scholarly journals Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-α induced apoptosis

2009 ◽  
Vol 8 (6) ◽  
pp. 763 ◽  
Author(s):  
Toshiyuki Ito ◽  
Shugo Oshita ◽  
Takakazu Nakabayashi ◽  
Fan Sun ◽  
Masataka Kinjo ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Tnf Α ◽  
Induced Apoptosis ◽  
Green Fluorescent

A novel yellowish-green fluorescent protein from the marine copepod, Chiridius poppei, and its use as a reporter protein in HeLa cells

Gene ◽  
2006 ◽  
Vol 372 ◽  
pp. 18-25 ◽  
Author(s):  
Hiromi Masuda ◽  
Yasuhiro Takenaka ◽  
Atsushi Yamaguchi ◽  
Satoshi Nishikawa ◽  
Hiroshi Mizuno
Keyword(s):  
Green Fluorescent Protein ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
Marine Copepod ◽  
Yellowish Green ◽  
Green Fluorescent

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 Green Fluorescent Protein with Anionic Tryptophan-Based Chromophore and Long Fluorescence Lifetime

2015 ◽  
Vol 109 (2) ◽  
pp. 380-389 ◽  
Author(s):  
Karen S. Sarkisyan ◽  
Alexander S. Goryashchenko ◽  
Peter V. Lidsky ◽  
Dmitry A. Gorbachev ◽  
Nina G. Bozhanova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Cortactin Is Necessary for F-Actin Accumulation in Pedestal Structures Induced by Enteropathogenic Escherichia coli Infection

2002 ◽  
Vol 70 (4) ◽  
pp. 2206-2209 ◽  
Author(s):  
Vlademir V. Cantarelli ◽  
Akira Takahashi ◽  
Itaru Yanagihara ◽  
Yukihiro Akeda ◽  
Kinichi Imura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Enteropathogenic Escherichia Coli ◽  
Escherichia Coli Infection ◽  
Green Fluorescent ◽  
Pedestal Formation

ABSTRACT Cortactin and the translocated intimin receptor, Tir, interacted with each other in pedestal formation in HeLa cells infected with enteropathogenic Escherichia coli (EPEC). Cortactin is shown to be necessary for organizing actin pedestals in response to EPEC, based on the expression of green fluorescent protein-fused cortactin derivatives in HeLa cells.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

scholarly journals A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Hideji Murakoshi ◽  
Akihiro C. E. Shibata ◽  
Yoshihisa Nakahata ◽  
Junichi Nabekura
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Response Signal ◽  
Dark Green ◽  
Green Fluorescent ◽  
Cell To Cell Variability

Abstract Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.

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