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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
PubMed1981

Ultrastructure of chicken cardiac muscle as studied by double immunolabeling in electron microscopy.

The ultrastructural localization of alpha-actinin and vinculin in chicken cardiac muscle was studied by double indirect immunoelectron microscopy, using ferritin and iron-dextran (Imposil) as the electron-dense markers conjugated to the secondary antibodies, on ultrathin frozen sections of fixed tissue. Fixation and immunolabeling procedures were developed that permitted maximal retention of the two proteins at their natural sites as well as their adequate labeling. alpha-Actinin was found both on the Z-bands, as expected, and near the fascia adherens of the intercalated discs, whereas vinculin was confined to the latter sites. At the fascia adherens, the double labeling results clearly showed that vinculin was situated closer to the membrane than was alpha-actinin. These results, coupled with earlier observations, suggest that vinculin may participate in the linkage of actin-containing microfilament bundles to membranes in a variety of cell types.

Actinin↗
PubMed1981

Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane.

The intracellular pathway of biogenesis of the vesicular stomatitis virus transmembrane glycoprotein was investigated in situ by using indirect immunofluorescence of whole infected Chinese hamster ovary cells and immunoelectron microscopy of ultrathin frozen sections of infected cells. Transport of the glycoprotein was synchronized by using the temperature-sensitive virus mutant Orsay-45 and a temperature shift-down protocol. Sequential appearance of the glycoprotein in the rough endoplasmic reticulum, Golgi apparatus, and plasmalemma was demonstrated. The potential of this system for further studies is discussed.

Animals↗
PubMed1981

Immunoelectron microscope studies of membrane-microfilament interactions: distributions of alpha-actinin, tropomyosin, and vinculin in intestinal epithelial brush border and chicken gizzard smooth muscle cells.

The ultrastructural localization of three cytoskeletal proteins, alpha-actinin, tropomyosin, and vinculin, in the brush border of epithelial cells of chicken small intestine and the smooth muscle cells of chicken gizzard was studied by immunofluorescence and immunonelectron microscope labeling of frozen sections of lightly fixed, intact tissues. In the immunoelectron microscope studies, a recently described new type of electron-dense antibody conjugate, imposil-antibody, has been successfully used, along with ferritin-antibody conjugates, in single and double immunolabeling experiments. In the intestinal brush border shows that vinvulin is sharply confined to the junctional complex close to the membrane region of the zonula adherens, in distinct contrast to the more diffuse distributions of the other two proteins. In the smooth muscle cells, the labeling patterns show that vinculin is sharply confined to the membrane-associated dense plaques, closer to the membrane than the alpha-Actinin is also present in the cytoplastic dense bodies, from which vinculin is absent. Tropomyosin is present diffusely distributed in the cytoplasm, but absent from both dense plaques and dense bodies. These findings with the muscle cells demonstrate, therefore, that the dense plaques and dense bodies are chemically and structurally distinct entities. The results with both tissues, along with those in previous papers (Geiger, 1979, Cell. 18:193-205.; Geiger et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:4127-4131), suggest that vinculin may play an important and widespread role in the linkage of actin-containing microfilament bundles to membranes.

Actinin↗
PubMed1980

The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts.

It has previously been shown by immunofluorescence experiments that the cross-linking of HLA antigens into patches (by antibody reagents directed to human beta 2--microglobulin) on the surfaces of cultured human fibroblasts leads to the lining up of the patches over the actomyosin-containing stress fibers lying immediately under the surface membrane. These experiments have now been extended to the resolution of the electron microscope by the use of ferritin-conjugated antibody. The results show that a substantial part of the HLA surface clusters that form by 5 min after the addition of the antibody reagents is found in small uncoated surface invaginations which are subsequently endocytosed and ultimately fuse with lysosomal bodies. At no stage in this process is there any indication that coated pits or coated vesicles participate. These and other results suggest, therefore, that there are at least two distinct mechanisms for the ligand-induced endocytosis and lysosomal processing of membrane components, one involving coated pits and the other the noncoated invaginations described in this paper. Transmembrane associations of clusters with intracellular actomyosin-containing structures may have a role in the endocytosis of these noncoated invaginations.

Antibodies↗
PubMed1980

Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus.

It was recently shown by combined immunofluorescence and interference reflection microscopy that a protein named vinculin, along with alpha-actinin, is concentrated at focal adhesion plaques inside cultured normal fibroblasts [Geiger, B. (1979) Cell 18, 193-205]. These plaques are the discrete, isolated sites of strong adhesions formed between the ventral surfaces of the cells and the substrata on which they are grown. We show that after transformation of fibroblasts by Rous sarcoma virus a majority of the cells have many fewer focal adhesion plaques and now exhibit a cluster of small patches that are immunolabelled for both vinculin and alpha-actinin. Such a cluster (rosette) is located near the ventral surface of the cell, usually partly under the nucleus. The significance that these altered distributions of vinculin and alpha-actinin may have for the rounding up and loss of adherence of transformed cells is discussed.

Actinin↗
PubMed1980

Fibronectin is not present in the focal adhesions formed between normal cultured fibroblasts and their substrata.

Fibronectin is an extracellular matrix protein that has been implicated in the spreading and adhesion of cultured fibroblasts to their substrata. In this paper, double immunoelectron microscopic labeling experiments for fibronectin and for concanavalin A-binding proteins on the cell surface were carried out on ultrathin frozen sections of cultures of embryonic chicken heart fibroblasts. On cross sections though the focal adhesions of the cell to the substratum there was substantial labeling for concanavalin A-binding proteins but no detectable labeling for fibronectin, whereas both the binding proteins and fibronectin were extensively labeled elsewhere on the cell surface and substratum. These results demonstrate that fibronectin is not present within the sites of focal adhesions. Therefore, the functions of fibronectin in cell spreading and adhesion are not directly mediated through its binding at focal adhesion sites. An alternative model is presented which can account for such fibronectin functions.

Animals↗
PubMed1980

Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes.

As intracellular protein of 130,000 molecular weight was recently isolated in this laboratory from chicken gizzard smooth muscle. By immunofluorescence observations of cultured chicken fibroblasts, it was shown to be concentrated on the ventral surfaces of the cells where they formed focal adhesions to the substratum [Geiger, B. (1979) Cell 18, 193-205]. Focal adhesions are sites where, inside the fibroblast, microfilament bundles are known to terminate at the cell membrane. The suggestion was made that this new protein (herein named "vinculin") might be involved in the linkage of the termini of microfilament bundles to membranes in various cell types. To explore this possibility, in the present study we examined several chicken tissues, including intestinal epithelium, gizzard smooth muscle, and cardiac striated muscle, by immunoelectron microscopic labeling for vinculin on ultrathin frozen sections of the specimens. In each case, the immunolabeling for vinculin was concentrated close to membrane sites where microfilament bundles terminate: at the zonula adherens in the junctional complex of the brush border of epithelial cells; at the membrane-associated sense plaques of smooth cells; and at the fascia adherens of the intercalated disk membranes of cardiac muscle cells. These results suggest therefore that vinculin may participate in the anchoring of microfilament bundles to specific membrane sites in various cells.

Animals↗
PubMed1980

Association of microtubules and intermediate filaments in chicken gizzard cells as detected by double immunofluorescence.

By double indirect immunofluorescence, using guinea pig and rabbit antibodies to tubulin and to desmin, we have simultaneously labeled microtubules and intermediate filaments in cultured chicken embryo gizzard cells. At the resolution of the light microscope there was extensive but not complete superposition of the labeling patterns for the two filamentous structures within cells in interphase and an essentially complete dissociation of the two labeling patterns in cells in mitosis. These results indicate that there is an extensive association of microtubules and intermediate filaments in these interphase muscle cells and suggest that this association is regulated metabolically.

Animals↗
PubMed1980

Interactions between the plasma membrane and cytoskeleton of cultured fibroblasts.

Observations of cultured cells made by double-fluorescence staining indicate that regions of the plasma membrane which are in close contact with actin-containing cytoplasmic fibers have characteristics different from other regions of the membrane. On fixed cells it is found that several integral membrane proteins are excluded from these regions of membrane-fiber apposition. If, however, these same integral proteins are clustered by their specific antibodies, the patches produced are rapidly lined up over the cytoplasmic fibers, resulting in a transmembrane linkage of clustered membrane proteins to the actin cytoskeleton. These observations have led us to predict the existence of a class of integral membrane proteins, X proteins, which are associated with actin fibers and are responsible for both the initial exclusion and then the transmembrane linkage of clusters of other integral membrane proteins. In an attempt to identify X proteins we have produced antisera against purified plasma membranes prepared from porcine intestinal brush borders. These antisera detect surface antigens on fixed human fibroblast cells which are initially lined up over actin fibers. These antigens are, thus, candidates for the hypothesized X proteins. Using the fluorescence microscope, we are attempting to isolate the potential X antigens with a staining absorption assay.

Actins↗
PubMed1979

Mechanochemical proteins, cell motility and cell-cell contacts: the localization of mechanochemical proteins inside cultured cells at the edge of an in vitro "wound".

We have examined the distribution of several mechanochemical proteins inside rat A10 cells in monolayer culture, both in sparse cultures and at the edges of in vitro "wounds" in confluent cultures. The proteins examined were actin, myosin, tropomyosin, alpha-actinin, filamin, and tubulin. In each experiment, a pair of these proteins (one of which was usually actin) were examined simultaneously by double fluorescence staining methods. Actin was specificially stained by double fluorescence staining methods. Actin was specifically stained by a method based on heavy meromyosin binding, while the other proteins were specifically stained by indirect immunofluorescence procedures. The most important of the various results described was obtained with cells moving out from the edge of an in vitro wound. Within the flat leading lamella of such a cell, there was an extended region in which myosin was severely depleted or absent compared to the proximal regions of the same cells. By contrast, the other proteins were abundantly present throughout the leading lamella, except for tropomyosin, which was somewhat depleted but not as extensively as myosin. In Nomarski optics, there was no detectable morphological differentiation between the region depleted of myosin and the more proximal portion of the same lamella. While the depletion of myosin from the motile regions of cells does not rule out the involvement of some form of an actomyosin sliding filament mechanism, it suggests that other molecular mechanisms for generating motility be seriously considered.

Actinin↗
PubMed1979

Molecular changes in the membranes of mouse erythroid cells accompanying differentiation.

The development of the mouse erythroblast to a mature erythrocyte is accompanied by changes in the composition and properties of the plasma membranes of these cells. Using double fluorescence techniques, we have simultaneously determined the distribution of lectin receptors and spectrin on the membranes of these cells. The lateral mobility of the lectin receptors in the membranes decreases as differentiation proceeds, and this is accompanied by an increasing concentration of spectrin associated with the membranes. The most significant concentration of spectrin occurs, however, during the enucleation of the late erythroblast, where we observe a complete segregation of the spectrin to the incipient reticulocyte, as well as a previously observed enrichment of receptors for concanavalin A into the plasma membrane surrounding the extruding nucleus. On the basis of these and other observations, we explore the possible molecular mechanisms involved in erythroblast enucleation and the role of spectrin in the regulation of protein mobility in erythroid cell membranes.

Actins↗
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↗