scholarly journals Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins

2005 ◽  
Vol 86 (4) ◽  
pp. 1223-1228 ◽  
Author(s):  
Tomas Canto ◽  
Peter Palukaitis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Subcellular Distribution ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Epidermal Cells ◽  
Green Fluorescent Proteins ◽  
Movement Proteins ◽  
Infected Cells ◽  
Green Fluorescent

The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubles.

scholarly journals Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

2015 ◽  
Author(s):  
Anton Khmelinskii ◽  
Matthias Meurer ◽  
Chi-Ting Ho ◽  
Birgit Besenbeck ◽  
Julia Fueller ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Proteasomal Degradation ◽  
Time Range ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Dependent Change ◽  
The Stability ◽  
Green Fluorescent

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far tFTs were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing greenFPs, while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT towards slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for design of new tFTs.

scholarly journals Red Fluorescent Protein (DsRed) as a Reporter inSaccharomyces cerevisiae

2001 ◽  
Vol 183 (12) ◽  
pp. 3791-3794 ◽  
Author(s):  
Fernando Rodrigues ◽  
Martijn van Hemert ◽  
H. Yde Steensma ◽  
Manuela Côrte-Real ◽  
Cecı́la Leão
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Protein ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Dsred Protein ◽  
Green Fluorescent

ABSTRACT We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

scholarly journals Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering

2019 ◽  
Vol 16 (152) ◽  
pp. 20180848 ◽  
Author(s):  
Maksym Golub ◽  
Virginia Guillon ◽  
Guillaume Gotthard ◽  
Dominik Zeller ◽  
Nicolas Martinez ◽  
...  
Keyword(s):  
Neutron Scattering ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cyan Fluorescent Protein ◽  
Green Fluorescent Proteins ◽  
Incoherent Neutron Scattering ◽  
Fluorescence Efficiency ◽  
Green Fluorescent ◽  
Molecular Bases ◽  
Incoherent Neutron

Cyan fluorescent proteins (CFPs) are variants of green fluorescent proteins in which the central tyrosine of the chromophore has been replaced by a tryptophan. The increased bulk of the chromophore within a compact protein and the change in the positioning of atoms capable of hydrogen bonding have made it difficult to optimize their fluorescence properties, which took approximately 15 years between the availability of the first useable CFP, enhanced cyan fluorescent protein (ECFP), and that of a variant with almost perfect fluorescence efficiency, mTurquoise2. To understand the molecular bases of the progressive improvement in between these two CFPs, we have studied by incoherent neutron scattering the dynamics of five different variants exhibiting progressively increased fluorescence efficiency along the evolution pathway. Our results correlate well with the analysis of the previously determined X-ray crystallographic structures, which show an increase in flexibility between ECFP and the second variant, Cerulean, which is then hindered in the three later variants, SCFP3A (Super Cyan Fluorescent Protein 3A), mTurquoise and mTurquoise2. This confirms that increasing the rigidity of the direct environment of the fluorescent chromophore is not the sole parameter leading to brighter fluorescent proteins and that increased flexibility in some cases may be helpful.

scholarly journals Tobacco mosaic virus (TMV) Replicase and Movement Protein Function Synergistically in Facilitating TMV Spread by Lateral Diffusion in the Plasmodesmal Desmotubule of Nicotiana benthamiana

2008 ◽  
Vol 21 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Dana Guenoune-Gelbart ◽  
Michael Elbaum ◽  
Guy Sagi ◽  
Amit Levy ◽  
Bernard L. Epel
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Integral Membrane Protein ◽  
Movement Proteins ◽  
Green Fluorescent ◽  
Cell Spread ◽  
Er Membrane

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus (TMVMP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that TMVMP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of TMVMP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.

scholarly journals A photoactivatable green-fluorescent protein from the phylum Ctenophora

2009 ◽  
Vol 277 (1685) ◽  
pp. 1155-1160 ◽  
Author(s):  
Steven H. D. Haddock ◽  
Nadia Mastroianni ◽  
Lynne M. Christianson
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Optical Probes ◽  
Fluorescent Emission ◽  
The Family ◽  
Fluorescent State ◽  
Green Fluorescent

Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the x YG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals.

scholarly journals A bright and high-performance genetically encoded Ca2+ indicator based on mNeonGreen fluorescent protein

2020 ◽  
Author(s):  
Landon Zarowny ◽  
Abhi Aggarwal ◽  
Virginia M.S. Rutten ◽  
Ilya Kolb ◽  
Ronak Patel ◽  
...  
Keyword(s):  
High Performance ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Fluorescent Proteins ◽  
Model Organisms ◽  
Green Fluorescent Proteins ◽  
Comparable Performance ◽  
Green Fluorescent

AbstractGenetically encodable calcium ion (Ca2+) indicators (GECIs) based on green fluorescent proteins (GFP) are powerful tools for imaging of cell signaling and neural activity in model organisms. Following almost two decades of steady improvements in the Aequorea victoria GFP (avGFP)-based GCaMP series of GECIs, the performance of the most recent generation (i.e., GCaMP7) may have reached its practical limit due to the inherent properties of GFP. In an effort to sustain the steady progression towards ever-improved GECIs, we undertook the development of a new GECI based on the bright monomeric GFP, mNeonGreen (mNG). The resulting indicator, mNG-GECO1, is 60% brighter than GCaMP6s in vitro and provides comparable performance as demonstrated by imaging Ca2+ dynamics in cultured cells, primary neurons, and in vivo in larval zebrafish. These results suggest that mNG-GECO1 is a promising next-generation GECI that could inherit the mantle of GCaMP and allow the steady improvement of GECIs to continue for generations to come.

scholarly journals Are Tubules Generated by the 3a Protein Necessary for Cucumber Mosaic Virus Movement?

1999 ◽  
Vol 12 (11) ◽  
pp. 985-993 ◽  
Author(s):  
Tomas Canto ◽  
Peter Palukaitis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Virus Movement ◽  
Wild Type ◽  
Gene Encoding ◽  
Green Fluorescent ◽  
Infected Tobacco

The 3a movement protein of cucumber mosaic virus (CMV), fused to the jellyfish green fluorescent protein (3a-GFP) generated surface punctate aggregates as well as tubules protruding from infected tobacco and Nicotiana benthamiana protoplasts. Fluorescent tubules also appeared on the surface of protoplasts prepared from transgenic tobacco plants expressing 3a-GFP, indicating that the 3a protein is the only viral component required for the formation of the tubules. CMV with a mutation in the gene encoding the 3a protein, M8 CMV, could infect tobacco systemically, but tubules were not detected protruding from infected protoplasts when the mutated 3a protein was fused to the GFP [(M8)3a-GFP]. This indicates that the ability of the 3a protein to generate tubules in the surface of protoplasts is not a function required for the spread of CMV in tobacco. On the other hand, the (M8)3a-GFP did not traffic through plasmodesmata interconnecting tobacco epidermal cells, in contrast to the wild-type 3a-GFP. This suggests that there may be a correlation between the ability of the 3a protein to assemble tubules in protoplasts and its ability to promote movement within certain tissues.

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