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Biomedical subjects

S J Singer

Publications and source records attributed to S J Singer.

At least 163 records · Page 9Linked to original sources
PubMed1977

Interaction of filamin with f-actin in solution.

Filamin is a major high-molecular-weight protein in smooth muscle which was recently identified and isolated [Wang, K., Ash, J. F. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4483-4486]. In the present studies, we shown that highly purified chicken gizzard filamin and muscle F-actin react in solution to form aggregates containing both proteins. Occasionally, these aggregates coagulate and contract into a dense gel in the absence of MgATP or CaATP. Immunofluorescence and electron microscopic studies suggest that the F-actin filaments are collected into fiber bundles and a crosslinked fiber meshwork by the binding of filamin molecules. These studies suggest that the function of filamin intact cells may be to regulate the ultrastructural state of F-actin filaments in a variety of dynamic cellular processes.

Actins↗
PubMed1977

Intracellular distributions of mechanochemical proteins in cultured fibroblasts.

We have used methods that have allowed simultaneous fluorescent staining of intracellular actin together with either myosin, filamin, or tubulin in normal rat kidney fibroblasts in monolayer culture. In the main portions of the cell body, the actin, myosin, and filamin are all present in two structures: in one, the three proteins are present in the same fiber bundles (stress fibers); in the other, there is a diffuse distribution of the three proteins. On portions of the cell periphery however-in the basal regions of microspikes, in ruffles, and in regions of cell-cell contact-actin and filamin are present, but myosin is severely depleted or absent. Microtubules are present in the cell body in a distribution independent of the stress fibers and are mostly absent from the cell periphery. Microspikes and ruffles are highly dynamic structures on the cell surface, and regions of cell-cell contact generally result from the association of ruffles on the two contacting cells. Therefore, the presence of filamin and actin but not myosin in these specialized regions on the cell surface, together with the recent demonstration [Wang, K. & Singer, S. J. (1977) Proc. Natl. Acad. Sci. USA 74, 2021-2025)] that pure filamin interacts with individual F-actin filaments in solution to form fiber bundles and sheet-like structures, suggest that in vivo filamin-actin interactions play an important role in the control of actin filament structure, in cell motility, and in the stabilization of cell-cell contacts.

Cell Line↗
PubMed1977

On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex.

Human erythrocyte ghosts have been shown, by scanning electron microscopy, to undergo ATP-dependent shape changes. Under appropriate conditions the ghosts prepared from normal disk-shaped intact cells adopt a highly crenated shape, which in the presence of Mg-ATP at 37 degrees C is slowly converted to the disk shape and eventually to the cup shape. These changes are not observed with other nucleotides or with 5'-adenylyl imidodiphosphate. Anti-spectrin antibodies, incorporated along with the Mg-ATP into the ghosts in amounts less than equivalent to the spectrin, markedly accelerate the shape changes observed with the Mg-ATP alone. The Fab fragments of these antibodies, however, have no effect. The conclusion is that the structural effect produced by the ATP is promoted by the cross-linking of spectrin by its antibodies, and may therefore itself be some kind of polymerization or network formation involving the spectrin complex on the cytoplasmic face of the membrane. The factors that contribute to the shape of the ghost and of the intact erythrocyte are discussed in the light of these findings.

Adenosine Triphosphate↗
PubMed1977

On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP.

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.

Adenosine Triphosphate↗
PubMed1976

Relationships of the spectrin complex of human erythrocyte membranes to the actomyosins of muscle cells.

Important similarities are reported between human smooth muscle actomyosin and the human erythrocyte spectrin complex, primarily components 1, 2, and 5 (Fairbanks G., Steck, T.L., and Wallach, D.F.H. (1971), Biochemistry 10, 2606). The actin-like protein, component 5, is identical with human uterine actin in its ability to form 50-70-A filaments to stimulate myosin ATPase activity, and to bind rabbit heavy meromyoson specit heavy meromyosin specifically. Antibodies to human smooth muscle myosin(uterine) were prepared which were monospecific. A weak but specific cross-reaction of these antisera with components 1 and/or 2 (spectrin) was characterized and at least 25% of the antimyosin antibodies showed a low affinity reaction iwth spectrin. Antibodies generated against a soluble complex of spectrin components 1 and 2 reacted only with component 1 and did not cross-react with myosin. In addition to these structural similarities between smooth muscle actomyosin and the spectrin complex, we have found that spectrin is involved in ATP-dependent erythrocyte shape changes (Sheetz, M.P., Painter, R.G., AND Singer, S.J. (1976B), Cold Spring Harbor Symp. Cell Motility (in press) and, therefore, the spectrin complex is also a mechanochemical protein system.

Actins↗
PubMed1976

Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus.

By the use of a rat kidney cell line infected with a temperature-sensitive Rous sarcoma virus, we have shown that, at permissive temperatures where the cells are transformed, concanavalin A induces a clustering of its cell membrane receptors into patches, and the intracellular smooth muscle myosin-like protein is in a disordered state. By contrast, with infected cells grown at nonpermissive temperatures, the addition of concanavalin A does not alter the uniform distribution of its receptors, and the smooth muscle myosin-like protein is arranged in an ordered filamentous structure. These results are consistent with the hypothesis that the myosin protein is part of an intracellular aggregating-disaggregating complex. In the normal cell it is in its aggregated state and inhibits the lateral mobility of the concanavalin A receptors in the membrane; in the transformed cell the complex is relatively disaggregated and permits the concanavalin A receptors to be mobile. The addition of protein synthesis inhibitors to infected cells grown at the permissive temperature causes the cell to change from the transformed phenotype to the normal. Removal of the reversible inhibitors causes the cells to revert to the transformed phenotype. These results show that (i) protein synthesis, presumably of an unstable product of the transforming gene of the temperature-sensitive virus, is required to maintain the transformed state in these infected cells at the permissive temperature; and (ii) protein synthesis is not required for the intracellular myosin-containing complex to revert from its disordered transformed state to its ordered normal state. This suggests that the product of the transforming gene directly or indirectly causes the disaggregation of the myosin-containing complex in the process of transformation.

Abrin↗
PubMed1976

Clustering and endocytosis of membrane receptors can be induced in mature erythrocytes of neonatal but not adult humans.

Concanavalin A (Con A) is taken up by endocytosis in mature erythrocytes of newborn humans but not in adult red cells. Thin sections of neonatal cells incubated with ferritin-conjugated Con A at 37 degrees show ferritin clusters on invaginations at the surface and in intracellular vesicles, but such invaginations and vesicles are absent with adult cells. The endocytosis induced by ferritin-conjugated Con A is inhibited at 0 degrees, and by methyl-alpha-D-mannopyranoside at 37 degrees. Succinylation of Con A, which is known to convert it from the tetrameric to dimeric form, renders Con A inactive in cell agglutination and endocytotic vesicle formation, presumably by reducing the number of oligosaccharide chains simultaneously bound by a single Con A molecule. Ferritin-conjugated succinyl Con A binds to neonatal erythrocytes but does not induce endocytosis; if, however, antibodies to ferritin are now added, endocytosis occurs. These results are consistent with a greater lateral mobility of at least a fraction of Con A recptors in the membrane of the intact neonatal erythrocyte compared to the adult. The results also support the hypothesis that the clustering of receptors is obligatory for endocytosis to occur. No discernible difference was found in the sodium dodecyl sulfate/polyacrylamide gel patterns of the membrane proteins of the neonatal and adult cells.

Adult↗
PubMed1976

Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin-containing filaments.

With normal rat kidney cells in monolayer culture, we have studied the distribution on the cell surface of receptors for concanavalin A, and the distribution of the smooth muscle myosin-like protein inside the same cell, using specific fluorescence microscopic methods. The concanavalin A receptors were initially uniformly dispersed over the cell surface, but 20 min after the addition of concanavalin A at 37 degrees, the receptors showed a variety of nonuniform surface distributions, including extended parallel linear arrays. These arrays of receptors were found to be superimposed on the linear arrays of the intracellular myosin-containing filaments, indicating that a transmembrane linkage of the receptors and the filaments had occurred. This linkage required a lateral redistribution of concanavalin A receptors, since it did not occur with succinylated concanavalin A, but was subsequently induced if the cells that had been reacted with succinylated concanavalin A were then treated with antibodies to concanavalin A. The redistributions of concanavalin A receptors on the surfaces of these normal rat kidney cells, however, were much less extensive than the patching that was induced on the surfaces of the same cells infected with, and transformed by, Rous sarcoma virus.

Cell Line↗
PubMed1976

An experiment eliminating the rotating carrier mechanism for the active transport of Ca ion in sarcoplasmic reticulum membranes.

An experiment has been carried out to test the rotating carrier mechanism of the translocation event in membrane transport. To the Ca-ATPase in intact sarcoplasmic reticulum membranes, 2,4-[3H]dinitrophenyl-cadaverine has been covalently attached by the action of the enzyme, transglutaminase. The binding of anti-2,4-dinitrophenyl antibodies to the 2,4-dinitrophenyl-modified membranes was found to have no effect on either the Ca-ATPase activity or the Ca ion transport rate of the membranes. These results rule out the rotating carrier mechanism in this system. A different scheme for the translocation process, the aggregate rearrangement mechanism, is discussed.

Adenosine Triphosphatases↗
PubMed1976

Biological membranes as bilayer couples. III. Compensatory shape changes induced in membranes.

We have previously proposed the hypothesis that asymmetric membranes behave like bilayer couples: the two layers of the bilayer membrane can respond differently to a particular perturbation. Such a perturbation, for example, can result in the expansion of one layer relative to the other, thereby producing a curvature of that membrane. In experiments with erythrocytes and lymphocytes, we now demonstrate that different membrane perturbations which have opposite effects on membrane curvature can compensate and neutralize one another, as expected from the bilayer couple hypothesis. This provides a rational basis, for example, for understanding the effects of amphipathic drugs on a variety of cellular phenomena which involve shape changes of membranes.

Adenosine Triphosphate↗
PubMed1976

Equilibrium and kinetic effects of drugs on the shapes of human erythrocytes.

We have previously proposed that if the two half-layers of a membrane are different in their protein and lipid compositions, they may respond differently to some membrane perturbation (the bilayer couple hypothesis). This hypothesis has been applied to explain the changes in shape of human erythrocytes that are produced by a variety of amphipathic compounds. These compounds are presumed to intercalate by their hydrophobic ends into the lipid portions of the membrane; if the compounds are anions, the binding is preferentially to the outer half of the bilayer, if cations, to the inner half. It is proposed that such preferential binding causes an expansion of one half-layer relative to the other, with a corresponding change in cell shape. The predicted sidedness of these shape changes is now demonstrated in experiments with methochlorpromazine and 2,4,6-trinitrophenol. Under appropriate nonequilibrium or equilibrium or equilibrium conditions, both of these compounds are shown to be either crenators or cup-formers of the intact erythrocyte, depending upon which side of the membrane they are concentrated in. These results therefore strongly support the bilayer couple hypothesis.

Chlorpromazine↗
PubMed1976

Improved procedures for immunoferritin labeling of ultrathin frozen sections.

In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

Antigens↗
PubMed1975

Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells.

A new high-molecular-weight protein, named filamin, was isolated from chicken gizzard. In chicken gizzard, filamin is present in an amount approximately 30-40% of that of myosin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of highly purified filamin revealed a single polypeptide of about 250,000 daltons. Rabbit antibody directed against purified chicken gizzard filamin did not crossreact with myosin purified from the same source. By the use of microcomplement fixation and indirect immunofluorescent staining with antibodies to chicken gizzard filamin, an antigenically similar or identical protein was found to be widely distributed both in other organs of the chicken and in cultured cells of other species, but not in chicken skeletal muscle. In cultured cells, filamin was found largely to be arranged as a filamentous array very similar to that found for myosin. These data imply that filamin is a widely occurring and chemically conserved component of filaments is smooth muscle and non-muscle cells.

Animals↗
PubMed1975

Detection and ultrastructural localization of human smooth muscle myosin-like molecules in human non-muscle cells by specific antibodies.

Spectrin, a protein complex which is peripherally attached to the cytoplasmic surface of the human erythrocyte membrane, cannot be detected (by complement fixation with anti-spectrin antibodies) in homogenates of several different human non-muscle cells studied. On the other hand, a protein antigenically identical or similar to human smooth muscle myosin was detected (by complement fixation with antibodies to uterine smooth muscle myosin) in these cells. In the case of human fibroblast line WI38, this smooth muscle myosin like component was shown (by ferritin-antibody experiments in electron microscopy) to be at least partly associated with cytoplasmic surface of the plasma membrane of the cell. It is proposed that the spectrin complex of the erythrocyte membrane and the smooth muscle myosin-like component of the fibroblast membrane play similar roles in regulating the translational mobilities of integral proteins in their respective membranes.

Antigen-Antibody Reactions↗