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S J Singer

Publications and source records attributed to S J Singer.

At least 55 records · Page 3Linked to original sources
PubMed1986

On the mechanism of unidirectional killing in mixtures of two cytotoxic T lymphocytes. Unidirectional polarization of cytoplasmic organelles and the membrane-associated cytoskeleton in the effector cell.

In mixtures of two CTL of the type a anti-b and b anti-c, only the latter is lysed; i.e., killing is unidirectional. Here, we show that two profound types of changes occur in the effector CTL but not in the target CTL upon formation of couples between them. One is that the microtubule organizing center (and presumably the Golgi apparatus that is invariably colocalized with it) is reoriented inside the effector CTL to face the bound target CTL. This unidirectional reorientation, it is proposed, serves to direct putative cytotoxic secretory components derived from the Golgi apparatus of the effector cell to the site of cell-cell binding. The second unidirectional change is in the membrane-associated cytoskeleton of the effector CTL in the area of target cell binding. The cytoskeletal protein talin, but not any of four other such proteins assayed, is highly concentrated at the contacting membrane of the effector CTL, while it is uniformly distributed over the entire membrane of the bound target CTL. This localized, massive cytoskeletal reorganization may reflect a mechanism to protect the membrane of the effector CTL from the effects of putative cytotoxic components secreted by the effector cell into the intercellular space between it and the target cell.

Animals↗
PubMed1986

Cytoskeletal organization, vinculin-phosphorylation, and fibronectin expression in transformed fibroblasts with different cell morphologies.

Neoplastic transformation of fibroblasts results in widely different cell morphologies. We have attempted to correlate cell morphology with cytoskeletal organization and fibronectin expression in murine and avian fibroblasts transformed by a diverse group of viral and chemical agents. The distribution of vinculin, alpha-actinin, actin, and surface fibronectin was studied, and, where appropriate, also the extent of phosphotyrosine modification of vinculin. Irrespective of the transforming agent we found that increased cell rounding was generally correlated with a reduction in vinculin-containing focal adhesions, a dissolution of microfilament bundles, and a reduction of extracellular fibronectin. In contrast, spindle-shaped fibroblasts expressed relatively high levels of surface fibronectin. Reorganization of vinculin, actin, and alpha-actinin into rosette-like structures was observed in polygonal or rounded cells transformed by viruses encoding tyrosine kinases, but was not seen in fibroblasts transformed by agents without associated tyrosine kinase activity or in spindle-shaped cells. No correlation was found between the extent of phosphotyrosine modification of vinculin and the extent of cell rounding. Irrespective of cell morphology, the extent of tyrosine phosphorylation of vinculin was high in all cells transformed by viruses carrying the src gene, but low in those transformed by viruses expressing the fps gene. Our results indicate that the morphology of a transformed cell is determined by a combination of several factors which are affected to different extents by different transforming agents.

Actin Cytoskeleton↗
PubMed1986

Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus.

We have examined the extent of tyrosine phosphorylation of talin, a component of the cytoskeleton localized in the focal adhesions and, therefore, a potential substrate of p60v-src, the transforming protein of Rous sarcoma virus. p60v-src is a tyrosine kinase that induces high levels of phosphotyrosine and the disorganization of the cytoskeleton in transformed cells. With a polyclonal antibody utilized in a previous study [Maher, P. A., Pasquale, E. B., Wang, J. Y. J. & Singer, S. J. (1985) Proc. Natl. Acad. Sci. USA 82, 6576-6580] for the detection of tyrosine-phosphorylated proteins, we have detected phosphotyrosine residues in talin molecules immunoprecipitated from Rous sarcoma virus-transformed, but not normal, chicken embryo fibroblasts. Phospho amino acid analysis of talin from the infected cells confirmed the presence of phosphotyrosine, in addition to phosphoserine and phosphothreonine. The extent of tyrosine modification in talin was compared to that in vinculin, the other focal adhesion component previously found to contain enhanced levels of phosphotyrosine in various retrovirus-transformed cells. A considerably (3 times) larger fraction of the talin than of the vinculin molecules was found to be phosphorylated on tyrosine. The phosphorylation of talin on tyrosine may be crucial for the expression of the abnormal morphology characteristic of cells transformed by Rous sarcoma virus.

Animals↗
PubMed1986

The specific direct interaction of helper T cells and antigen-presenting B cells.

Cell couples have been formed by mixing an antigen- and Ia-specific cloned helper T-cell line with a B-cell hybridoma presenting the antigen. By immunofluorescence observations, we have shown that the microtubule-organizing center (MTOC) inside the helper T cell, but not in the bound antigen-presenting cell, becomes oriented to face the area of specific cell-cell contact. This MTOC orientation is antigen- and Ia-specific, and thus provides direct evidence for the specific interaction of a helper T cell with a B cell. It is presumed that the function served by this MTOC orientation, which is accompanied by the coordinate reorientation of the Golgi apparatus, is to target Golgi apparatus-derived secretory vesicles, containing putative lymphokines and/or growth factors, from the helper T cell directly to the antigen-presenting cell.

Animals↗
PubMed1986

Disulfide bonds and the translocation of proteins across membranes.

We are concerned with the mechanisms whereby hydrophilic proteins synthesized in the cytoplasm are translocated across one or two membranes into different cellular organelles. On the basis of a model of the translocation process to be described elsewhere, we propose an explanation of previous findings that the in vitro translocation across the endoplasmic reticulum of secretory proteins of higher eukaryotic cells appears to be obligatorily co-translational (i.e., occurs only while the polypeptide chain is being synthesized on the ribosome). We suggest that in vitro the intrachain disulfide bonds of the polypeptide rapidly form after it is released from the ribosome; the three-dimensional conformation of the chain is thereby stabilized and cannot undergo the unfolding that is required for post-translational translocation. In accord with this proposal, we show that the secretory preprotein human preprolactin, after translation and release from the ribosome, can indeed undergo translocation across endoplasmic reticulum membranes in vitro if the medium is sufficiently reducing. Those polypeptides that, in the absence of reducing agents, can be post-translationally translocated in vitro across bacterial, mitochondrial, and other types of membranes may generally lack intrachain disulfide bonds.

Biological Transport↗
PubMed1986

Immunocytochemical studies of endothelial cells in vivo. I. The presence of desmin only, or of desmin plus vimentin, or vimentin only, in the endothelial cells of different capillaries of the adult chicken.

It is currently believed that the intermediate filaments of endothelial cells contain vimentin subunits exclusively. This inference, however, is derived from studies of only a few types of endothelial cells. By double indirect immunofluorescence and immunoelectron microscopy, we have now examined the endothelial cells of the micro- and macrovasculature of a variety of tissues and organs of adult chicken in vivo for their content of desmin and vimentin. Endothelial cells of the peritubular capillary in the renal cortex, the hepatic sinusoid, and the splenic sinusoid were found to contain only desmin; those of the exocrine pancreas capillary contained both desmin and vimentin; and the endothelial cells of the macrovasculatures and of all the other microvasculatures examined, including the vasa recta of the renal medulla, contained only vimentin. Such heterogeneity suggests that different types of adult chicken endothelial cells may have different embryological origins. To the extent that desmin and vimentin intermediate filaments may be functionally distinct, these results also suggest that different capillary endothelial cells may have different functional properties.

Animals↗
PubMed1986

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence.

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

Animals↗
PubMed1985

Reorientation of the Golgi apparatus and the microtubule-organizing center inside macrophages subjected to a chemotactic gradient.

Mouse peritoneal macrophages subjected to gradients of activated mouse serum were found by immunofluorescence observations to have their Golgi apparatus and their microtubule-organizing center largely oriented in the direction of the gradient. By analogy with similar results obtained with motile fibroblasts, it is proposed that these two organelles are rapidly and coordinately reoriented inside the macrophages in order to direct the insertion of new membrane mass, via vesicles derived from the Golgi apparatus, into the leading edge of the cell. Consistent with the importance of such membrane insertion to cell migration, we found that the ionophore monensin, an inhibitor of Golgi functions, inhibited cell motility in the chemostactic gradient. It was further shown that several inhibitors of chemotaxis (monensin, cytochalasin D, cycloheximide) did not inhibit the reorientation of the Golgi apparatus/microtubule-organizing center in cells exposed to a chemotactic gradient, and that the reorientation required extracellular Ca+2.

Animals↗
PubMed1985

Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells.

We have used a high-affinity polyclonal antibody directed against phosphotyrosine (P-Tyr) to localize P-Tyr-containing proteins in normal and transformed cells in culture by immunofluorescence microscopy experiments. The distribution of the proteins with modified tyrosine was compared with that of F-actin in these cells. Cells infected with Abelson murine leukemia virus were found to contain elevated levels of P-Tyr, as expected. Various permanent lines of fibroblastic and epithelial cells exhibited lower, but easily detectable, levels of P-Tyr. The P-Tyr in fibroblasts was concentrated at the focal contacts at the termini of actin-containing microfilament bundles and, in the epithelial cells examined, at the intercellular junctions. Early passages of primary cultures of chicken embryo fibroblasts and chicken embryo heart cells also showed detectable levels of P-Tyr in focal contacts and cell-cell junctions. However, P-Tyr was not detectable in later passages of chicken embryo fibroblasts. The concentration of P-Tyr-containing proteins in intercellular junctions in normal cells suggests that these are sites of significant biochemical regulatory activities which may be important in the control of normal cell adhesivity, motility, and shape.

Actin Cytoskeleton↗
PubMed1985

3-Hydroxy-3-methylglutaryl-coenzyme A reductase is present in peroxisomes in normal rat liver cells.

The location inside rat liver parenchymal cells of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), the key regulatory enzyme in cholesterol biosynthesis, has been examined by immunoelectron microscopy and by subcellular fractionation. Although HMG-CoA reductase is generally thought to be exclusively a microsomal enzyme, we find that a substantial portion of cellular HMG-CoA reductase is localized in peroxisomes. Immunoelectron microscopic labeling of ultrathin frozen sections of normal rat liver, using two monoclonal antibodies to purified HMG-CoA reductase, showed that the enzyme is present in the peroxisomes at a higher concentration than at any other site inside the hepatocytes. Subcellular fractionation studies using Percoll and metrizamide gradients demonstrated a close correspondence of peaks of HMG-CoA reductase activity and of catalase activity, again revealing the presence of the reductase enzyme in peroxisomes. HMG-CoA reductase is therefore localized in peroxisomes in addition to being in the microsomal fraction.

Animals↗
PubMed1985

Anomalous interaction of the acetylcholine receptor protein with the nonionic detergent Triton X-114.

Integral membrane proteins that form water-filled channels through membranes often exist as aggregates of similar or identical subunits spanning the membrane. It has been suggested that the insertion into the membrane of the channel-forming domains of the subunits may impart unusual structural features to the membrane-intercalated portions of the protein. To test this proposal, we have investigated the interaction of a multisubunit channel-forming integral membrane protein, the acetylcholine receptor protein, with the nonionic detergent Triton X-114. Whereas non-channel-forming integral membrane proteins that have heretofore been studied form mixed micelles with the detergent, the acetylcholine receptor was excluded from the Triton X-114 micelles. The structural implications of this result are discussed.

Animals↗
PubMed1985

Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study.

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.

Animals↗
PubMed1985

An integral glycoprotein associated with the membrane attachment sites of actin microfilaments.

An integral membrane protein associated with sites of microfilament-membrane attachment has been identified by a newly developed IgG1 monoclonal antibody. This antibody, MAb 30B6, was derived from hybridoma fusion experiments using intact mitotic cells of chick embryo fibroblasts as the immunization vehicle as well as the screening probe for cell surface antigens. In immunofluorescent experiments with fixed cells, MAb 30B6 surface labeling is uniquely correlated with microfilament distributions in the cleavage furrow region of dividing chick embryo fibroblasts and cardiac myocytes in culture. The MAb 30B6 antigen in addition is associated with microfilament-membrane attachment sites in interphase fibroblasts at the dorsal surface, the adhesion plaque region at the ventral surface, and at junction-like regions of cell-cell contact. It is also found co-localized with the membrane-dense plaques of smooth muscle. The MAb 30B6 antigen is expressed in a wide number of chicken cell types (particularly smooth muscle cells, platelets, and endothelial cells), but not in erythrocytes. Some of the molecular characteristics of the MAb 30B6 antigen have been determined from immunoblotting, immunoaffinity chromatography, immunoprecipitation, cell extraction, and charge shift electrophoresis experiments. It is an integral sialoglycoprotein with an apparent molecular mass of 130 kD (reduced form)/107 kD (nonreduced form) in SDS PAGE. Another prominent glycoprotein species with an apparent molecular mass of 175 kD (reduced form)/165 kD (nonreduced form) in SDS PAGE is co-isolated on MAb 30B6 affinity columns, but appears to be antigenically distinct since it is not recognized by MAb 30B6 in immunoblotting or immunoprecipitation experiments. By virtue of its surface distributions relative to actin microfilaments and its integral protein character, we propose that the MAb 30B6 antigen is an excellent candidate for the function of directly or indirectly anchoring microfilaments to the membrane.

Actins↗
PubMed1985

Zeugmatin: a new high molecular weight protein associated with Z lines in adult and early embryonic striated muscle.

Monoclonal antibodies were generated to a purified preparation of the fascia adherens domains of the intercalated discs of chicken cardiac cell membranes. One of these antibodies, McAb 20, immunofluorescently labeled the Z lines of adult skeletal muscle, the Z lines and intercalated discs of adult cardiac muscle, and the dense bodies and dense plaques of adult gizzard smooth muscle. In addition, McAb 20 was found to label regenerating muscle cells in a cross-striated pattern much like that of Z lines in 24-h muscle cell cultures before the appearance of Z lines was detectable by phase or Nomarski optics and before the concentration of alpha-actinin occurred at the Z lines. Thus, McAb 20 appears to be directed against an antigen involved in early myofibrillar organization. Preliminary biochemical characterization of the antigen recognized by McAb 20 indicates that it is a high molecular weight doublet of over 5 X 10(5) kD that is highly susceptible to proteolysis. By virtue of its presence in Z lines, and its possible role in the end-on attachment of microfilaments to Z lines and membranes, we have named this protein zeugmatin (xi epsilon nu gamma mu alpha identical to yoking).

Aging↗
PubMed1985

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues.

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.

Animals↗