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  • Book
    Stephanie C. Weber.
    The cellular interior is crowded with macromolecules that form highly organized but dynamic structures. While these properties of the cytoplasm have become well known, their physical consequences on intracellular processes such as transport and gene expression are still not well understood. The goal of this dissertation is to begin to elucidate these physical consequences and to develop a new model for macromolecular motion in vivo. Tracking of fluorescently labeled chromosomal loci in live bacterial cells reveals a robust subdiffusive scaling of the mean square displacement (MSD). To identify the source of anomalous behavior, we developed analytical theory and Brownian dynamics simulations of polymers under various physical environments. Specifically, we examined the consequences of confinement, self-interaction, viscoelasticity and random waiting on monomer motion. We found that neither confinement nor self-interaction alter the fundamental Rouse mode relaxations of a polymer. However, viscoelasticity, modeled by fractional Langevin motion (fLm), and random waiting, modeled with a continuous time random walk (CTRW), led to significant and distinct deviations from the classic polymer dynamics model. We propose that the subdiffusive motion of chromosomal loci observed in vivo arises from relaxation of the Rouse modes of the DNA polymer within the viscoelastic environment of the cytoplasm. The time-averaged and ensemble-averaged MSD of chromosomal loci exhibit ergodicity, and the velocity autocorrelation function is negative at short time lags. These observations are most consistent with fLm and rule out a CTRW model as an explanation for anomalous motion in vivo. In contrast to the subdiffusive scaling exponent, the apparent diffusion coefficient is variable across species and sensitive to biological perturbations. When ATP synthesis is inhibited in E. coli, the apparent diffusion coefficient decreases 2-fold while the scaling exponent remains constant. This observation suggests that metabolic activity contributes to the magnitude of locus motion, but does not affect the subdiffusive mechanism (or the viscoelastic properties of the cytoplasm). Furthermore, the temperature-dependence of locus motion is steeper in untreated cells than in ATP-depleted cells. These results suggest that untreated cells have an additional source of molecular agitation that increases with temperature. Such ATP-dependent fluctuations are likely mechanical, as the heat dissipated from glycolysis and oxidative phosphorylation is insufficient to account for the difference in the apparent diffusion coefficient between untreated and ATP-depleted cells. We are currently investigating the dynamic contributions of several molecular candidates. Our data indicate that ATP-dependent enzymatic activity, in addition to thermal fluctuations, may contribute to the molecular agitation driving (sub)diffusion in the cell.