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S J Singer

Publications and source records attributed to S J Singer.

At least 127 records · Page 7Linked to original sources
PubMed1979

The participation of alpha-actinin in the capping of cell membrane components.

By means of double fluorescence staining experiments, intracellular alpha-actinin was found to accumulate under caps and patches induced in several cells by a variety of ligands. This phenomenon was demonstrated in lymphocytes and lymphoma cells treated with anti-H-2 sera; spleen lymphocytes treated with concanavalin A or anti-immunoglobulin antibodies, and VSV-infected mouse fibroblast line MC57 treated with antiserum against viral antigens. It occurred during both rapid and slow capping processes, and could be obtained by either direct or indirect ligand-induced redistribution. These observations were carried out on whole cells. For other cytoskeletal proteins such as filamin, tropomyosin and myosin, a similar accumulation under caps was not readily apparent using whole cell mounts, although earlier experiments with frozen-sectioned cells had shown such an enrichment of myosin (as well as actin). The enrichment of alpha-actinin under the clustered surface molecules was already apparent in early stages (patching) of the capping process, with or without 10 mM sodium azide present. Prolonged incubation of the cells with the different ligands resulted in endocytosis of the ligand-receptor complex. alpha-Actinin was not associated with the inernalized complex, however, suggesting that it may dissociate from the patched or capped surface structures at some stage during endocytosis.

Actinin↗
PubMed1979

Evidence for a large internal pressure in biological membranes.

We have developed a method (hygroscopic desorption) for measuring the binding of small molecules to membranes. With this method, we have found that the binding of the amphipathic compounds chlorpromazine, 2,4-dinitrophenol, and 1-decanol to various cell membranes is remarkably low, with partition coefficients, Kp, no larger than about 0.1. On the other hand, with phospholipid vesicles of large or small diameters, Kp values for these compounds were much larger. The results suggest that there exists in membranes a large internal pressure that excludes the amphipaths from the membranes and that does not exist in phospholipid vesicles.

B-Lymphocytes↗
PubMed1979

Immunocytochemical localization of alpha-actinin in intestinal epithelial cells.

alpha-Actinin was localized in chicken intestinal epithelial cells by immunofluorescence and immunoferritin labeling of thin frozen sections. Most of the label of the brush border was confined to the terminal web area. The label there was concentrated mainly along the "roots" of the microvilli core microfilaments and in the vicinity of the zonula adherens. In the latter structure, the narrow electron-dense zones adjacent to the cell membranes, however, were not significantly labeled. This suggests that alpha-actinin does not mediate directly the association of the transverse terminal web microfilaments to the membrane at the zonula adherens. Sparse ferritin labeling was found near the tight junction, whereas the staining associated with the spot desmosome was negligible. The microvilli were not significantly labeled by either immunofluorescence or immunoferritin staining unless the sections were previously treated with detergent. Moreover, alpha-actinin (or a structurally related protein) was not detected in preparations of purified microvillar vesicles, suggesting the possibility that the alpha-actinin staining in the microvilli may be an artificial due to its translocation by the detergent from the terminal web onto the microvilli. The possible roles of alpha-actinin in the organization and function of the brush border are discussed.

Actinin↗
PubMed1979

Selective and unidirectional membrane redistribution of an H-2 antigen with an antibody-clustered viral antigen: relationship to mechanisms of cytotoxic T-cell interactions.

We have studied the co-redistribution of vesicular stomatitis virus (VSV) antigen and of individual H-2 antigens on the surfaces of mouse cells, and in parallel we have also used these VSV-infected cells as targets in cytotoxic T-cell killing experiments. Antibody-induced patching and capping of the VSV antigen caused an extensive co-patching and co-capping of the H-2Kb antigen but not of the H-2Db antigen. In reciprocal experiments, the antibody-induced patching of the H-2Kb or H-2Db antigen did not result in a co-patching of the VSV antigen. Radioimmunoassays showed that the relative numbers of H-2Kb, H-2Db, and VSV antigens on the surfaces of the cells exhibiting such nonreciprocal co-redistributions were closely similar. Furthermore, the H-2 restricted cytotoxic T-cell lysis of these target cells showed a marked preference for H-2Kb compared to H-2Db compatibility. We propose that the VSV and H-2 antigens are molecularly independent entities in the unpreturbed target cell membrane but that the antibody-induced clustering of the VSV antigen causes a selective and unidirectional co-redistribution (which we designate as syn-capping) of H-2Kb with the VSV antigen clusters. It is suggested that such a T-cell-induced syn-capping process involving an antigen and an H-2 molecule on the target cell may play a critical role in the mechanism of cytotoxic T-cell killing.

Antibodies, Viral↗
PubMed1979

Domains of receptor mobility and endocytosis in the membranes of neonatal human erythrocytes and reticulocytes are deficient in spectrin.

It has previously shown (Schekman, R., and S.J. Singer, Proc. Natl. Acad. Sci. U.S.A. 73:4075-4079) that receptors in the membranes of neonatal human erythrocytes show a restricted degree of lateral mobility, whereas in adult human erythrocytes the receptors are essentially immobile. This restricted mobility is exhibited, for example, when concanavalin A (Con A) induces a limited clustering of its receptors in the neonatal erythrocyte membrane, resulting in the formation of invaginations and endocytic vesicles. This does not happen with adult cells. By the use of indirect immunoferritin labeling of ultrathin frozen sections of Con A-treated neonatal blood cells, we now show that the invaginations and endocytotic vesicles do not stain for spectrin, whereas the adjacent unperturbed membrane is heavily stained. The reticulocytes in the neonatal cell population undergo substantially more Con A-induced invagination and endocytosis than do the erythrocytes. These results lend strong support to the hypothesis that specialized discrete domains exist, or are induced, in the membranes of these neonatal cells, in which receptors are laterally mobile, whereas in the remaining (and predominant) part of the membrane the receptors are immobile. Such mobile domains are characterized by an absence of spectrin. During the maturation of the neonatal reticulocyte to erythrocyte, it is proposed that these domains are in large part, but not completely, eliminated.

Concanavalin A↗
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↗