Regulation of Cdc42-Dependent Signaling Pathways Throughout the Cell Cycle

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The long-term objective of this project is to understand the signal-transduction mechanisms that control cellular morphogenesis during the eukaryotic cell cycle. Normal morphogenesis, the process by which the complex, three-dimensional organization of the cell is developed and maintained, is essential for the fidelity of cellular differentiation and reproduction. The goal of this project is to decipher the signal-transduction mechanisms controlling cell polarity process in the yeast Saccharomyces cerevisiae. Polarized growth in response to different signals during the yeast cell cycle can result in the generation of several different morphological structures, such as buds, mating projections, and pseudohyphae. Two protein components of the cell polarity apparatus in yeast; the Ras-related GTPase Cdc42p and its guanine-nucleotide exchange factor (GEF) Cdc24p have previously been characterized in this laboratory. These components are integral parts of a signal-transduction pathway that leads to the generation of cell polarity during the cell cycle. These components have conserved counterparts in other eukaryotes, suggesting that common signal-transduction mechanisms controlling cell polarity may exist. The specific aims of this project are directed at understanding the regulation of the Cdc42p GTPase and how it interfaces with different cell-cycle regulatory processes. The hypotheses that will be tested with these experiments are: 1). Initiation of cell polarity signaling depends on localized targeting of Cdc24p but not Cdc42p; and 2). Cdc42p is activated and de-activated during the cell cycle. Cdc24p is targeted to the nucleus and sites of polarized growth at specific times in the cell cycle. However, recent data suggest a paradigm shift in which Cdc42p is localized around the entire periphery of the cell throughout the cell cycle and enriched at sites of polarized growth after activation. To better understand this critical regulatory process, the targeting mechanisms for both Cdc24p and Cdc42p will be determined using green fluorescent protein (GFP)-tagged wild-type and mutant proteins. S. cerevisiae Cdc42p is activated by the Cdc24p GEF and de-activated through the action of the Bem3p, Rga1p, and Rga2p GTPase-activating proteins (GAPs). Recent studies from this laboratory suggest that Cdc24p activates Cdc42p at multiple stages of the cell cycle, most probably prior to bud emergence in G 1 phase and cytokinesis in M phase. If so, then Cdc42p is likely to be de-activated in the intervening time frames by the action of its GAPs. The cell-cycle specific localization of individual GAPs will be investigated using GFP-tagged proteins. The answers to these questions will not only be relevant to the basic understanding of signal-transduction mechanisms in cell biology, but also to the understanding of the cellular morphogenesis process in yeast as well as in other eukaryotes.