The development and implementation of microscopy strategies for investigating protein diffusion and chromatin binding Public Deposited

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  • March 22, 2019
Creator
  • Tycon, Michael August
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Nearly all cellular processes, notably transcription, translation, and genomic repair, are enacted by multiprotein complexes that coalesce into functional assemblies in response to constantly fluctuating cellular demands. A complex interplay of endogenous and exogenous cellular cues regulates the assembly and activity of these complexes by both active and passive mechanisms, with a current fundamental dilemma in the field of molecular biology being the elucidation of the mechanisms governing the assembly of these supramolecular complexes. Such complexes arise through two processes, the nucleation of macromolecular assemblies and target binding site recognition. Collectively, this phenomenon is anthropomorphized asprotein recruitment, yet this term conceals the underlying physical interactions that govern the spatiotemporal formation of such assemblies, turning protein activity into a series of black boxes with prescribed functions. In response to this overarching question, microscopy technologies were tailored to investigate the mechanisms of these two inextricable facets of protein recruitment. Thus, during my tenure in the Fecko Laboratory, I have been concerned with the big picture while simultaneously looking at the very small. Methods were developed enabling the observation of model systems of complex recruitment dynamics and have been used to illustrate paradigms of biological function. An initial effort was focused on designing optical systems for observation of DNA repair protein diffusion. The ability to generate user-defined DNA photolesions in real time, a highly characterized binding site of many classes of DNA repair proteins, creates opportunities for optical imaging experiments in which protein behavior before and after a biological perturbation can be observed. To this end a two-photon DNA damage method was developed, which enabled the production of UV-type DNA photolesions by blue light and is highly compatible with conventional laser-scanning optical microscopy configurations. This visible light damage method was compared to alternative damage induction processes, and the advantages of the two-photon method enumerated. Continuing towards an integrated system for observing protein diffusion, a popular single-molecule imaging DNA immobilization and visualization technique was characterized. In this work, the extent of optically-induced DNA binding site artifacts was established with a unique pairing of a widefield microscopy based single-molecule and gel electrophoresis based ensemble biochemical DNA damage assays. The results indicated that many commonly used DNA visualization practices, from imaging parameters through fluorescent intercalaters, lead to extensive photodamage and can perturb native DNA-protein interactions. Later work shifted away from single molecule investigations and towards studying the diffusion dynamics of large macromolecular complexes in vivo. A unique two-photon FRAP microscopy and image processing technique was developed and used to characterize the diffusion of RNA Polymerase II subunits in live cell nuclei. The findings substantiate a hybrid model of macromolecular assembly in which a broad distribution of macromolecular species allow for mechanistic flexibility in the assembly of transcription complexes. This provides evidence for further speculation on mechanisms controlling gene expression.
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  • In Copyright
Advisor
  • Fecko, Christopher J.
Degree
  • Doctor of Philosophy
Graduation year
  • 2013
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