scholarly journals Antibody to AP1B Adaptor Blocks Biosynthetic and Recycling Routes of Basolateral Proteins at Recycling Endosomes

2007 ◽  
Vol 18 (12) ◽  
pp. 4872-4884 ◽  
Author(s):  
Jorge Cancino ◽  
Carolina Torrealba ◽  
Andrea Soza ◽  
María Isabel Yuseff ◽  
Diego Gravotta ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Transferrin Receptor ◽  
Fluorescent Protein ◽  
Ldl Receptor ◽  
Recycling Endosomes ◽  
Basolateral Plasma Membrane ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Rat Thyroid ◽  
Golgi Network

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit μ1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3–5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in ∼45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.

scholarly journals Matrix protein of Vesicular stomatitis virus harbours a cryptic mitochondrial-targeting motif

2006 ◽  
Vol 87 (11) ◽  
pp. 3379-3384 ◽  
Author(s):  
Brian D. Lichty ◽  
Heidi McBride ◽  
Stephen Hanson ◽  
John C. Bell
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
M Protein ◽  
Mitochondrial Targeting ◽  
M Gene ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

Vesicular stomatitis virus (VSV) is a rhabdovirus that has attracted attention of late as an oncolytic virus and as a vaccine vector. Mutations in the matrix (M) gene of VSV yield attenuated strains that may be very useful in both settings. As a result of this interest in the M protein, this study analysed various M–green fluorescent protein (GFP) fusion constructs. Remarkably, fusion of the N terminus of the M protein to GFP targeted the fluorescent protein to the surface of mitochondria. Mutational analysis indicated that a mitochondrial-targeting motif exists within aa 33–67. Expression of these fusion proteins led to loss of mitochondrial membrane permeability and to an alteration in mitochondrial organization mirroring that seen during viral infection. In addition, a portion of the M protein present in infected cells co-purified with mitochondria. This work may indicate a novel function for this multifunctional viral protein.

scholarly journals Formation of Vesicular Stomatitis Virus Pseudotypes Bearing Surface Proteins of Hepatitis B Virus

2005 ◽  
Vol 79 (19) ◽  
pp. 12566-12574 ◽  
Author(s):  
Manujendra N. Saha ◽  
Atsushi Tanaka ◽  
Atsushi Jinno-Oue ◽  
Nobuaki Shimizu ◽  
Kazushi Tamura ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Lines ◽  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Surface Proteins ◽  
B Virus ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Short Time

ABSTRACT It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVΔG*-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVΔG*(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.

Human Na+/H+ exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria

2002 ◽  
Vol 282 (5) ◽  
pp. C1031-C1041 ◽  
Author(s):  
Christopher L. Brett ◽  
Ying Wei ◽  
Mark Donowitz ◽  
Rajini Rao
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Phylogenetic Analyses ◽  
Human Homolog ◽  
Recycling Endosomes ◽  
Mitochondrial Markers ◽  
Vacuolar Compartment ◽  
Green Fluorescent ◽  
Golgi Network

Since the discovery of the first intracellular Na+/H+exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na+ into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na+/H+ exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na+/H+ exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis.

scholarly journals Nested Genes CDA12 and CDA13 Encode Proteins Associated with Membrane Trafficking in the Ciliate Tetrahymena thermophila

2009 ◽  
Vol 8 (6) ◽  
pp. 899-912 ◽  
Author(s):  
Erica Zweifel ◽  
Joshua Smith ◽  
Daniel Romero ◽  
Thomas H. Giddings ◽  
Mark Winey ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Protein Localization ◽  
Recycling Endosomes ◽  
Nested Genes ◽  
Membrane Bound ◽  
Green Fluorescent ◽  
Golgi Network ◽  
Antisense Genes

ABSTRACT We describe a novel pair of nested genes, CDA12 and CDA13, from Tetrahymena thermophila. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and their neighboring Golgi networks. The expression of antisense CDA12 RNA in transformants produces defects in cytokinesis, macronuclear segregation, and the processing of pinosomes to downstream compartments. Antisense CDA13 RNA expression produces a conjugation phenotype, resulting in the failure of mating pairs to separate, as well as failures in postconjugation cytokinesis and macronuclear fission. This study offers insight into the membrane trafficking events linking endosome and Golgi network activities, cytokinesis, and karyokinesis and the unique membrane-remodeling events that accompany conjugation in the ciliate T. thermophila. We also highlight an unusual aspect of genome organization in Tetrahymena, namely, the existence of nested, antisense genes.

scholarly journals Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells Is Dependent on both Microtubules and Actin Filaments

2016 ◽  
Vol 90 (13) ◽  
pp. 6159-6170 ◽  
Author(s):  
Shalane K. Yacovone ◽  
Amanda M. Smelser ◽  
Jed C. Macosko ◽  
George Holzwarth ◽  
David A. Ornelles ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Distribution Method ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Time Required

ABSTRACTThe distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments.IMPORTANCENucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments play a larger role than microtubules in incorporation of nucleocapsids into virions.

scholarly journals The p14 FAST Protein of Reptilian Reovirus Increases Vesicular Stomatitis Virus Neuropathogenesis

2008 ◽  
Vol 83 (2) ◽  
pp. 552-561 ◽  
Author(s):  
Christopher W. Brown ◽  
Kyle B. Stephenson ◽  
Stephen Hanson ◽  
Michael Kucharczyk ◽  
Roy Duncan ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Syncytium Formation ◽  
Gene Encoding ◽  
Fast Proteins ◽  
Bona Fide ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

ABSTRACT The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.

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