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Biomedical subjects

S J Singer

Publications and source records attributed to S J Singer.

At least 145 records · Page 8Linked to original sources
PubMed1979

Concanavalin A-induced endocytosis in rabbit reticulocytes, and its decrease with reticulocyte maturation.

Concanavalin A (Con A) was taken up to a limited extent by endocytosis in rabbit reticulocytes but not in rabbit erythrocytes. This process was observed by the use of ferritin-labeled Con A and transmission electron microscopy of thin sections of plastic-embedded cells. Furthermore, the extent of endocytosis among the reticulocytes decreased with the extent of their maturation, reticulocyte age being measured by ribosome configurations. These results are consistent with the proposal that there are domains in the membranes of reticulocytes in which the Con A receptors are laterally mobile, and can be clustered and endocytosed. These mobile domains exist, or are formed, within a larger framework of immobile membrane. During reticulocyte maturation, these domains are gradually eliminated, eventually disappearing upon formation of the mature erythrocyte. Possible molecular mechanisms for this proposed elimination process are discussed.

Animals↗
PubMed1978

Localization of T25 glycoprotein in wild-type and Thy 1- mutant cells by immunofluorescence and immunoelectron microscopy.

The wild-type BW5147 (Thy 1+) cell line and its Thy 1- mutant derivative BW5147 (Thy 1-a) were examined by immunofluorescence and immunoelectron microscopy for the presence of T25, the glycoprotein which bears the Thy 1 alloantigen. The wild-type cell had T25 predominantly localized on the cell surface. In the mutant cell line, T25 accumulated intracellularly and was present in a clustered distribution throughout the cytoplasm. T25 was not present on the surface of the mutant cell line in significant amount.

Animals↗
PubMed1978

The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections.

Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 1 micrometer thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F-actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.

Actins↗
PubMed1978

Transmembrane interactions and the mechanisms of transport of proteins across membranes.

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin- and myosin-containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand-induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin- and myosin-containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane-associated actin- and myosin-containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism. To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin.

Actins↗
PubMed1978

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

Normal rat kidney cells infected with a temperature-sensitive mutant (LA23) of Rous sarcoma virus exhibit the transformed phenotype when grown at 33 degrees and the normal phenotype at 39 degrees. We have previously shown [Ash, J.F., Vogt, P.K. & Singer, S.J. (1976) Proc. Natl. Acad. Sci. USA 73, 3603-3607] that the addition of protein synthesis inhibitors to LA23-infected cells grown at 33 degrees causes them to revert, over a period of 12 hr, to the normal phenotype with respect to morphological and cytoskeletal characteristics. We now show that reversion of the metabolic characteristics of the transformed phenotype to those of the normal also occurs under these conditions. LA23-infected cells show an increased rate of aerobic glycolysis at 33 degrees compared to that at 39 degrees. They also show a different sensitivity of that rate to dinitrophenol and oligomycin at 33 degrees compared to 39 degrees. Such cells grown at 33 degrees in the presence of cycloheximide or abrin rapidly recover the aerobic glycolysis characteristics of the normal phenotype. These results support the thesis that transformation by the src gene of the Rous sarcoma virus is a pleiotypic and reversible process, such as is involved in a pleiotypic enzymic modification reaction and its reversal.

Aerobiosis↗
PubMed1978

Participation of histocompatibility antigens in capping of molecularly independent cell surface components by their specific antibodies.

The antibody-induced capping of several cell surface components has been investigated by immunofluorescence methods using two mouse cell lines, a parental C58 thymoma line and a mutant derived from it lacking TL and H-2 antigens. Other cell surface components were present in approximately equal amounts on the two cells. Parental cells treated with rabbit antibodies to T200, a major surface glycoprotein, rapidly formed caps containing T200, but the mutant cells similarly treated showed a uniform surface distribution of T200. On the other hand, with a secondary antibody treatment, the T200 on both cells capped equally well. When the indirect T200 caps were examined using a second immunofluorescent stain for H-2, TL, or Thy-1 antigens, it was found that on parental cells all three of these antigens were co-capped with T200; on mutant cells no staining was found for H-2 or TL, as expected, and essentially uniform distribution of Thy-1 was observed. The co-capping of H-2, TL, and Thy-1 antigens with T200 on the parent cell is remarkable, because the first three components are known to be molecularly independent in lymphocyte cell surfaces. The indirect capping of the viral glycoprotein gp 69/71 similarly induced a co-capping of H-2 and TL antigens on the parent cell. These results demonstrate that H-2 and related molecules may co-cap with a variety of independent cell surface antigens. Such co-capping of histocompatibility components could play an important role in a proposed dual recognition mechanism for cell-mediated cytotoxicity reactions and other immunologically important cell-cell interactions.

Antibodies, Neoplasm↗
PubMed1978

Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431.

The binding and internalization of epidermal growth factor (EGF) in human epithelioid carcinoma cells (A-431), which have approximately 2.6 X 10(6) receptors per cell, has been followed with 125I-labeled EGF and by fluorescence microscopy. We have prepared a fluorescent derivative of EGF that is biologically active and retains substantial binding affinity for cell receptors. After binding of this derivative to cells at 6 degrees, the cellular borders were prominently stained and the fluorescence on the remainder of the membrane was uniform. Upon warming of these cells to 37 degrees for 10 min, the surface fluorescence diminished and randomly distributed endocytotic vesicles appeared in the cytoplasm. After 20 min at 37 degrees these fluorescent vesicles formed a perinuclear ring. The binding of EGF to the surface of these cells was also visualized by immunofluorescence using rabbit antibodies to EGF and rhodamine-labeled goat anti-rabbit antibodies. We did not detect large fluorescent clusters or cap formation in these experiments. These data provide direct confirmation of the previous biochemical data that suggested that cell membrane-bound EGF is rapidly internalized.

Biological Transport↗
PubMed1978

Association of mitochondria with microtubules in cultured cells.

By indirect immunofluorescence techniques, microtubules and mitochondria were localized in normal rat kidney cells, human WI38 fibroblasts, mouse peritoneal macrophages, and a putative smooth muscle rat cell line, in monolayer culture. The mitochondria were found to be arranged along the cytoplasmic microtubules in each cell type. Disruption of the microtubules with colcemid caused a redistribution of the mitochondria in these cells. There was no correlation between the location of the mitochondria and actin-containing filaments. This evidence suggests that mitochondria are directly or indirectly associated with microtubules in these cells.

Actins↗
PubMed1977

Fluidity in the membranes of adult and neonatal human erythrocytes.

Several antigens and receptors are mobile in the plane of the membrane of the intact neonatal human erythrocyte but not in the membrane of the normal adult cell. In this report, measurements of the fluorescence polarization of perylene dissolved in isolated erythrocyte membranes are reported, which are indistinguishable for the two kinds of cells. This result indicates that the viscosities of the membrane interiors of the two cells are the same. The observed mobility differences, therefore, cannot be attributed to different lipid viscosities in the two membranes.

Adult↗
PubMed1977

Thermodynamics, the structure of integral membrane proteins, and transport.

Membranes are structures whose lipid and protein components are at, or close to, equilibrium in the plane of the membrane, but are not at equilibrium across the membrane. The thermodynamic tendency of ionic and highly polar molecules to be in contact with water rather than with nonpolar media (hydrophilic interactions) is important in determining these equilibrium and nonequilibrium states. In this paper, we speculate about the structures and orientations of integral proteins in a membrane, and about how the equilibrium and nonequilibrium features of such structures and orientations might be influenced by the special mechanisms of biosynthesis, processing, and membrane insertion of these proteins. The relevance of these speculations to the mechanisms of the translocation event in membrane transport is discussed, and specific protein models of transport that have been proposed are analyzed.

Biological Transport, Active↗
PubMed1977

Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands.

The mechanism of capping of cell surface receptors has been examined by a double fluorescence staining procedure that permitted simultaneous observations of the distribution of a surface-bound ligand together with intracellular actin or myosin. At an early stage in the capping of the T-25 antigen or the H2 histocompatibility antigens on mouse splenic T lymphocytes, or of concanavalin A receptors on HeLa cells, when the specific receptors in question were collected into patches that were distributed over the entire cell surface, the intracellular membrane-associated actin or myosin was also accumulated into patches that were located directly under the receptor patches. These and other results have led us to propose a general molecular mechanism for the process of capping, in which actin and myosin are directly involved. It is suggested that membrane-associated actin is directly or indirectly bound to an integral protein or class of proteins, X, in the plasma membranes of eukaryotic cells. When any receptor in the membrane is aggregated by an external multivalent ligand, the aggregate binds effectively to X, whereas unaggregated receptors do not bind to X. The receptor aggregates, linked to actin (and myosin) through X, are then actively collected into a cap by an analogue of the actin--myosin sliding filament mechanism of muscle contraction.

Actins↗
PubMed1977

Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts.

The surface distributions of three different membrane integral proteins, beta2-microglobulin (part of the histocompatibility antigen complex), aminopeptidase (alpha-aminoacyl-peptide hydrolase; EC 3.4.11.2), and the Na+,K+-ATPase (ATP phosphohydrolase; EC 3.6.1.3) on human fibroblasts grown in monolayer culture have been studied with their specific antibodies by immunofluorescence. On the same cells, the distribution of intracellular actin was observed by a spectrally distinct fluorescent staining procedure. If each of the antibody reagents was permitted to cluster its specific protein in the plane of the membrane, these clusters apparently became linked, through the membrane, to actin- and myosin-containing filaments (stress fibers) underneath the membrane, and were thereby immobilized. From these and other experiments, it appears that most, if not all, integral proteins can, upon clustering, form such transmembrane linkages to actin and myosin. A molecular mechanism for the formation of these linkages is proposed which postulates that actin is associated with the cytoplasmic surface of plasma membranes by peripheral attachment to a ubiquitous integral protein X in the membrane; when other integral proteins are induced to form clusters, they become bound to X and hence to actin (and myosin). The possible physiological role of these transmembrane linkages is briefly discussed.

Actomyosin↗
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↗