pre-activated keratinocytes, like the precursor beta 1 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a approximately 10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa beta 1 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte alpha subunits associated with beta 1. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.

Animals↗
PubMed1992

Contact stimulation of cell migration.

Mass migrations of dense cell populations occur periodically during embryonic development. It is known that extracellular matrices, through which the cells migrate, facilitate locomotion. However, this does not explain how cells, such as neural crest, can migrate as a dense cohort of cells in essentially continuous contact with one another. We report here that unique behavioral characteristics of the migrating cells may contribute to cohesive migration. We used time-lapse video microscopy to analyze the migration of quail neural crest cells and of two crest derivatives, human melanoma cells and melanocytes. These cells migrated poorly, if at all, when isolated, but could be stimulated up to 200-fold to travel following contact with migrating cells. This phenomenon, which we have termed "contact-stimulated migration," appeared to activate and sustain migration of the mass of cells. Cells that became dissociated from the others ceased directional migration, thereby limiting aberrant cell dispersion. Fibroblasts were minimally responsive to this novel phenomenon, which may be crucial for major, mass cell migrations.

Animals↗
PubMed1990

Novel function for beta 1 integrins in keratinocyte cell-cell interactions.

We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.

Actins↗
PubMed1979

Partial purification and characterization of the messenger RNA for cell fibronectin.

Fibronectin mRNA has been partially purified by guanidine extraction, oligo-(dT)-cellulose chromatography and sucrose density gradient centrifugation. We obtain a fraction which programs a wheat germ in vitro translation system to synthesize a polypeptide species which co-electrophoreses with fibronectin in SDS-polyacrylamide gels and which is immunoprecipitated with affinity purified fibronectin-specific IgG. Analysis of this RNA fraction by methyl mercury hydroxide-agarose gel electrophoresis reveals the presence of a band accounting for 30 percent to 50 percent of the ethidium bromide-staining material in the fraction. The RNA of this band has an estimated molecular weight of about 3 million daltons and is greatly reduced in the corresponding RNA fraction from RSV transformed CEF. This RNA has been tentatively identified as fibronectin mRNA.

Animals↗
PubMed1979

Characterization of factor(s) in culture supernatants affecting cell social behavior.

Treatment of embryonic chick heart fibroblast cultures with 0.2 M urea reversibly increases cellular overlap. The increase in cellular overlapping over that in control cultures may be quantitated by the overlap ratio (R), the ratio of the number of superimposed nuclei observed, to the number expected to occur when cells are assumed to be distributed randomly over the culture substratum (R = observed/expected overlaps). Reversal of the urea-induced increase in R is blocked by 0.2 micrograms/ml cycloheximide. In the presence of cycloheximide, normal (low) overlap ratios are restored to urea-treated cultures by adding non-dialyzable material recovered by washing fibroblast monolayers with serum-free medium. The overlap ratio assay revealed no effect of supernatant material added either to urea-treated cultures in the continued presence of urea, or to untreated cultures. Although unfiltered supernatants were shown by SDS-polyacrylamide gel electrophoresis to contain fibronectin (CSP; LETS; MWappar. = 220,000 d) and smaller proteins, the ability to reverse the urea-induced increase in overlap ratio was present in Diaflo and Millipore filtrates of culture supernatants in which fibronectin was greatly depleted or absent. In contrast, purified fibronectin preparations failed to lower urea-induced increases in overlap-ratio. Partially purified, biologically active supernatants, prepared from 14C-leucine or 125I-labeled cultures, contained several macromolecules smaller than fibronectin that were labeled by both radioisotopes. In particular, one band (MWappar. = 58--60,000 d) was present in polyacrylamide gels of active supernatant and also depleted in gels of homogenates from urea-treated cultures. These results indicate that external macromolecules other than fibronectin are synthesized by cultured fibroblasts and can affect cell social behavior or culture morphology.

Animals↗
PubMed1979

Enhanced cellular fibronectin accumulation in chondrocytes treated with vitamin A.

Chick sternal chondrocytes cultured at high cell density lack fibronectin as a surface protein, while vitamin A-treated chondrocytes contain it as the major cell surface protein. We investigated the mechanism of fibronectin accumulation under these conditions. Control chondrocytes synthesized nearly as much fibronectin as vitamin A-treated chondrocytes, but it was secreted primarily into culture medium. Althought the fibronectin of control chondrocytes was of a slightly lower apparent molecular weight than the fibronectin synthesized by the treated cells, it bound as effectively to the cell layer of both normal and treated cells. In contrast, the vitamin A-treated cultures were 2.7 fold more effective in binding fibronectin synthesized by either control or treated cells. Thus in chondrocytes, vitamin A appears to regulate the cellular accumulation of fibronectin by increasing the ability of the cell layer to bind fibronectin rather than by altering its synthesis or its adhesivity for the cell layer.

Animals↗
PubMed1979

Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin.

Using tunicamycin, we have investigated the role of glycoproteins in membrane transport. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues of glycoproteins. Inhibition of protein glycosylation in chick embryo fibroblasts by tunicamycin or other inhibitors of glycosylation resulted in defective transport of glucose, uridine, and amino acid analogs (alpha-aminoisobutyrate and cycloleucine). The defect in glucose transport is accompanied by decreased glucose metabolism, as determined by rates of CO2 and lactate production. In contrast, tunicamycin treatment did not affect other membrane-associated processes, such as secretion of fibronectin and procollagen, uptake of glucose by passive diffusion, Na+/K+ ATPase and adenylate cyclase activities, or stimulation of adenylate cyclase by prostaglandin and cholera toxin. Two glucose/glycosylation-regulated membrane proteins with apparent subunit molecular weights of 95,000 and 75,000 were induced by tunicamycin treatment. Our results indicate that glycoprotein glycosylation is required for membrane transport.

Amino Acids↗
PubMed1979

Identification and isolation of a collagen-binding fragment of the adhesive glycoprotein fibronectin.

We have identified and purified a polypeptide region containing the collagen-binding site of the adhesive glycoprotein fibronectin. Chicken cellular fibronectin isolated from cultured embryonic fibroblasts was permitted to bind to gelatin coupled to agarose beads and was then digested extensively with chymotrypsin. A prominent 40,000-dalton fragment of fibronectin consisting of a single polypeptide chain was detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of material remaining bound to the gelatin-agarose. This fragment appeared within 10 min after the digestion was initiated and persisted for more than 20 hr. This proteolytic fragment was isolated in electrophoretically pure form and retained its affinity for collagen. Plasma fibronectins from chicken and human blood also contained collagen-binding proteolytic fragments of similar size. This finding suggest that the collagen-binding sites of cellular and plasma fibronectins are homologous.

Animals↗