SIGNALING MECHANISMS IN CELL POLARITY IN YEAST

我的长期目标是使用遗传上易处理的真核生物酿酒酵母来确定Cdc 42 p，一种进化上保守的GTdR，如何控制肌动蛋白细胞骨架和septins的组织。 最近的研究表明，Cdc 42 p通过两条平行的途径控制肌动蛋白的组织：一条涉及Msb 3 p，Msb 4p和Bnilp;另一条涉及Gic 1 p，Gic 2 p和Bem 4p。 在本研究中，这些途径将进一步探讨，最初集中在Msb 3 p和Msb 4p的作用。Msb 3 p和Msb 4p是酵母中一对高度同源的蛋白质，在将Cdc 42 p连接到肌动蛋白细胞骨架中起冗余作用。 它们属于一个蛋白质家族，包括果蝇细胞粘附分子Pollux和人类癌蛋白Tre 17。 因此，研究Msb 3 p和Msb 4p的功能将对该蛋白家族的功能有重要意义。 具体来说，本提案中的研究将解决以下问题：什么蛋白质将Msb 3 p和Msb 4p连接到上游Cdc 42 p和下游肌动蛋白细胞骨架？ Msb介导的通路中的蛋白质在分子水平上是如何组织的？ Msb 3 p和Msb 4p中推定的结构域（高度保守的PTM结构域、跨膜结构域和脂质修饰位点）的功能是什么？ Msb 3 p和Msb 4p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、细胞学和生物化学方法的结合来回答。为了解释我们的假设，参与调节Cdc 42 p活性或特定效应途径的其他基因将通过遗传筛选和亲和层析与串联质谱联用来鉴定。Cdc 42 p的同源物参与多种细胞过程，例如细胞极性、细胞迁移和细胞生长控制。此外，哺乳动物中Cdc 42 p活性的失调与严重疾病如癌症有关。 因此，研究Cdc 42 p在酵母中的信号转导机制是基础科学和临床科学之间的重要桥梁。

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.