scholarly journals Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant-adapted rhabdovirus

2007 ◽  
Vol 88 (6) ◽  
pp. 1810-1820 ◽  
Author(s):  
Michael M. Goodin ◽  
Romit Chakrabarty ◽  
Sharon Yelton ◽  
Kathleen Martin ◽  
Anthony Clark ◽  
...  
Keyword(s):  
Protein Function ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
M Protein ◽  
N Protein ◽  
Glycoprotein G ◽  
Putative Site ◽  
Infected Cells ◽  
Green Fluorescent

Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells.

scholarly journals Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

2013 ◽  
Vol 79 (20) ◽  
pp. 6481-6490 ◽  
Author(s):  
Wout Overkamp ◽  
Katrin Beilharz ◽  
Ruud Detert Oude Weme ◽  
Ana Solopova ◽  
Harma Karsens ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Streptococcus Pneumoniae ◽  
Lactococcus Lactis ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Experimental Conditions ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTGreen fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localizationin vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically forBacillus subtilisandStreptococcus pneumoniaeand have benchmarked them against five other previously available variants of GFP inB. subtilis,S. pneumoniae, andLactococcus lactis, using promoter-gfpfusions. Surprisingly, the best-performing GFP under our experimental conditions inB. subtiliswas the one codon optimized forS. pneumoniaeandvice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

scholarly journals Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM

2003 ◽  
Vol 84 (10) ◽  
pp. 2781-2793 ◽  
Author(s):  
Adeline Catteau ◽  
Olga Kalinina ◽  
Marie-Christine Wagner ◽  
Vincent Deubel ◽  
Marie-Pierre Courageot ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Fluorescent Protein ◽  
Apoptotic Cell ◽  
Host Cells ◽  
M Protein ◽  
Apoptotic Pathway ◽  
Apoptosis Protein ◽  
Infected Cells ◽  
Human Hepatoma Hepg2 Cells ◽  
Green Fluorescent

The induction of apoptotic cell death is a prominent cytopathic effect of dengue (DEN) viruses. One of the key questions to be addressed is which viral components induce apoptosis in DEN virus-infected cells. This study investigated whether the small membrane (M) protein was involved in the induction of apoptosis by DEN virus. This was addressed by using a series of enhanced green fluorescent protein-fused DEN proteins. Evidence is provided that intracellular production of the M ectodomains (residues M-1 to M-40) of all four DEN serotypes triggered apoptosis in host cells such as mouse neuroblastoma Neuro 2a and human hepatoma HepG2 cells. The M ectodomains of the wild-type strains of Japanese encephalitis, West Nile and yellow fever viruses also had proapoptotic properties. The export of the M ectodomain from the Golgi apparatus to the plasma membrane appeared to be essential for the initiation of apoptosis. The study found that anti-apoptosis protein Bcl-2 protected HepG2 cells against the death-promoting activity of the DEN M ectodomain. This suggests that the M ectodomain exerts its cytotoxic effects by activating a mitochondrial apoptotic pathway. The cytotoxicity of the DEN M ectodomain reflected the intrinsic proapoptotic properties of the nine carboxy-terminal amino acids (residues M-32 to M-40) designated ApoptoM. Residue M-36 was unique in that it modulated the death-promoting activity of the M ectodomain. Defining the ApoptoM-activated signalling pathways leading to apoptosis will provide the basis for studying how the M protein might play a key role in the fate of the flavivirus-infected cells.

scholarly journals Single-strand DNA Aptamers as Probes for Protein Localization in Cells

2003 ◽  
Vol 51 (6) ◽  
pp. 797-808 ◽  
Author(s):  
Kristi K.H. Stanlis ◽  
J. Richard McIntosh
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Freeze Substitution ◽  
Accurate Localization ◽  
Cell Structures ◽  
Specific Proteins ◽  
General Protein ◽  
Green Fluorescent ◽  
Very High

The accurate localization of proteins in fixed cells is important for many studies in cell biology, but good fixation is often antagonistic to good immunolabeling, given the density of well-preserved cells and the size of most labeled antibody probes. We therefore explored the use of single-stranded oligonucleotides (aptamers), which can bind to proteins with very high affinity and specificity but which are only ∼10 kD. To evaluate these probes for general protein localization, we sought an aptamer that binds to a widely used protein tag, the green fluorescent protein (GFP). Although this quest was not successful, we were able to solve several practical problems that will confront any such labeling effort, e.g., the rates at which oligonucleotides enter fixed cells of different kinds and the extent of nonspecific oligonucleotide binding to both mammalian and yeast cell structures. Because such localization methods would be of particular value for electron microscopy of optimally fixed material, we also explored the solubility of aptamers under conditions suitable for freeze-substitution fixation. We found that aptamers are sufficiently soluble in cold organic solvents to encourage the view that this approach may be useful for the localization of specific proteins in context of cellular fine structure.

scholarly journals A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

2009 ◽  
Vol 9 (1) ◽  
pp. 224-226 ◽  
Author(s):  
Chengda Zhang ◽  
James B. Konopka
Keyword(s):  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Codon Usage ◽  
Signal Intensity ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Protein Variant ◽  
Green Fluorescent ◽  
Green Fluorescent Protein Variant

ABSTRACT Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

scholarly journals Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

2009 ◽  
Vol 83 (6) ◽  
pp. 2611-2622 ◽  
Author(s):  
Subash C. Das ◽  
Debasis Panda ◽  
Debasis Nayak ◽  
Asit K. Pattnaik
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Protein ◽  
Dynamic Imaging ◽  
Viral Envelope ◽  
M Protein ◽  
Recombinant Viruses ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

Confocal Analysis of the Distribution and Persistence of Sindbis Virus (TaV-GFP) Infection in Midguts of Aedes aegypti Mosquitoes

2020 ◽  
Vol 26 (2) ◽  
pp. 267-274
Author(s):  
Jason J. Saredy ◽  
Florence Y. Chim ◽  
Zoë L. Lyski ◽  
Yani P. Ahearn ◽  
Doria F. Bowers
Keyword(s):  
Aedes Aegypti ◽  
Sindbis Virus ◽  
Fluorescent Protein ◽  
Blood Feeding ◽  
Target Tissue ◽  
Secondary Target ◽  
Posterior Midgut ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Global Threat

AbstractBiological transmission of arthropod-borne viruses (arboviruses) to vertebrate hosts by hematophagous insects poses a global threat because such arboviruses can result in a range of serious public health infectious diseases. Sindbis virus (SINV), the prototype Alphavirus, was used to track infections in the posterior midgut (PMG) of Aedes aegypti adult mosquitoes. Females were fed viremic blood containing a virus reporter, SINV [Thosea asigna virus-green fluorescent protein (TaV-GFP)], that leaves a fluorescent signal in infected cells. We assessed whole-mount PMGs to identify primary foci, secondary target tissues, distribution, and virus persistence. Following a viremic blood meal, PMGs were dissected and analyzed at various days of post blood-feeding. We report that virus foci indicated by GFP in midgut epithelial cells resulted in a 9.8% PMG infection and a 10.8% dissemination from these infected guts. The number of virus foci ranged from 1 to 3 per individual PMG and was more prevalent in the PMG-middle > PMG-frontal > PMG-caudal regions. SINV TaV-GFP was first observed in the PMG (primary target tissue) at 3 days post blood-feeding, was sequestered in circumscribed foci, replicated in PMG peristaltic muscles (secondary target tissue) following dissemination, and GFP was observed to persist in PMGs for 30 days postinfection.

Sign in / Sign up

Export Citation Format

Share Document