scholarly journals Estimation of the Size of Genetic Bottlenecks in Cell-to-Cell Movement of Soil-Borne Wheat Mosaic Virus and the Possible Role of the Bottlenecks in Speeding Up Selection of Variations in trans-Acting Genes or Elements

2009 ◽  
Vol 84 (4) ◽  
pp. 1828-1837 ◽  
Author(s):  
Shuhei Miyashita ◽  
Hirohisa Kishino
Keyword(s):  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Rna Viruses ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Cell Movement ◽  
Essential Element ◽  
Growth Conditions ◽  
Genetic Bottlenecks ◽  
Selection Of

ABSTRACT Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil -b orne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 ± 0.22 on average and 5.02 ± 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans-acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

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.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Actin Cytoskeleton Is Involved in Targeting of a Viral Hsp70 Homolog to the Cell Periphery

2005 ◽  
Vol 79 (22) ◽  
pp. 14421-14428 ◽  
Author(s):  
Alexey I. Prokhnevsky ◽  
Valera V. Peremyslov ◽  
Valerian V. Dolja
Keyword(s):  
Actin Cytoskeleton ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Fluorescent Proteins ◽  
Cell Movement ◽  
Plant Viruses ◽  
Cell Periphery ◽  
Virus Particles ◽  
Shock Proteins

ABSTRACT The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular ∼70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

2015 ◽  
Vol 7 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Kevin M. Dean ◽  
Jennifer L. Lubbeck ◽  
Lloyd M. Davis ◽  
Chola K. Regmi ◽  
Prem P. Chapagain ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Microfluidic Platform ◽  
Red Fluorescent Proteins ◽  
New Variant ◽  
Selection Of

We use a high-throughput microfluidic platform that sorts cells on the basis of fluorescent protein photostability to identify a new variant with improved photon output.

scholarly journals Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus

2001 ◽  
Vol 75 (18) ◽  
pp. 8712-8723 ◽  
Author(s):  
Diane M. Lawrence ◽  
A. O. Jackson
Keyword(s):  
Subcellular Localization ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Barley Stripe Mosaic Virus ◽  
Helicase Domain ◽  
Chenopodium Amaranticolor ◽  
Gene Block ◽  
Green Fluorescent

ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

scholarly journals High-Throughput Analysis of Protein with Tandem Fluorescent Protein Timers

2022 ◽  
pp. 85-100
Author(s):  
Jia Jun Fung ◽  
Karla Blöcher-Juárez ◽  
Anton Khmelinskii
Keyword(s):  
High Throughput ◽  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Growth Conditions ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
Instrument Choice ◽  
Fluorescence Measurements ◽  
Genome Wide

AbstractTandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays. We describe guidelines on optimization of experimental design with regard to the layout of colony arrays, growth conditions, and instrument choice. Combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, this approach can be used to compare protein turnover across the proteome and to identify regulators of protein turnover genome-wide.

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