Forward genetic analysis of lysosome-related organelle formation in Tetrahymena thermophila

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The overall project objective is to develop a deeper understanding of the cellular mechanisms that have evolved to allow cells to interact, in sophisticated ways, with their surroundings. In particular, the project will investigate a feature of eukaryotic cells called lysosome-related organelles (LROs), by using genetic approaches in a single celled organism called Tetrahymena thermophila. The project will significantly enhance the tools that are available to perform advanced genetics using Tetrahymena, and will allow students at several different levels to be trained in using those tools. While the project is focused on using these genetic tools to understand one aspect of cellular organization in Tetrahymena, the findings will be highly relevant to broad questions about how cells in general have become specialized to perform their many functions. In addition, the tools that are being developed can be applied in the future to probing a wide array of cellular features. Findings from this project may lead to patenting and commercialization of genetic modifications to Tetrahymena, to make this organism a more efficient tool for commercial production of proteins and lipids. The project will involve students from a wide variety of backgrounds, including students from groups that are currently under-represented in science. The project will hone a forward genetic approach to dissecting the biosynthesis of LROs. LROs are ubiquitous and heterogeneous eukaryotic organelles that provide a very wide range of functions in cellular physiology and intercellular communication, and whose evolution appears to have been a major driver of diversification in the secretory pathway. The project will enhance the understanding of mechanisms involved in LRO formation, by identifying genes required for this pathway in Tetrahymena. The approach takes advantage of a high-throughput screen to isolate Mendelian mutants, following chemical mutagenesis, that are defective in LRO formation. Those mutants will be characterized using cell biological and classical genetic approaches. Following that analysis, the mutant genomes will be sequenced, after employing an outcross and backcross strategy to generate pools of F2 meiotic segregants from each initial mutant. By taking this strategy, one can greatly simplify the problem of identifying the causative mutation in each mutant, and thereby identifying the gene required for LRO formation in that strain. It is anticipated that approximately 10 mutants will be analyzed in this fashion, to the level of identifying the causative genetic lesions.