Linda Huang, Ph.D. - US grants

DESCRIPTION (provided by applicant): Although most eukaryotic cells contain similar parts, these parts are differentially arranged in different cell types. Furthermore, cells are dynamic, rearranging their internal structures in response to the environment and during developmental processes. Cellular architecture is important for cellular function. This application proposes to use S. cerevisiae to examine how cellular organization is established and maintained by studying the complex internal rearrangements that occur during spore morphogenesis, when a single diploid cell is transformed into an ascus containing four haploid spores. The long-term goal is to understand how cells regulate their internal architecture. This research seeks to understand how the structures built during spore development are organized. The proposal focuses on understanding the function of SPO71, which encodes a PH domain-containing protein that is essential for proper spore wall morphogenesis. SPO71 is thought to be important for the organization rather than the production of the spore wall because spore wall components are made in spo71 mutants but fail to localize correctly. The study of SPO71 should yield insights into how spatial organization is established and maintained during sporulation. We will understand how SPO71 contributes to spore morphogenesis by further characterizing the defects seen in cells lacking SPO71 through the use of molecular markers that allow for the visualization of structures inside sporulating yeast cells. Experiments are also proposed to examine the potential role of the Spo71p PH domains and N-terminus in controlling Spo71p function. Finally, this proposal contains experiments designed to identify molecules that work in concert with SPO71 to generate proper spatial organization during spore development. The experiments in this proposal should provide information about the organization of spore morphogenesis and insights into how a developmentally regulated PH domain containing protein is utilized to regulate cellular architecture. PUBLIC HEALTH RELEVANCE: Understanding how cells regulate their internal architecture is important for understanding cellular function. This is important for human health because cells in disease states frequently take on morphologies distinct from healthy cells and these morphological changes frequently underlie pathological diagnosis.

DESCRIPTION (provided by applicant): The goal of this project is to understand how membrane morphology is regulated by the septin cytoskeleton and by lipid composition. To study this question, we use the S. cerevisiae prospore membrane as a model. During sporulation, four new cells are generated within the mother cell as the prospore membrane grows to encapsulate the meiotic nuclei. Ultimately, the prospore membrane will act as the template for the deposition of the spore wall, and its inner leaflet will become the plasma membrane for the new haploid cells. Synthesis of the prospore membrane is essential for yeast to form functional, environmentally resistant spores. Our long-term goal is to understand the regulation of cellular architecture. The proposed research will contribute to our understanding of the molecular mechanisms important for shaping a cellular structure (the prospore membrane) important for the integrity and form of the yeast's meiotic products. The work in this proposal wil provide insights into fundamental aspects of cell biology concerning how cells and cellular structures acquire their shapes, and will also contribute to our understanding of septins, which are filament- forming proteins associated with membranes found in fungi and animals. In the previous funding period, the Spo71 protein was found to be important for determining the size of the prospore membrane and to be required for proper localization of septins along the prospore membrane. Preliminary studies suggest SPO71 is important in the elongation phase of prospore membrane development. The proposed experiments specifically examine the role of septins and septin regulators in prospore membrane development, and also examine how membrane lipid composition is regulated and contributes to prospore membrane morphogenesis. These studies will distinguish models for how these proteins work together to regulate PSM size and shape. As septins are an evolutionarily conserved protein family vital for a variety of cellular processes (including cell division, immune function, and neuronal development) and because the effect of membrane composition on membrane morphology is a fundamental issue in cell biology, the findings from these proposed studies should be of interest to a wide range of biologists.

Project Summary: This project will examine the cellular events that occur at the end of meiosis, to better understand how these events are coordinated and regulated. Meiosis is a specialized cell division used to produce haploid cells from diploid cells. Although some aspects of meiosis are regulated as in mitotic cells, some parts of meiosis are regulated using distinct mechanisms. In the budding yeast Saccharomyces cerevisiae, meiosis occurs during sporulation, where a diploid mother cell will remodel its interior through meiosis and spore morphogenesis to create four haploid spores. As cells prepare to complete meiosis II, spindle disassembly and cytokinesis must be coordinated. In the budding yeast, meiotic cytokinesis takes place through the closure of the prospore membrane, a membrane that is synthesized during sporulation and grows to surround the newly formed 1N DNA products of meiosis. Meiosis II spindle disassembly and timely prospore membrane closure are regulated by Cdc15 (a Hippo-like kinase) acting upstream of Sps1 (a STE20-family GCKIII kinase). The regulation of exit from meiosis II is distinct from the regulation of exit from mitosis. This project will examine how cytokinesis and spindle disassembly are coordinated in meiosis II, and will also identify additional component important for regulating the exit of meiosis II. The experiments in this proposal will use yeast molecular genetics, biochemistry, and imaging studies to provide a more detailed understanding of meiosis II spindle disassembly, how this is coordinated with timely prospore membrane closure, and the molecular targets used to regulate these processes. The work in the proposal will also begin to define other components involved in regulating exit of meiosis II, and will provide a better understanding of the the signaling pathway using Cdc15 and Sps1. The knowledge gained from this project will contribute to the fundamental understanding of meiotic cell cycle regulation and how cellular processes are coordinated. These studies will also contribute to our knowledge of fungal biology, which is important because fungi are commensal organisms (as part of the fungal microbiome) and can also become pathogenic.