scholarly journals Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling Events

2004 ◽  
Vol 15 (11) ◽  
pp. 4911-4925 ◽  
Author(s):  
Vilja Pietiäinen ◽  
Varpu Marjomäki ◽  
Paula Upla ◽  
Lucas Pelkmans ◽  
Ari Helenius ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Rna Virus ◽  
Brefeldin A ◽  
Cell Entry ◽  
Viral Pathogens ◽  
Caveolin 1 ◽  
Cellular Cytoskeleton ◽  
Green Fluorescent ◽  
Viral Capsid Proteins

Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the α2β1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.

scholarly journals Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

2002 ◽  
Vol 76 (17) ◽  
pp. 8808-8819 ◽  
Author(s):  
C. Ritzenthaler ◽  
C. Laporte ◽  
F. Gaire ◽  
P. Dunoyer ◽  
C. Schmitt ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Replication ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rna Virus ◽  
Lipid Synthesis ◽  
Cell Movement ◽  
Brefeldin A ◽  
Cell Periphery ◽  
Green Fluorescent

ABSTRACT Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

scholarly journals The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial Bud

1998 ◽  
Vol 142 (3) ◽  
pp. 613-623 ◽  
Author(s):  
Koji Okamoto ◽  
Philip S. Perlman ◽  
Ronald A. Butow
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Mitochondrial Matrix ◽  
Outer Membranes ◽  
Marker Proteins ◽  
The Matrix ◽  
Green Fluorescent ◽  
Mitochondrial Reticulum

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

scholarly journals Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

2006 ◽  
Vol 17 (7) ◽  
pp. 3085-3094 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Peter Schweinsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Cortical Granule ◽  
Major Protein ◽  
Dynamic Regulation ◽  
Caveolin 1 ◽  
Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

scholarly journals Fate of Salmonella Typhimurium in laboratory-scale drinking water biofilms

2013 ◽  
Vol 11 (4) ◽  
pp. 629-635 ◽  
Author(s):  
L. M. Schaefer ◽  
V. S. Brözel ◽  
S. N. Venter
Keyword(s):  
Drinking Water ◽  
Green Fluorescent Protein ◽  
Health Risk ◽  
Salmonella Typhimurium ◽  
Fluorescent Protein ◽  
Laboratory Scale ◽  
Green Fluorescent ◽  
In Situ Detection

Investigations were carried out to evaluate and quantify colonization of laboratory-scale drinking water biofilms by a chromosomally green fluorescent protein (gfp)-tagged strain of Salmonella Typhimurium. Gfp encodes the green fluorescent protein and thus allows in situ detection of undisturbed cells and is ideally suited for monitoring Salmonella in biofilms. The fate and persistence of non-typhoidal Salmonella in simulated drinking water biofilms was investigated. The ability of Salmonella to form biofilms in monoculture and the fate and persistence of Salmonella in a mixed aquatic biofilm was examined. In monoculture S. Typhimurium formed loosely structured biofilms. Salmonella colonized established multi-species drinking water biofilms within 24 hours, forming micro-colonies within the biofilm. S. Typhimurium was also released at high levels from the drinking water-associated biofilm into the water passing through the system. This indicated that Salmonella could enter into, survive and grow within, and be released from a drinking water biofilm. The ability of Salmonella to survive and persist in a drinking water biofilm, and be released at high levels into the flow for recolonization elsewhere, indicates the potential for a persistent health risk to consumers once a network becomes contaminated with this bacterium.

Important role for the V-type H+-ATPase and the Golgi apparatus in the recycling of PTH/PTHrP receptor

2004 ◽  
Vol 286 (5) ◽  
pp. E704-E710 ◽  
Author(s):  
Hesham A. W. Tawfeek ◽  
Abdul B. Abou-Samra
Keyword(s):  
Golgi Apparatus ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Receptor Recycling ◽  
Bafilomycin A1 ◽  
Coated Pits ◽  
Concanamycin A ◽  
Hydrogen Atpase ◽  
Green Fluorescent ◽  
Pthrp Receptor

Our previous studies demonstrated that a green fluorescent protein-tagged parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor stably expressed in LLCPK-1 cells undergoes agonist-dependent internalization into clathrin-coated pits. The subcellular localization of the internalized PTH/PTHrP receptor is not known. In the present study, we explored the intracellular pathways of the internalized PTH/PTHrP receptor. Using immunofluorescence and confocal microscopy, we show that the internalized receptors localize at a juxtanuclear compartment identified as the Golgi apparatus. The receptors do not colocalize with lysosomes. Furthermore, whereas the internalized receptors exhibit rapid recycling, treatment with proton pump inhibitors (bafilomycin-A1 and concanamycin A) or brefeldin A, Golgi disrupting agents, reduces PTH/PTHrP receptor recycling. Together, these data indicate an important role for the vacuolar-type hydrogen-ATPase and the Golgi apparatus in postendocytic PTH/PTHrP receptor recovery.

In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members

1998 ◽  
Vol 64 (2) ◽  
pp. 721-732 ◽  
Author(s):  
Søren Møller ◽  
Claus Sternberg ◽  
Jens Bo Andersen ◽  
Bjarke Bak Christensen ◽  
Juan Luis Ramos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microbial Communities ◽  
Pure Culture ◽  
Fluorescent Protein ◽  
Confocal Laser ◽  
Specific Gene ◽  
Community Members ◽  
Metabolic Interactions ◽  
Green Fluorescent

ABSTRACT Microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. Among the community members studied, the focus was in particular on Pseudomonas putida and a strain of anAcinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. The upper-pathway promoter (Pu) and themeta-pathway promoter (Pm) from the TOL plasmid were fused independently to the gene coding for the green fluorescent protein (GFP), and expression from these promoters was studied inP. putida, which was a dominant community member. Biofilms were cultured in flow chambers, which in combination with scanning confocal laser microscopy allowed direct monitoring of promoter activity with single-cell spatial resolution. Expression from thePu promoter was homogeneously induced by benzyl alcohol in both community and pure-culture biofilms, while the Pmpromoter was induced in the mixed community but not in a pure-culture biofilm. By sequentially adding community members, induction ofPm was shown to be a consequence of direct metabolic interactions between an Acinetobacter species and P. putida. Furthermore, in fixed biofilm samples organism identity was determined and gene expression was visualized at the same time by combining GFP expression with in situ hybridization with fluorescence-labeled 16S rRNA targeting probes. This combination of techniques is a powerful approach for investigating structure-function relationships in microbial communities.

scholarly journals Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo

2018 ◽  
Vol 2 (20) ◽  
pp. 2589-2606 ◽  
Author(s):  
Chris Moore ◽  
Joanna L. Richens ◽  
Yasmin Hough ◽  
Deniz Ucanok ◽  
Sunir Malla ◽  
...  
Keyword(s):  
Cell Fate ◽  
Fluorescent Protein ◽  
Erythroid Differentiation ◽  
Transcriptional Repressors ◽  
Rna Seq ◽  
Promote Cell Proliferation ◽  
Cell Fate Decisions ◽  
Maturation Defect ◽  
Green Fluorescent

Abstract The transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebrafish, prRBCs express 2 of 3 zebrafish Gfi1/1b paralogs, Gfi1aa and Gfi1b. The recently identified zebrafish gfi1aa gene trap allele qmc551 drives erythroid green fluorescent protein (GFP) instead of Gfi1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of Gfi1b in gfi1aaqmc551 homozygous embryos. To study the transcriptome of the Gfi1aa/1b double-depleted cells, we performed an RNA-Seq experiment on GFP-positive prRBCs sorted from 20-hour-old embryos that were heterozygous or homozygous for gfi1aaqmc551, as well as wt or morphant for gfi1b. We subsequently confirmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combined, the data showed that in the absence of Gfi1aa, the synchronously developing prRBCs were delayed in activating late erythroid differentiation, as they struggled to suppress early erythroid and endothelial transcription programs. The latter highlighted the bipotent nature of the progenitors from which prRBCs arise. In the absence of Gfi1aa, Gfi1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed Gfi1aa-depleted prRBCs even further, showing that Gfi1aa and Gfi1b together set the pace for prRBC differentiation from hemangioblasts.

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

2003 ◽  
Vol 284 (5) ◽  
pp. H1647-H1654 ◽  
Author(s):  
Jean-Philippe Fortin ◽  
Johanne Bouthillier ◽  
François Marceau
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Brefeldin A ◽  
Metabolic Inhibitors ◽  
Maximal Effect ◽  
Hek 293 ◽  
B1 Receptors ◽  
Green Fluorescent

We hypothesized that the inducible kinin B1 receptor (B1R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B1Rs and related B2 receptors (B2Rs) was investigated. Endocytosis of the B1R-yellow fluorescent protein (YFP) conjugate was more intense than that of B2R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B1R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B2R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B1R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B2R-GFP remained constant. Wild-type B1Rs were also cleared faster than B2Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B1Rs are more rapidly eliminated than B2Rs (decreased maximal effect of agonist over 2 h). The highly regulated B1R is rapidly degraded, relative to the constitutive B2R.

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