Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biology

Supervisor

Karagiannis, Jim

Abstract

The mechanisms that ensure cytokinetic fidelity are important for cellular proliferation and the maintenance of genomic integrity. To better understand these mechanisms, the actin depolymerizing drug, LatA, was previously used to screen for fission yeast genes required to prevent cytokinesis failure. This screen identified the pna1 polyamine-N-acetyltransferase. Here, I show that pna1Δ mutants display severe growth defects when exposed to even low doses of LatA. Furthermore, LatA-treated pna1Δ cells display increased rates of cytokinesis failure characterized by fragmentation of the cytokinetic actomyosin ring. Surprisingly, Pna1-GFP fusion proteins form cytoplasmic filaments that may represent cytoophidia (meaning "cellular snakes"), a class of structures implicated in metabolic regulation via compartmentalization. Interestingly, Pna1p filament length is dependent on growth phase, but is not affected by inhibition of the TOR pathway. I suggest that fission yeast respond to LatA-induced cytoskeletal perturbations through modulating polyamine interaction with actin via a mechanism involving Pna1p-mediated polyamine acetylation.

Summary for Lay Audience

Cells division is essential for the growth and development of living organisms. The process of cell division has many steps, the last being "cytokinesis", which involves the physical separation of a mother cell into two independent daughter cells. When cytokinesis fails, it leads to the formation of cells with an abnormal number of nuclei. This can result in genetic instability, which in turn contributes to tumorigenesis. Hence, understanding cytokinesis is of fundamental importance. One way to understand this process is to disrupt cytokinesis and observe how the cell reacts to the disturbance. For example, a drug called LatA can be used as a tool to perturb the cytokinetic machinery and to identify genes that are required to counter this stress. Using this strategy, a group of genes required for growth in the presence of LatA was identified in fission yeast (a simple, single-celled model organism that is often utilized by researchers to better understand basic biological processes). One gene from this group, pna1 encodes a protein that modifies an important class of cellular molecules called polyamines, which are known to interact with a variety of negatively charged molecules, including actin.

To better understand the role of the Pna1p protein in responding to LatA-induced celldivision stress, I assayed cytokinesis failure rates using fluorescence microscopy. Unlike normal cells, cells lacking Pna1p display dramatically increased rates of failure. Furthermore, time-lapse imaging of live cells revealed that this failure was due to a severe fragmentation of the actomyosin ring (a contractile structure crucial for cytokinesis). Surprisingly, I also show that Pna1p forms unusual cytoplasmic filaments that may represent a novel example of a class of structures termed cytoophidia (meaning "cellular snakes"). These structures have been identified in organisms ranging from bacteria to humans and may play a role in metabolic regulation via the compartmentalization of proteins. Interestingly, the length of Pna1p-cytoophidia are dependent on growth phase, as I show that cells that are not actively growing display longer filaments than those that are actively growing. The continued characterization of this structure and its role in cytokinesis may provide further insight into the cellular mechanisms preventing cytokinesis failure.

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