[Show abstract][Hide abstract] ABSTRACT: The three-dimensional organization of nuclear compartments within living cells determines genome function and yet their underlying self-organizing principles are unclear. We visualize in real-time transcriptionally active compartments (TCs) by the transient enrichment of fluorescently-labeled uridine 5'-triphosphate molecules within living cells. These TCs partially colocalize with active RNA-Pol II in the cell nucleus. Fluorescence anisotropy maps of chromatin compaction evidences a more open chromatin structure at the TCs. Using live-cell timelapse imaging, heterogeneity in the dynamic behavior of TCs has been revealed which falls into three distinct classes: subdiffusive, super-diffusive, and normal diffusive behavior. In contrast, the mobility of a candidate gene locus, either in the repressed or activated state, undergoes a differential restricted motion that is coupled to TC movement. Further TC dynamics is directly affected by small molecule chromatin structure modulators and adenosine triphosphate depletion. This heterogeneous behavior in TC dynamics within living cells could provide an interesting paradigm to explore the spatiotemporal dimension to gene transcription control.
Full-text · Article · Oct 2008 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: Trichostatin-A (TSA), a histone deacetylase (HDAC) inhibitor, results in enhanced acetylation of core histones thereby disrupting chromatin organization within living cells. We report on changes in chromatin organization and the resultant alteration in nuclear architecture following treatment with TSA using fluorescence imaging. TSA triggers an expected increase in the euchromatin fraction which is accompanied by a significant increase in nuclear volume and alterations in chromatin compaction mapped using fluorescence anisotropy imaging. We observe differential changes in the mobility of core and linker histones as measured by fluorescence recovery after photo-bleaching (FRAP) and fluorescence correlation spectroscopy (FCS) methods. Further TSA induces a differential increase in linker histone transcription and increased phosphorylation of linker histone proteins accompanying an expected increase in core histone acetylation patterns. Thus subtle feedback responses triggered by changes in chromatin configurations impinge selectively on linker histone mobility and its expression. These observations have implications for understanding the role of HDAC in the dynamic maintenance of chromatin organization.
Preview · Article · Dec 2007 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Local chromatin compaction undergoes dynamic perturbations to regulate genetic processes. To address this, the direct measurement of the fluidity of chromatin structure is carried out in single live cells using steady-state anisotropy imaging and polarization modulation microscopy. Fluorescently tagged core and linker histones are used to probe different structural aspects of chromatin compaction. A graded spatial heterogeneity in compaction is observed for the chromatin besides the distinct positional ordering of core and linker histones. These spatio-temporal features are maintained by active processes and perturbed during death. With cell cycle, the distribution in compaction heterogeneity continually changes maximizing during M-G1 transition where it displays bimodal behavior. Such measurements of spatio-temporal chromatin fluidity could have broader implications in understanding chromatin remodeling within living cells.
Full-text · Article · Oct 2006 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: Gene expression noise results in protein number distributions ranging from long-tailed to Gaussian. We show how long-tailed distributions arise from a stochastic model of the constituent chemical reactions and suggest that, in conjunction with cooperative switches, they lead to more sensitive selection of a subpopulation of cells with high protein number than is possible with Gaussian distributions. Single-cell-tracking experiments are presented to validate some of the assumptions of the stochastic simulations. We also examine the effect of DNA looping on the shape of protein distributions. We further show that when switches are incorporated in the regulation of a gene via a feedback loop, the distributions can become bimodal. This might explain the bimodal distribution of certain morphogens during early embryogenesis.
Full-text · Article · Apr 2005 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: We have constructed a confocal fluorescence microscope combined with force measurements. Our method allows for simultaneous measurements of fluorescence anisotropy, energy transfer and correlation. The methodology and the sensitivity of the set-up using enhanced green fluorescent protein are described. We present results on (a) the detection of mRNA polymerization during in vitro transcription using fluorescence correlation spectroscopy and anisotropy, (b) detection of in vivo protein-DNA interactions using fluorescence anisotropy and (c) nanomanipulation of polytene chromosomes using the micropippete force sensor. Such a combined method allows for probing novel structure- function relationship underlying gene-expression.
[Show abstract][Hide abstract] ABSTRACT: We report the results of operator state fluctuations in gene expression for the entire bacterial growth cycle, using single-cell analysis and synthetic unregulated and negative-feedback transcription regulatory gene circuits. In the unregulated circuit, during the cell cycle, we observe a crossover from log-normal-to-normal distribution of expressed proteins and an unusual linear dependence of their standard deviation on the mean gene expression levels. With negative-feedback circuits we find the existence of bimodality as the cell cycle progresses. We suggest that such long-tail and bimodal distributions may be used as selection mechanisms in developmental switches and for assigning cell identity.
Preview · Article · Jun 2004 · Biophysical Journal