scholarly journals Short Tetracysteine Tags to β-Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging

2004 ◽  
Vol 15 (12) ◽  
pp. 5616-5622 ◽  
Author(s):  
Martin Andresen ◽  
Rita Schmitz-Salue ◽  
Stefan Jakobs
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Open Reading Frames ◽  
Host Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent ◽  
Β Tubulin

Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein (GFP) were fused by integrative epitope tagging to the C terminus of β-tubulin (Tub2) in the budding yeast Saccharomyces cerevisiae. The increasing tag size correlated with functional interference to the host protein. Tub2 tagged with either 1×TetCys (10 amino acids [aa]) or 2×TetCys (20 aa) was able to substitute Tub2 in haploid cells. In contrast, C-terminal tagging of Tub2 with 3×TetCys (29 aa) or with GFP (244 aa) resulted in nonviable haploid cells. Cells expressing Tub2-1×TetCys or Tub2-2×TetCys were stained with FlAsH, which selectively binds to the TetCys-tag. The stained cells displayed dynamic FlAsH-labeled microtubules and low cellular background fluorescence. The presented approach to tag open reading frames (ORFs) at their native loci with very small TetCys-tags and the subsequent visualization of the tagged proteins in vivo can be extended in principle to any ORF in S. cerevisiae.

scholarly journals Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins

2018 ◽  
Vol 19 (12) ◽  
pp. 3778 ◽  
Author(s):  
Nina Bozhanova ◽  
Mikhail Baranov ◽  
Nadezhda Baleeva ◽  
Alexey Gavrikov ◽  
Alexander Mishin
Keyword(s):  
Green Fluorescent Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Protein Labeling ◽  
Cell Protein ◽  
Gfp Chromophore ◽  
Green Fluorescent ◽  
Derivatives Of

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.

scholarly journals Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts

2007 ◽  
Vol 71A (10) ◽  
pp. 827-834 ◽  
Author(s):  
Michael Halter ◽  
Alex Tona ◽  
Kiran Bhadriraju ◽  
Anne L. Plant ◽  
John T. Elliott
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Degradation ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Green Fluorescent

scholarly journals A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici

2015 ◽  
Vol 79 ◽  
pp. 125-131 ◽  
Author(s):  
S. Kilaru ◽  
M. Schuster ◽  
D. Studholme ◽  
D. Soanes ◽  
C. Lin ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Zymoseptoria Tritici ◽  
Green Fluorescent

scholarly journals Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kazuki Harada ◽  
Takami Chihara ◽  
Yuki Hayasaka ◽  
Marie Mita ◽  
Mai Takizawa ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Live Cell ◽  
Trace Detection ◽  
Lactate And Pyruvate ◽  
Lactate Levels ◽  
Green Fluorescent

Abstract Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC50 for trace detection of metabolic molecules and live cell imaging and named them “Green Lindoblum” and “Green Pegassos,” respectively. Green Lindoblum (EC50 of 30 µM for lactate) and Green Pegassos (EC50 of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca2+ in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.

scholarly journals Live-Cell Imaging of Rhabdovirus-Induced Morphological Changes in Plant Nuclear Membranes

2005 ◽  
Vol 18 (7) ◽  
pp. 703-709 ◽  
Author(s):  
Michael Goodin ◽  
Sharon Yelton ◽  
Debasish Ghosh ◽  
Stephanie Mathews ◽  
Judith Lesnaw
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Live Cell ◽  
Live Cells ◽  
Nuclear Membranes ◽  
Yellow Dwarf Virus ◽  
Study Sites ◽  
Green Fluorescent

Potato yellow dwarf virus (PYDV) and Sonchus yellow net virus (SYNV) belong to the genus Nucleorhabdovirus. These viruses replicate in nuclei of infected cells and mature virions accumulate in the perinuclear space after budding through the inner nuclear membrane. Infection of transgenic Nicotiana benthamiana 16c plants (which constitutively express green fluorescent protein (GFP) targeted to endomembranes) with PYDV or SYNV resulted in virusspecific patterns of accumulation of both GFP and membranes within nuclei. Using immunolocalization and a lipophilic fluorescent dye, we show that the sites of the relocalized membranes were coincident with foci of accumulation of the SYNV nucleocapsid protein. In contrast to the effects of PYDV and SYNV, inoculation of 16c plants with plusstrand RNA viruses did not result in accumulation of intranuclear GFP. Instead, such infections resulted in accumulation of GFP around nuclei, in a manner consistent with proliferation of the endoplasmic reticulum. We propose that the relocalization of GFP in 16c plants can be used to study sites of rhabdovirus accumulation in live cells. This study is the first to use live-cell imaging to characterize the effects of rhabdoviruses on plant nuclear membranes.

scholarly journals Red-shifted iLOV mutants have decreased brightness disrupting their use in live cell imaging

2021 ◽  
Author(s):  
Pierre Wehler ◽  
Mehmet Ali Oeztuerk
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Emission Spectra ◽  
Live Cell ◽  
Red Shift ◽  
Flavin Mononucleotide ◽  
Low Oxygen ◽  
Triple Mutant ◽  
Green Fluorescent

iLOV is a flavin mononucleotide (FMN)-binding fluorescent protein (FbFP) with excitation and emission spectra similar to those of the green fluorescent protein (GFP). Importantly, contrary to GFP, iLOV fluoresces independently of molecular oxygen, making its usage in low-oxygen conditions possible. Moreover, iLOV is smaller than GFP, increasing the likelihood of retaining full functionality when creating fusions with proteins of interest. Nonetheless, GFP remains to date the most widely used FP in molecular biology, also due to the availability of multiple spectrally tuned variants allowing multi-color imaging experiments. To expand the range of applications of iLOV, spectrally tuned red-shift variants are desirable to have reduced phototoxicity and better tissue penetration. Here we experimentally tested two iLOV mutants, iLOV L470T/Q489K and iLOV V392K/F410V/A426S, which were previously computationally proposed to have red-shifted excitation and emission spectra. We found that only the triple mutant has moderately red-shifted excitation and emission spectra. Both mutants exhibit strongly decreased brightness, which impedes their employment in live cell imaging. Finally, we show that the single V392K mutation suffices to red-shift the emission spectrum as well as abolishes fluorescence of iLOV.

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