Biomedical subjects

G V Shivashankar

Publications and source records attributed to G V Shivashankar.

10 recordsLinked to original sources

PubMed2024

Remodeling of cytoskeleton, chromatin, and gene expression during mechanical rejuvenation of aged human dermal fibroblasts.

Aging is associated with a progressive decline in cellular function. To reset the aged cellular phenotype, various reprogramming approaches, including mechanical routes, have been explored. However, the epigenetic mechanisms underlying cellular rejuvenation are poorly understood. Here, we studied the cytoskeletal, genome-wide chromatin and transcriptional changes in young, aged, and mechanically rejuvenated fibroblasts using immunofluorescence, RNA sequencing, and Hi-C experiments. The mechanically rejuvenated aged fibroblasts, that had partially reset their transcription to a younger cell state, showed a local reorganization of the interchromosomal contacts and lamina-associated domains. Interestingly, the observed chromatin reorganization correlated with the transcriptional changes. Immunofluorescence experiments in the rejuvenated state confirmed increased actomyosin contractility like younger fibroblasts. In addition, the rejuvenated contractile properties were maintained over multiple cell passages. Overall, our results give an overview of how changes in the cytoskeleton, chromatin, and gene activity are connected to aging and rejuvenation.

Humans↗

PubMed2006

Direct measurement of local chromatin fluidity using optical trap modulation force spectroscopy.

Chromatin assembly is condensed by histone tail-tail interactions and other nuclear proteins into a highly compact structure. Using an optical trap modulation force spectroscopy, we probe the effect of tail interactions on local chromatin fluidity. Chromatin fibers, purified from mammalian cells, are tethered between a microscope coverslip and a glass micropipette. Mechanical unzipping of tail interactions, using the micropipette, lead to the enhancement of local fluidity. This is measured using an intensity-modulated optically trapped bead positioned as a force sensor on the chromatin fiber. Enzymatic digestion of the histone tail interactions of tethered chromatin fiber also leads to a similar increase in fluidity. Our experiments show that an initial increase in the local fluidity precedes chromatin decompaction, suggesting possible mechanisms by which chromatin-remodeling machines access regulatory sites.

Chromatin↗

PubMed2006

EGFP-tagged core and linker histones diffuse via distinct mechanisms within living cells.

The effect of chromatin organization on EGFP-tagged histone protein dynamics within the cell nucleus has been probed using fluorescence correlation and recovery measurements on single living HeLa cells. Our studies reveal that free fraction of core-particle histones exist as multimers within the cell nucleus whereas the linker histones exist in monomeric forms. The multimeric state of core histones is found to be invariant across mammalian and polytene chromosomes and this is ATP dependent. In contrast, the dynamics of the linker histones exhibits two distinct diffusion timescales corresponding to its transient binding and unbinding to chromatin governed by the tail domain residues. Under conditions of chromatin condensation induced by apoptosis, the free multimeric fraction of core histones is found to become immobile, while the monomeric linker histone mobility is partially reduced. In addition, we observe differences in nuclear colocalization of linker and core particle histones. These results are validated through Brownian dynamics simulation of core and linker histone mobility. Our findings provide a framework to understand the coupling between the state of chromatin assembly and histone protein dynamics that is central to accessing regulatory sites on the genome.

Adenosine Triphosphate↗

PubMed2006

Chromatin structure exhibits spatio-temporal heterogeneity within the cell nucleus.

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.

Adenosine Triphosphate↗

PubMed2005

Stochastic simulations of the origins and implications of long-tailed distributions in gene expression.

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.

Animals↗

PubMed2004

Tracking operator state fluctuations in gene expression in single cells.

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.

Anisotropy↗

PubMed2004

Dynamics of membrane nanotubulation and DNA self-assembly.

A localized point-like force applied perpendicular to a vesicular membrane layer, using an optical tweezer, leads to membrane nanotubulation beyond a threshold force. Below the threshold, the force-extension curve shows an elastic response with a fine structure (serrations). Above the threshold the tubulation process exhibits a new reversible flow phase for the multilamellar membrane, which responds viscoelastically. Furthermore, with an oscillatory force applied during tubulation, broad but well-resolved resonances occur in the flow phase, presumably matching the time scales associated with the vesicle-nanotubule coupled system. These nanotubules, anchored to the optical tweezer also provide, for the first time, a direct probe of the real-time dynamics of DNA self-assembly on membranes. Our studies are a step in the direction of analyzing the dynamics of membrane self-assembly and artificial nanofluidic membrane networks.

Biosensing Techniques↗

PubMed2003

Single particle tracking of correlated bacterial dynamics.

Pattern formation in 3D random media has been a topic of interest in soft matter and biological systems. However, the onset of long-range microscopic ordering has not been explored in randomly moving self-propelled particles due to a lack of model systems as well as local probe techniques. In this article, we report on a novel experiment, using motile Escherichia coli bacteria as a model system, to study the onset of dynamic correlation and collective movement in three-dimension. We use fluctuation of an optically trapped micron-size bead as a detector of correlated bacterial motion, and further study this behavior by analyzing the motility of fluorescent bacteria in a confocal volume. We find evidence of dynamic correlation at very low volume fractions (0.01). We show that the magnitude of this correlation strongly depends on the interbacterial distances and their coupling modes. This opens up possibilities to probe long-range pattern formation in actively propelled cells or organisms coupled through hydrodynamics and/or chemical signaling.

Escherichia coli↗

PubMed2000

Method for linking a synthesized protein to its mRNA-DNA complex.

A nascent protein remains in a complex with its ribosome and mRNA if the stop codon is deleted from the mRNA. In the same manner, mRNA forms a stable complex with DNA if the transcription termination is blocked. In principle, if both mRNA translation and DNA transcription termination are prevented, the protein should stay in a complex with its mRNA and DNA. A method is designed to test these possibilities. Using an immobilized luciferase gene sequence, a functional luciferase protein is produced that remains associated through its mRNA with its DNA, confirming the feasibility of the proposed scheme. It has potential application for in vitro synthesis of proteins and protein micro-arrays.

DNA↗

PubMed1999

RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis.

The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.

Adenosine Triphosphate↗