Experimental Study of the Molecular Basis for Chromatin Motion in Live Cells and Model Systems

University Of California-Riverside, Riverside CA

Investigators

Abstract

In the nucleus of a live cell, small structural fluctuations in the DNA-containing chromatin provide transient opportunities for protein binding, and thus comprise the initial chemical steps in gene expression. It is therefore important to characterize the small-scale motions of chromatin inside the nuclei of live cells, with the ultimate aim of being able to observe in vivo transcriptional activity in real time. To accomplish this, a combination of standing wave microscopy and two-photon patterned photobleaching is employed to attain high spatial sensitivity to translational motion inside living cells. Using this method, diffusive displacements of less than 100 nm can be measured, and the motion of intranuclear DNA labeled with the fluorophore Hoechst 33342 has been observed in live cells. The goal is to correlate the amount of mesoscopic, 100 nm motion with the presence of specific nanoscale biochemical processes like histone binding and unbinding, which are important for determining the transcriptional availability of DNA. To relate the two types of dynamics, a two channel diffusion experiment will be developed to measure the dynamics of chromatin and GFP-labeled histone proteins in parallel. In addition, the effects of other environmental factors like acetylation and the presence of other types of nuclear proteins will be assessed. Finally, the effects of chromatin motion on the diffusion of other macromolecular solutes in the nucleus will be measured. This will be done by using inert probes and measuring their diffusivity as a function of chromatin conformational freedom. By studying chromatin dynamics in both live cells and isolated nuclei under a variety of conditions, it should be possible to bridge the gap between in vitro studies of molecular-level nucleosome dynamics and in vivo studies of chromatin diffusion on mesoscopic lengthscales. This project has as its goal enabling investigators to see the movements of chromatin and proteins during transcription; this ability should greatly increase their understanding of gene transcription and its regulation. In order to make the research accessible to the general public, a portable demonstration will be created that will allow the visualization of single, giant DNA molecules in a classroom environment, with the aim of introducing beginning science students to experimental biophysical research, both at the college and high school levels. The outreach project, which involves the construction of a specialized fluorescence microscope, will be carried out by a team of undergraduate science majors under the supervision of the principal investigator. This research is being jointly funded by the Physics Division and the Office of Multidiscipinary Activities in the Mathematical and Physical Sciences Directorate, and by the Molecular and Cellular Biosciences Division in the Biological Sciences Directorate.