Fluorescent microscopy in the nucleus: Investigating protein diffusion and binding in live cells.

摘要

One of the open questions in biophysics is the process by which DNA-binding proteins, transcription and repair proteins, find very specific binding sites that are minuscule in comparison to the size of the genome. In vitro results have provided some insight into the search mechanism; however, these studies simulate neither the complicated DNA topography nor the crowded macromolecular environment present inside live cells. The focus of this dissertation is the development of microscope technology and experiments that build toward the ultimate goal of probing DNA-binding protein transport and binding in live cells.;Fluorescence recovery after photobleaching (FRAP) is used to study anomalous diffusion of unconjugated green fluorescence protein (GFP) in the polytene cells of Drosophila larval salivary glands. Polytene nuclei contain optically resolvable chromosomes, permitting FRAP experiments that focus separately on chromosomal or interchromosomal regions. GFP exhibits anomalous diffusion in the chromosomal regions, but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin is the source of anomalous diffusion in polytene nuclei and likely other cells. In vitro studies of GFP diffusion in artificial crowding environments confirm normal diffusive behavior in crowded environments similar to the interchromatin space. The diffusion dynamics of two RNA Polymerase II subunits in the interchromatin space exhibit anomalous diffusion in direct contrast to the normal diffusion of unconjugated GFP. This apparent anomalous diffusion in both unengaged subunits is a result of the subunits' incorporation into a broad distribution of complexes, with sizes ranging from free proteins to fully assembled gene transcription units. The broad distribution of macromolecular species allows for mechanistic flexibility in the recruitment of RNA Polymerase II.;Perturbations of the DNA environment with an optical microscope, such as generating well-defined regions of ultraviolet (UV)-like photolesions, assist investigations into the spatiotemporal dynamics of a class of DNA-binding proteins, DNA repair proteins. The production of thymine cyclopyrimidine dimers, the primary UV DNA photoproduct, is demonstrated using multiphoton excitation of DNA in live cells with visible light pulses. The spatiotemporal recruitment of GFP-tagged topoisomerase I to sites of localized DNA damage is investigated through this method.