SIGNALING MECHANISMS IN CELL POLARITY IN YEAST

我的长期目标是使用遗传上易驯化的真核生物酿酒酵母来确定CDC42P，一种进化上保守的GTP酶，如何控制肌动蛋白细胞骨架和间隔蛋白的组织。最近的工作表明，CDC42p通过两条平行的途径控制肌动蛋白的组织：一条涉及Msb3p、Msb4p和Bnilp；另一条涉及Gic1p、Gic2p和Bem4p。在目前的研究中，这些途径将被进一步探索，最初的重点是Msb3p和Msb4p的作用。Msb3p和Msb4p是酵母中一对高度同源的蛋白质，它们在连接CDC42P和肌动蛋白细胞骨架的过程中扮演着多余的角色。它们属于一个蛋白质家族，包括果蝇细胞黏附分子Pollux和人类癌蛋白Tre17。因此，研究Msb3p和Msb4p的功能将有助于了解这一蛋白家族的功能。具体地说，这项提案的研究将解决以下问题：是什么蛋白质将Msb3p和Msb4p与上游的CDC42p和下游的肌动蛋白细胞骨架联系起来？MSB介导的途径中的蛋白质是如何在分子水平上组织起来的？Msb3p和Msb4p中可能的结构域(高度保守的PTM结构域、跨膜结构域和脂质修饰位点)有什么功能？Msb3p和Msb4p在细胞周期中的定位是如何调节的？这些问题将通过遗传学、细胞学和生化方法的组合来回答。为了验证我们的假设，我们将通过基因筛选和亲和层析与串联质谱仪相结合的方法来鉴定参与调控CDC42P活性或特定效应途径的其他基因。CDC42P的同系物参与多种细胞过程，如细胞极性、细胞迁移和细胞生长控制。此外，哺乳动物中CDC42p活性的解除调控与癌症等严重疾病有关。因此，研究酵母中CDC42P的信号转导机制是连接基础科学和临床科学的重要桥梁。

My long-term objective is to use the genetically tractable eukaryote Saccharomyces cerevisiae to determine how Cdc42p, an evolutionarily conserved GTPase, controls the organization of the actin cytoskeleton and of the septins. Recent work suggests that Cdc42p controls the actin organization by two parallel pathways: one involving Msb3p, Msb4p, and Bnilp; the other involving Gic1p, Gic2p, and Bem4p. In the present studies, these pathways will be explored further, focusing initially on the roles of Msb3p and Msb4p. Msb3p and Msb4p, a pair of highly homologous proteins in yeast, play a redundant role in linking Cdc42p to the actin cytoskeleton. They belong to a family of proteins that includes the Drosophila cell adhesion molecule, Pollux, and the human oncoprotein, Tre17. Thus, studying the function of Msb3p and Msb4p will shed significant light on the function of this family of proteins. Specifically, research in this proposal will address the following questions: What proteins link Msb3p and Msb4p to the upstream Cdc42p, and to the downstream actin cytoskeleton? How are the proteins in the Msb-mediated pathways organized at the molecular level? What are the functions of the putative domains in Msb3p and Msb4p (the highly conserved PTM domain, the membrane-spanning domains, and a lipid modification site)? How are the localization of Msb3p and Msb4p regulated in the cell cycle? These questions will be answered with a combination of genetic, cytological, and biochemical approaches. To exapnd our hypothesis, additional genes that are involved in regulating Cdc42p activity or a specific effector pathway will be identified by genetic screens, and affinity chromatography coupled with tandem mass spectrometry. Homologs of Cdc42p are involved in diverse cellular processes, such as cell polarity, cell migration, and cell growth control. In addition, deregulation of Cdc42p activity in mammals is associated with serious diseases, such as cancer. Thus, studying the signaling mechanisms of Cdc42p in yeast represents an important bridge between basic and clinical sciences.