Dynamic Organization of Transcription Compartments Is Dependent on Functional Nuclear Architecture

Maharana, Shovamayee; Sharma, Divya; Shi, Xinli; Shivashankar, G. V.

doi:10.1016/j.bpj.2012.06.036

Cited by 15 publications

(21 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The bead diffusion data were in good agreement with single point confocal measurements (see Fig. S4 and Table S3; setup as described in Maharana et al (28)). Furthermore, the corresponding cumulative distribution function (CDF) for the fitted diffusion coefficients at room temperature ( Fig.…”

Section: Sospim-fcs Characterization and Validationsupporting

confidence: 82%

3D Protein Dynamics in the Cell Nucleus

Singh

Galland

Finch-Edmondson

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.

show abstract

Section: Sospim-fcs Characterization and Validationsupporting

confidence: 82%

3D Protein Dynamics in the Cell Nucleus

Singh

Galland

Finch-Edmondson

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

show abstract

“…The mean cage size is around nm and the TCs move through regions of low chromatin compaction (data not shown). This large scale movement of TCs was shown to be similarly affected by ATP depletion and perturbations to chromatin remodeling [5], [31]. Also, the movement characteristics of the chromatin itself has been shown to be affected by ATP depletion [35].…”

Section: Resultsmentioning

confidence: 97%

“…Cells were transfected at confluency in coverslip-bottomed 35 mm plates with of media. Under these conditions Lamin B1 decreased by after [2], [31]. Cells treated with the Lamin B1 siRNA were used after of transfection.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Passive and Active Particles in the Cell Nucleus

2012

View full text Add to dashboard Cite

Inspite of being embedded in a dense meshwork of nuclear chromatin, gene loci and large nuclear components are highly dynamic at C. To understand this apparent unfettered movement in an overdense environment, we study the dynamics of a passive micron size bead in live cell nuclei at two different temperatures ( and C) with and without external force. In the absence of a force, the beads are caged over large time scales. On application of a threshold uniaxial force (about 10 pN), the passive beads appear to hop between cages; this large scale movement is absent upon ATP-depletion, inhibition of chromatin remodeling enzymes and RNAi of lamin B1 proteins. Our results suggest that the nucleus behaves like an active solid with a finite yield stress when probed at a micron scale. Spatial analysis of histone fluorescence anisotropy (a measure of local chromatin compaction, defined as the volume fraction of tightly bound chromatin) shows that the bead movement correlates with regions of low chromatin compaction. This suggests that the physical mechanism of the observed yielding is the active opening of free-volume in the nuclear solid via chromatin remodeling. Enriched transcription sites at C also show caging in the absence of the applied force and directed movement beyond a yield stress, in striking contrast with the large scale movement of transcription loci at C in the absence of a force. This suggests that at physiological temperatures, the loci behave as active particles which remodel the nuclear mesh and reduce the local yield stress.

show abstract

“…The addressed topics include site specific interactions between nucleic acids and proteins [80,81], RNA interference mechanisms [85,86], transcrip-tion kinetics [82,89] or mechanisms of enzymatic DNA cleavage [87]. The addressed topics include site specific interactions between nucleic acids and proteins [80,81], RNA interference mechanisms [85,86], transcrip-tion kinetics [82,89] or mechanisms of enzymatic DNA cleavage [87].…”

Section: Protein-nucleic Acid Interactionsmentioning

confidence: 99%

“…Although not as numerous as FCS studies of protein-protein interactions, recent work using FCS to investigate protein-nucleic acid interactions demonstrate the broad range of question related to nucleic acids, which FCS can help to answer. The addressed topics include site specific interactions between nucleic acids and proteins [80,81], RNA interference mechanisms [85,86], transcrip-tion kinetics [82,89] or mechanisms of enzymatic DNA cleavage [87].…”

Section: Protein-nucleic Acid Interactionsmentioning

confidence: 99%

Recent applications of fluorescence correlation spectroscopy in live systems

2014

View full text Add to dashboard Cite

Fluorescence correlation spectroscopy (FCS) is a widely used technique in biophysics and has helped address many questions in the life sciences. It provides important advantages compared to other fluorescence and biophysical methods. Its single molecule sensitivity allows measuring proteins within biological samples at physiological concentrations without the need of overexpression. It provides quantitative data on concentrations, diffusion coefficients, molecular transport and interactions even in live organisms. And its reliance on simple fluorescence intensity and its fluctuations makes it widely applicable. In this review we focus on applications of FCS in live samples, with an emphasis on work in the last 5 years, in the hope to provide an overview of the present capabilities of FCS to address biologically relevant questions.

show abstract

Dynamic Organization of Transcription Compartments Is Dependent on Functional Nuclear Architecture

Cited by 15 publications

References 35 publications

3D Protein Dynamics in the Cell Nucleus

3D Protein Dynamics in the Cell Nucleus

Dynamics of Passive and Active Particles in the Cell Nucleus

Recent applications of fluorescence correlation spectroscopy in live systems

Contact Info

Product

Resources

About