scholarly journals The Vaccinia Virus B5 Protein Requires A34 for Efficient Intracellular Trafficking from the Endoplasmic Reticulum to the Site of Wrapping and Incorporation into Progeny Virions

2007 ◽  
Vol 82 (5) ◽  
pp. 2161-2169 ◽  
Author(s):  
Amalia K. Earley ◽  
Winnie M. Chan ◽  
Brian M. Ward
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Subcellular Fractionation ◽  
Gfp Fluorescence ◽  
Virion Morphogenesis ◽  
Green Fluorescent

ABSTRACT The glycoproteins encoded by the vaccinia virus A34R and B5R genes are involved in intracellular envelope virus formation and are highly conserved among orthopoxviruses. A recombinant virus that has the A34R gene deleted and the B5R gene replaced with a B5R gene fused to the enhanced green fluorescent protein (B5R-GFP) gene was created (vB5R-GFP/ΔA34R) to investigate the role of A34 during virion morphogenesis. Cells infected with vB5R-GFP/ΔA34R displayed GFP fluorescence throughout the cytoplasm, which differed markedly from that seen in cells infected with a normal B5R-GFP-expressing virus (vB5R-GFP). Immunofluorescence and subcellular fractionation demonstrated that B5-GFP localizes with the endoplasmic reticulum in the absence of A34. Expression of either full-length A34 or a construct consisting of the lumenal and transmembrane domains restored normal trafficking of B5-GFP to the site of wrapping in the juxtanuclear region. Coimmunoprecipitation studies confirmed that B5 and A34 interact through their luminal domains, and further analysis revealed that in the absence of A34, B5 is not efficiently incorporated into virions released from the cell.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

2000 ◽  
Vol 278 (5) ◽  
pp. E825-E831 ◽  
Author(s):  
Aimee W. Kao ◽  
Chunmei Yang ◽  
Jeffrey E. Pessin
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Splice Variants ◽  
Wild Type ◽  
Cell Cytoplasm ◽  
Functional Specificity ◽  
Glut 4 ◽  
Green Fluorescent ◽  
Dynamin 2

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.

scholarly journals mCLCA4 ER processing and secretion requires luminal sorting motifs

2008 ◽  
Vol 295 (1) ◽  
pp. C279-C287 ◽  
Author(s):  
Chunlei Huan ◽  
Kai Su Greene ◽  
Bo Shui ◽  
Gwendolyn Spizz ◽  
Haitao Sun ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chloride Transport ◽  
Proteolytic Processing ◽  
Regulatory Sequences ◽  
Cellular Processing ◽  
Green Fluorescent

Ca+-activated Cl− channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH2- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

scholarly journals Mucin Granules are in Close Contact with Tubular Elements of the Endoplasmic Reticulum

2005 ◽  
Vol 53 (10) ◽  
pp. 1305-1309 ◽  
Author(s):  
Juan Perez-Vilar ◽  
Carla M. Pedrosa Ribeiro ◽  
Wendy C. Salmon ◽  
Raean Mabolo ◽  
Richard C. Boucher
Keyword(s):  
Endoplasmic Reticulum ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Close Contact ◽  
Biological Significance ◽  
Goblet Cells ◽  
Mucin Secretion ◽  
Green Fluorescent

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

scholarly journals Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1547
Author(s):  
Anastasia V. Mamontova ◽  
Aleksander M. Shakhov ◽  
Konstantin A. Lukyanov ◽  
Alexey M. Bogdanov
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Important Determinant ◽  
Physicochemical Characteristics ◽  
High Brightness ◽  
Short Lifetime ◽  
Green Fluorescent

The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions.

scholarly journals Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

2003 ◽  
Vol 77 (16) ◽  
pp. 9008-9019 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1H79G, a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with α-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.

scholarly journals Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

2001 ◽  
Vol 12 (7) ◽  
pp. 2031-2046 ◽  
Author(s):  
Nina Tolonen ◽  
Laura Doglio ◽  
Sibylle Schleich ◽  
Jacomine Krijnse Locker
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Nuclear Envelope Assembly ◽  
Green Fluorescent Protein Tagging ◽  
Green Fluorescent

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.

scholarly journals Marker Fusion Tagging, a New Method for Production of Chromosomally Encoded Fusion Proteins

2010 ◽  
Vol 9 (5) ◽  
pp. 827-830 ◽  
Author(s):  
Julian Lai ◽  
Seng Kah Ng ◽  
Fang Fang Liu ◽  
Rajesh Narhari Patkar ◽  
Yanfen Lu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Spindle Pole ◽  
Hygromycin B ◽  
Content Type ◽  
Spindle Pole Bodies ◽  
New Gene ◽  
Green Fluorescent

ABSTRACT A new gene-tagging method (marker fusion tagging [MFT]) is demonstrated for Neurospora crassa and Magnaporthe oryzae. Translational fusions between the hygromycin B resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. This method can produce tags at any position and create deletion alleles that maintain N- and C-terminal sequences. We show the utility of MFT by producing enhanced green fluorescent protein (EGFP) tags in proteins localized to nuclei, spindle pole bodies, septal pore plugs, Woronin bodies, developing septa, and the endoplasmic reticulum.

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