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S J Singer

Publications and source records attributed to S J Singer.

At least 73 records · Page 4Linked to original sources
PubMed1987

On the transfer of integral proteins into membranes.

We have earlier proposed a molecular mechanism for the translocation of hydrophilic proteins across membranes that accounts for the experimental facts and meets the restrictions that we stipulate for such a mechanism. In particular, the restrictions are that translocation occurs by successive segments of the polypeptide chain and that the ionic groups of the polypeptide remain in contact with water throughout the translocation process. The evidence indicates that the transfer of integral proteins into membranes very likely uses the same molecular machinery as does the translocation of hydrophilic proteins across membranes. Here we show how the mechanism we have proposed for translocation can also be utilized in the intercalation of known types of integral proteins, accounting for their specific topologies in the membrane.

Biological Transport↗
PubMed1987

Atlantoaxial distance in patients with Down syndrome: standardization of measurement.

Atlantoaxial subluxation is common in persons with Down syndrome. For radiologic screening to be valid, accurate measurement of the atlantoaxial distance (AAD) must be obtained from radiographs of the cervical spine. A radiopaque millimeter scale has been designed to simplify accurate measurement of the AAD from these radiographs.

Atlanto-Axial Joint↗
PubMed1987

Immunocytochemical studies of desmin and vimentin in pericapillary cells of chicken.

The composition of intermediate filaments in pericytes was examined by immunofluorescent and immunoelectron microscopic labeling of frozen sections of various chicken microvascular beds in situ. Pericytes in capillaries of cardiac muscle, exocrine pancreas, and kidney (peritubular capillary) were found to contain both desmin and vimentin. In some capillaries where pericytes do not exist, cells apposed to endothelial cells--the Ito cell in the hepatic sinusoid and the reticular cell in the splenic sinusoid--were shown to contain both of the intermediate filament proteins. In contrast, podocytes and mesangial cells around renal glomerular capillaries contained only vimentin. The presence of desmin supports the hypothesis that pericytes may have a contractile apparatus similar to that of vascular smooth muscle cells. Our results also revealed that even in microvascular beds where pericytes are not found, cells having both desmin and vimentin exist next to endothelial cells and may assume similar functions to pericytes.

Animals↗
PubMed1987

The microtubule-organizing complex and the Golgi apparatus are co-localized around the entire nuclear envelope of interphase cardiac myocytes.

In most animal cells, the microtubule-organizing centre (MTOC) and the Golgi apparatus (GA) are co-localized on one side of the nucleus, an arrangement that allows these cells to acquire a functional polarity. An exception has been reported in the skeletal muscle myotube, where the MTOC and GA exhibit a circumnuclear distribution. We wished to determine if this unusual distribution of the MTOC and GA was peculiar to syncytial myotubes or reflected a pattern found in muscle cells generally. Immunofluorescence microscopic studies of cultured chicken skeletal muscle, cardiac muscle and gizzard smooth muscle cells were carried out using preimmune sera that recognized the pericentriolar material, anti-tubulin antibodies to label the MTOC, and fluorescent wheat-germ agglutinin to label the GA. These studies have shown that cardiac myocytes possess a circumnuclear distribution of their MTOC and GA as do skeletal myotubes, but smooth muscle cells exhibit the centrosomal MTOC and GA distribution found in most other cells. The circumnuclear MTOC/GA distribution therefore is associated with striated muscle cells. We also found that as embryonic cardiac myocytes pass through the cell cycle the microtubule-organizing activity in these cells switches from a circumnuclear distribution in interphase to the conventional centrosomal location during mitosis. Thus, cardiac myocytes provide a rare example of mononucleated animal cells that do not display a centrosomal MTOC or a polarized GA, and also reveal a system in which the MTOC structure can be reversibly altered in a cell cycle-dependent manner.

Animals↗
PubMed1987

Direct morphological demonstration of the coexistence of vimentin and desmin in the same intermediate filaments of vascular smooth muscle cells.

The stable coexistence of the intermediate filament proteins desmin and vimentin in vascular smooth muscle cells raises questions about the relative amounts of the two proteins in different individual cells, and the distribution of the two proteins in individual intermediate filaments within each cell. These questions have been explored by double immunofluorescence microscopy and double immunoelectron microscopy on semi-thin and ultrathin frozen sections of chicken aorta. The former studies indicate that there is a surprisingly wide variation in the desmin/vimentin ratio in adjacent smooth muscle cells. The latter studies show that both proteins are present in individual intermediate filaments, in clustered arrays rather than uniformly distributed. These findings extend earlier related results, and suggest that the turnover of intermediate filaments may involve the remodelling of existing filaments rather than their de novo polymerization.

Animals↗
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↗