scholarly journals The rubella virus E2 and E1 spike glycoproteins are targeted to the Golgi complex.

1993 ◽  
Vol 121 (2) ◽  
pp. 269-281 ◽  
Author(s):  
T C Hobman ◽  
L Woodward ◽  
M G Farquhar
Keyword(s):  
Membrane Proteins ◽  
Golgi Complex ◽  
Rubella Virus ◽  
Cell Types ◽  
Vero Cells ◽  
Type I ◽  
Surface Transport ◽  
Golgi Stack ◽  
Golgi Region ◽  
Different Cell Types

Rubella virus (RV) has been reported to bud from intracellular membranes in certain cell types. In this study the intracellular site of targeting of RV envelope E2 and E1 glycoproteins has been investigated in three different cell types (CHO, BHK-21 and Vero cells) transfected with a cDNA encoding the two glycoproteins. By indirect immunofluorescence, E2 and E1 were localized to the Golgi region of all three cell types, and their distribution was disrupted by treatment with BFA or nocodazole. Immunogold labeling demonstrated that E2 and E1 were localized to Golgi cisternae and indicated that the glycoproteins were distributed across the Golgi stack. Analysis of immunoprecipitates obtained from stably transfected CHO cells revealed that E2 and E1 become endo H resistant and undergo sialylation without being transported to the cell surface. Transport of RV glycoproteins to the Golgi complex was relatively slow (t1/2 = 60-90 min). Coprecipitation experiments indicated that E2 and E1 form a heterodimer in the RER. E1 was found to fold much more slowly than E2, suggesting that the delay in transport of the heterodimer to the Golgi may be due to the slow maturation of E1 in the ER. These results indicate that RV glycoproteins behave as integral membrane proteins of the Golgi complex and thus provide a useful model to study targeting and turnover of type I membrane proteins in this organelle.

scholarly journals Polymorphism and Structural Maturation of Bunyamwera Virus in Golgi and Post-Golgi Compartments

2003 ◽  
Vol 77 (2) ◽  
pp. 1368-1381 ◽  
Author(s):  
Iñigo J. Salanueva ◽  
Reyes R. Novoa ◽  
Pilar Cabezas ◽  
Carmen López-Iglesias ◽  
José L. Carrascosa ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Vero Cells ◽  
Productive Infection ◽  
Tubular Structures ◽  
Enveloped Viruses ◽  
Golgi Stack ◽  
Golgi Region ◽  
Viral Particles ◽  
Infected Cells ◽  
Bunyamwera Virus

ABSTRACT The Golgi apparatus is the assembly site for a number of complex enveloped viruses. Using high-preservation methods for electron microscopy, we have detected two previously unknown maturation steps in the morphogenesis of Bunyamwera virus in BHK-21 cells. The first maturation takes place inside the Golgi stack, where annular immature particles transform into dense, compact structures. Megalomicin, a drug that disrupts the trans side of the Golgi complex, reversibly blocks transformation, showing that a functional trans-Golgi is needed for maturation. The second structural change seems to take place during the egress of viral particles from cells, when a coat of round-shaped spikes becomes evident. A fourth viral assembly was detected in infected cells: rigid tubular structures assemble in the Golgi region early in infection and frequently connect with mitochondria. In Vero cells, the virus induces an early and spectacular fragmentation of intracellular membranes while productive infection progresses. Assembly occurs in fragmented Golgi stacks and generates tubular structures, as well as the three spherical viral forms. These results, together with our previous studies with nonrelated viruses, show that the Golgi complex contains key factors for the structural transformation of a number of enveloped viruses that assemble intracellularly.

scholarly journals Role of Rubella Virus Glycoprotein Domains in Assembly of Virus-Like Particles

1999 ◽  
Vol 73 (5) ◽  
pp. 3524-3533 ◽  
Author(s):  
Mike Garbutt ◽  
Lok Man J. Law ◽  
Honey Chan ◽  
Tom C. Hobman
Keyword(s):  
Golgi Complex ◽  
Rubella Virus ◽  
Transmembrane Domain ◽  
Viral Envelope ◽  
Structural Proteins ◽  
Type I ◽  
Cytoplasmic Domains ◽  
Virus Like Particles ◽  
Positive Strand Rna

ABSTRACT Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7–20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670–7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses.

scholarly journals The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. I. Observations in hepatocytes.

1981 ◽  
Vol 88 (1) ◽  
pp. 1-15 ◽  
Author(s):  
G Bennett ◽  
D O'Shaughnessy
Keyword(s):  
Plasma Membrane ◽  
Sialic Acid ◽  
Cell Types ◽  
Mouse Cell ◽  
Time Intervals ◽  
Light Reaction ◽  
Golgi Stack ◽  
Golgi Region ◽  
Grain Distribution ◽  
Silver Grains

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of [3H]N-acetylmannosamine (8 mCi) and then sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the silver grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. Meanwhile, the proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h and over 55% at 4 and 24 h; two-thirds of these grains lay over the sinusoidal membrane, and the remainder were equally divided over the lateral and bile canalicular membranes. Many silver grains also appeared over lysosomes at the 4- and 24-h time intervals, accounting for 15-17% of the total. At 3 and 9 d after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling such that by 9 d only a very light reaction remained. Because our biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), the interpretation of these results is that sialic acid is incorporated into these molecules in the Golgi apparatus and that the latter then migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. It is possible that some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.

scholarly journals Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.

1995 ◽  
Vol 129 (5) ◽  
pp. 1421-1432 ◽  
Author(s):  
J Rossert ◽  
H Eberspaecher ◽  
B de Crombrugghe
Keyword(s):  
Transgenic Mice ◽  
Type I Collagen ◽  
Cell Types ◽  
Proximal Promoter ◽  
Type I ◽  
High Level Expression ◽  
Cis Acting ◽  
High Level ◽  
Different Cell Types ◽  
Beta Galactosidase

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell-specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis-acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.

scholarly journals Unfolding and identification of membrane proteins from native cell membranes

2019 ◽  
Author(s):  
Nicola Galvanetto ◽  
Sourav Maity ◽  
Nina Ilieva ◽  
Zhongjie Ye ◽  
Alessandro Laio ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Single Cells ◽  
Total Content ◽  
Cell Types ◽  
Single Cell Level ◽  
Cell Level ◽  
Single Molecule Force Spectroscopy ◽  
Native Cell ◽  
Different Cell Types

AbstractIs the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or can it provide useful information even for real biological samples? Here, we describe a pipeline for analyzing native membranes based on high throughput single-molecule force spectroscopy. The protocol includes a technique for the isolation of the plasma membrane of single cells. Afterwards, one harvests hundreds of thousands SMSF traces from the sample. Finally, one characterizes and identifies the embedded membrane proteins. This latter step is the cornerstone of our approach and involves combining, within a Bayesian framework, the information of the shape of the SMFS Force-distance which are observed more frequently, with the information from Mass Spectrometry and from proteomic databases (Uniprot, PDB). We applied this method to four cell types where we classified the unfolding of 5-10% of their total content of membrane proteins. The ability to mechanically probe membrane proteins directly in their native membrane enables the phenotyping of different cell types with almost single-cell level of resolution.

Computer Reconstructions of Normal Human Alveoli From Serial Sections

1985 ◽  
Vol 43 ◽  
pp. 312-313
Author(s):  
Saundra C. Parra ◽  
Ricky Burnette ◽  
Timothy Takaro
Keyword(s):  
Cell Types ◽  
Type I ◽  
Type Ii ◽  
Serial Sections ◽  
Single Plane ◽  
Random Sections ◽  
Normal Human ◽  
Connective Tissue Matrix ◽  
Different Cell Types ◽  
Tissue Matrix

Portions of two adjacent normal human alveoli were reconstructed from serial sections in order to examine normal alveolar organization, including anatomical relationships among the different cell types, the connective tissue matrix and gaps in the alveolar septum. Computer reconstructions were prepared from montaged electron micrographs of serial sections. Rotation of these reconstructions in the x, y or z axes allowed examination of the alveoli from many different aspects other than the actual plane of sectioning. Anatomical relationships “between Type I and Type II epithelial cells, alveolar macrophages, and pores of Kohn that could not he deduced from a single plane of the section (random sections) were revealed.

scholarly journals Detachment of cells from culture substrate by soluble fibronectin peptides.

1985 ◽  
Vol 100 (6) ◽  
pp. 1948-1954 ◽  
Author(s):  
E G Hayman ◽  
M D Pierschbacher ◽  
E Ruoslahti
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Cell Types ◽  
Soluble Form ◽  
Detectable Effect ◽  
Type I ◽  
Recognition Mechanism ◽  
Synthetic Cell ◽  
Attachment Mechanism ◽  
Different Cell Types

The synthetic cell attachment-promoting peptides from fibronectin (Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.)., 309:30-33) were found to detach cultured cells from the substratum when added to the culture in a soluble form. Peptides ranging in length from tetrapeptide to heptapeptide and containing the active L-arginyl-glycyl-L-aspartic acid (Arg-Gly-Asp) sequence had the detaching activity, whereas a series of different peptides with chemically similar structures had no detectable effect on any of the test cells. The Arg-Gly-Asp-containing peptides caused detachment of various cell lines of different species and histogenetic origin. Studies with defined substrates showed that the active peptides could inhibit the attachment of cells to vitronectin in addition to fibronectin, indicating that vitronectin is recognized by cells through a similar mechanism as fibronectin. The peptides did not inhibit the attachment of cells to collagen. However, cells cultured on collagen-coated plastic for 24-36 h, as well as cells with demonstrable type I or type VI collagen in their matrix, were susceptible to the detaching effect of the peptides. These results indicate that the recognition mechanism(s) by which cells bind to fibronectinand vitronectin plays a major role in the substratum attachment of cells and that collagens may not be directly involved in cell-substratum adhesion. Since vitronectin is abundant in serum, it is probably an important component in mediating the attachment of cultured cells. The independence of the effects of the peptide on the presence of serum and the susceptibility of many different cell types to detachment by the peptide show that the peptides perturb an attachment mechanism that is intrinsic to the cells and fundamentally significant to their adhesion.

Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells

2011 ◽  
Vol 106 (10) ◽  
pp. 693-704 ◽  
Author(s):  
Valeria Bozzi ◽  
Emanuele Panza ◽  
Serena Barozzi ◽  
Cristian Gruppi ◽  
Marco Seri ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Cell Types ◽  
Type I ◽  
Wild Type ◽  
Mutant Proteins ◽  
Wild Type Counterpart ◽  
Platelet Release ◽  
Myosin Iia ◽  
Different Cell Types

SummaryMYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations in the gene for the heavy chain of nonmuscle myosin-IIA (NMMHC-IIA). Recent in vitro studies led to the hypothesis that thrombocytopenia of MYH9-RD derives from an ectopic platelet release by megakaryocytes in the osteoblastic areas of bone marrow (BM), which are enriched in type I collagen, rather than in vascular spaces. SDF-1-driven migration of megakaryocytes within BM to reach the vascular spaces is a key mechanism for platelet biogenesis. Since myosin-IIA is implicated in polarised migration of different cell types, we hypothesised that MYH9 mutations could interfere with this mechanism. We therefore investigated the SDF-1-driven migration of a megakaryoblastic cell line, Dami cells, on type I collagen or fibrinogen by a modified transwell assay. Inhibition of myosin-IIA ATPase activity suppressed the SDF-1-driven migration of Dami cells, while over-expression of NMMHC-IIA increased the efficiency of chemotaxis, indicat- ing a role for NMMHC-IIA in this mechanism. Transfection of cells with three MYH9 mutations frequently responsible for MYH9-RD (p.R702C, p.D1424H, or p.R1933X) resulted in a defective SDF-1-driven migration with respect to the wild-type counterpart and in increased cell spreading onto collagen. Analysis of differential localisation of wild-type and mutant proteins suggested that mutant NMMHC-IIAs had an impaired cytoplasmic re-organisation in functional cytoskeletal structures after cell adhesion to collagen. These findings support the hypothesis that a defect of SDF-1-driven migration of megakaryocytes induced by MYH9 mutations contributes to ectopic platelet release in the BM osteoblastic areas, resulting in ineffective platelet production.

scholarly journals Ste20-related Protein Kinase LOSK (SLK) Controls Microtubule Radial Array in Interphase

2008 ◽  
Vol 19 (5) ◽  
pp. 1952-1961 ◽  
Author(s):  
Anton V. Burakov ◽  
Olga N. Zhapparova ◽  
Olga V. Kovalenko ◽  
Liudmila A. Zinovkina ◽  
Ekaterina S. Potekhina ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Golgi Complex ◽  
Cell Types ◽  
Vero Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cellular Regulation ◽  
Microtubule Organization ◽  
Microtubule Stabilization ◽  
Interphase Cells

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

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