Cell Size Control in Fission Yeast

Among all living organisms, there is almost much variety in cell size as there is for cell function and cell type. However, within each cell type, cells stay remarkably faithful to a defined size over generations. Many factors have been found to influence this ability to specify and maintain cell size, yet clear mechanisms have yet to be elucidated. The fission yeast Schizosaccharomyces pombe is an ideal model organism whose simple but conserved cell biology has led to the identification of many important cell size regulators common to all eukaryotes. In this thesis, I have quantitatively analyzed the dynamics and localization of several key players of cell size regulation, which lead to a new physical model on cell size regulation based on the localization and accumulation of a size sensing kinase cdr2p. In this model, cdr2p molecules accumulate in proportion to cell size into complexes called midsomes, which localize to the cortex at the central section of the cell. Upon reaching the desired cell size, cdr2p accumulation surpasses a concentration threshold and the cell will divide. This accumulation is partly facilitated by the key negative regulator pom1p, which prevents midsome formation at the cell tip. Evidence also suggests that the ER serves a role in confining midsome localization to the medial plasma membrane, perhaps by providing a physical link to the nucleus. Together, this work elucidates a mechanistic understanding of how cell size can be determined and controlled

A Computational Study of Elongation Factor G (EFG) Duplicated Genes: Diverged Nature Underlying the Innovation on the Same Structural Template

BACKGROUND: Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG's evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif "RGITI"; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV. CONCLUSIONS/SIGNIFICANCE: Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes

Nonabelian Hodge Theory in Characteristic p

Given a scheme in characteristic p together with a lifting modulo p^2, we construct a functor from a category of suitably nilpotent modules with connection to the category of Higgs modules. We use this functor to generalize the decomposition theorem of Deligne-Illusie to the case of de Rham cohomology with coefficients.Comment: Revised version. Chapter 3 is almost completely ne

Protein folding in crowded environments and living cells

Biomolecular dynamics and stability are predominantly investigated in vitro, and extrapolated to explain function in the living cell. In this thesis, we attempt to bridge this divide by performing studies of protein folding in in vitro crowded environments and in living cells. We begin by investigating the thermodynamic and kinetic behavior of the proteins in protein/carbohydrate matrices, and find significant differences compared to dilute, buffer solutions. We then develop Fast Relaxation Imaging (FReI) as a novel imaging technique to investigate protein folding dynamics inside living cells with millisecond temporal resolution and micrometer spatial resolution. We study the folding of the metabolic enzyme phosphoglycerate kinase (PGK) in U2OS (human osteosarcoma) cells and observe variations in folding kinetics within a single cell. FReI experiments carried out on an ensemble of 30 cells reveal large variations in folding time from cell to cell, which are analyzed to estimate the folding diffusion coefficient in vivo. Finally, we use short peptide tags to target PGK to the nucleus and endoplasmic reticulum of live cells and discern differential folding stability and kinetics in these organelles compared to protein folding in the cytoplasm