Showing papers by "David D. Sabatini published in 1984"
TL;DR: Observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides, and it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.
Abstract: Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus [VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins of the two viruses are synthesized simultaneously and assembled into virions at their characteristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane of the cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi cisternae. This indicates that the pathways of the two proteins towards the plasma membrane do not diverge before passage through the Golgi apparatus and therefore that critical sorting steps must take place during or after passage of the glycoproteins through this organelle. After its passage through the Golgi, the HA accumulated primarily at the apical membrane, where influenza virion assembly occurred. A small fraction of HA did, however, appear on the lateral surface and was incorporated into the envelope of budding VSV virions. Although predominantly found on the basolateral surface, significant amounts of G protein were observed on the apical plasma membrane well before disruption of the tight junctions was detectable. Nevertheless, assembly of VSV virions was restricted to the basolateral domain and in doubly infected cells the G protein was only infrequently incorporated into the envelope of budding influenza virions. These observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.
222 citations
TL;DR: In vitro translation of mRNA extracted from free and boundpolysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes, and the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglobin D are consistent with the notion that the biosynthetic pathway of the ribophOrins does not require a stage of passage through the Golgi apparatus.
Abstract: Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 31) and in cell-free synthesis experiments In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s) Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus
73 citations
TL;DR: The transient existence of communicating junctions observed in monolayers of MDCK cells is similar to that described in the literature for embryo tissues during development.
Abstract: We explore the existence of cell-to-cell communication in monolayers of MDCK cells plated at high densities so that they form a continuous monolayer in a few minutes. Lucifer Yellow CH is injected in the cytoplasm of a given cell by using a glass microelectrode with a fine tip (ca. 100 MΩ) and passing square pulses of current of 1.0 nA that last 10 msec, every 20 msec, during 1 to 3 min. We then examine the monolayer with fluorescence microscopy. In 27 out of 111 cells injected during the first 4 to 15 hr after plating, the dye was transferred to neighboring cells. Electron micrographs of freeze-fracture replicas prepared at this time, show that 20 to 25% of the lateral surfaces present the aggregates of intramembrane particles typical of gap junctions. These early hours correspond to the formation of occluding junctions and polarization into an apical and a basolateral domain of the plasma membrane (Cereijido, Meza & Martinez-Palomo, 1981). Cell-to-cell coupling then decreases sharply and, in the period between the 1st and 3rd day (mature monolayers), only 4 out of 49 injected cells were able to transfer the dye to their neighbors in the monolayers. No image of gap junctions was found in freeze-fracture replicas of mature monolayers. The degree of coupling between cells, as well as the number of cells coupled to the injected one, were highly variable. The lack of coupling between cells in mature monolayers observed in this article with Lucifer Yellow CH and electron microscopy is in keeping with the absence of electrical coupling observed in a previous work (Stefani & Cereijido, 1983). The transient existence of communicating junctions observed in monolayers of MDCK cells is similar to that described in the literature for embryo tissues during development.
34 citations
TL;DR: This chapter introduces immunolabeling techniques at the EM level, which enabled to localize viral glycoproteins inside the cell as well as on its surface and to obtain information concerning the pathways followed by these proteins from their sites of synthesis to their site of assembly into virions.
Abstract: Publisher Summary This chapter introduces immunolabeling techniques at the electron microscope (EM) level. The efficacy of immunolabeling procedures designed for the EM level largely depends on the ability of the immunoreagents to gain access to their corresponding antigens within the tissue or the cell. There are two general strategies for immunolabeling to overcome this limitation and to exploit the specificity of the antibody-antigen interaction and the ultrastructural resolution of the EM. One group includes those protocols that rely on the partial permeabilization of the cellular membranes by detergents, such as saponin to give IgGs and EM markers access to intracellular compartments and the other consists of those that employ thin or thick sectioned material to provide direct exposure of intracellular antigens to the immunoreagents. Immunolabeling of thin sections has been applied with varying levels of success to specimens embedded in methacrylate, polyethylene glycol (PEG), Epon, Lowicryl, serum albumin, and sucrose. To study the biogenesis of epithelial cell polarity, immunolabeling technique is applied to ultrathin frozen sections prepared from virally infected MDCK cells. This method enabled to localize viral glycoproteins inside the cell as well as on its surface and to obtain information concerning the pathways followed by these proteins from their site of synthesis to their site of assembly into virions.
7 citations