Large-scale Analysis of Filamentous Growth in Saccharomyces cerevisiae and the Related Human fungal Pathogen, Candida albicans.

Abstract

Many diverse fungal species are dimorphic, shifting between a cellular yeast-like form and a filamentous invasive form in response to specific cellular and environmental cues. My research is directed towards identifying the molecular mechanisms driving this dimorphism in the budding yeast Saccharomyces cerevisiae and in the related human pathogen Candida albicans. In S. cerevisiae, nitrogen stress initiates a developmental program characterized by the formation of filamentous chains of cells, called pseudohyphae. Pseudohyphal growth is regulated transcriptionally and post-transcriptionally, and regulated protein localization is an important mechanism for the post-transcriptional control of gene function. To determine the extent to which protein compartmentalization is regulated during pseudohyphal growth, I constructed a unique set of 125 protein kinase-yellow fluorescent protein fusions and screened these proteins for differential localization during pseudohyphal growth. In total, we identified six cytoplasmic kinases (Bcy1p, Fus3p, Ksp1p, Kss1, Sks1p, and Tpk2p) that localize predominantly to the nucleus during filamentous growth. These kinases form part of an interdependent, localization-based regulatory network, since deletion of each individual kinase disrupts the nuclear translocation of at least two other kinases. In a separate set of experiments, I further investigated the regulation of kinase function in the related yeast Candida albicans by performing a synthetic genetic screen for interactors of the kinase Cbk1p. Cbk1p is a critical kinase in the RAM network (Regulation of Ace2p and Morphogenesis). Specifically, a C. albicans strain heterozygous at CBK1 was subjected to transposon mutagenesis in order to generate double heterozygotes. These double mutants were screened for an exaggerated decrease in hyphal formation relative to the cbk1 mutant, indicative of a genetic interaction with CBK1. From this large-scale analysis of 6,528 mutants, we identified a set of 44 genes that genetically interact with CBK1. This gene set encompasses 17 putative targets (12 of which are new) of the Cbk1p-dependent transcription factor Ace2p and a large cohort of cAMP-PKA regulated genes, indicating that the RAM and cAMP-PKA pathways may interact during hyphal development. Collectively, my research provides fundamental insight into the regulatory mechanisms governing fungal dimorphism and a set of interesting filamentous growth genes for additional follow-up analysis.