Visualizing Transcription Factor Binding on Mitotic Chromosomes Using Single-Molecule Live-Cell Imaging

2019 ◽  
pp. 239-250
Author(s):  
James Z. J. Kwan ◽  
Thomas F. Nguyen ◽  
Sheila S. Teves
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Transcription Factor Binding ◽  
Live Cell ◽  
Mitotic Chromosomes ◽  
Factor Binding

scholarly journals Autofluorescent Proteins in Single-Molecule Research: Applications to Live Cell Imaging Microscopy

2001 ◽  
Vol 80 (5) ◽  
pp. 2396-2408 ◽  
Author(s):  
Gregory S. Harms ◽  
Laurent Cognet ◽  
Piet H.M. Lommerse ◽  
Gerhard A. Blab ◽  
Thomas Schmidt
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell

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.

Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting

2021 ◽  
Author(s):  
Ineke Brouwer ◽  
Emma Kerklingh ◽  
Fred van Leeuwen ◽  
Tineke L Lenstra
Keyword(s):  
Transcription Factor ◽  
Chromatin Remodeling ◽  
Single Molecule ◽  
Binding Sites ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Nucleosome Remodeling ◽  
Epistasis Analysis ◽  
Transcriptional Bursting ◽  
Kinetics Of

Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in budding yeast to measure how transcriptional bursting changes upon single and double perturbations of chromatin remodeling factors, the transcription factor Gal4 and preinitiation complex (PIC) components. Using dynamic epistasis analysis, we reveal how remodeling of different nucleosomes regulates individual transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC acts synergistically with Gal4 binding to facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. In the absence of remodelers, nucleosomes at canonical TATA boxes are displaced by TBP binding to allow for transcription activation. Overall, our results reveal how promoter nucleosome remodeling, together with transcription factor and PIC binding regulates the kinetics of transcriptional bursting.

scholarly journals Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Methods ◽  
2017 ◽  
Vol 123 ◽  
pp. 76-88 ◽  
Author(s):  
Diego M. Presman ◽  
David A. Ball ◽  
Ville Paakinaho ◽  
Jonathan B. Grimm ◽  
Luke D. Lavis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Molecule ◽  
Transcription Factor Binding ◽  
Live Cells ◽  
Single Molecule Level ◽  
Factor Binding

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance

2017 ◽  
Vol 46 (1) ◽  
pp. 23-35 ◽  
Author(s):  
Harshad Ghodke ◽  
Han Ho ◽  
Antoine M. van Oijen
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Live Cell Imaging ◽  
Spatial Organization ◽  
Cell Imaging ◽  
Live Cell ◽  
Live Cells ◽  
Temporal Regulation ◽  
Repair Processes ◽  
Wide Range

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA–repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA–repair processes in prokaryotes.

Sign in / Sign up

Export Citation Format

Share Document