Signaling mechanisms in cell polarity in yeast

描述(由申请人提供)：细胞极性对发育和分化是必不可少的，它在细胞迁移、营养物质跨上皮运输和神经元传递等基本过程中发挥重要作用。细胞极性的缺陷通常与癌症等严重的人类疾病有关。我们的长期目标是使用遗传上易驯化的真核生物酿酒酵母来阐明细胞极化的原理。在这个提案中，我们将解决两个重要的问题，即CDC42P，一种进化上保守的小GTP酶，在极性发育中的核心作用。在AIM1中，我们将确定在形成单一偏振域的过程中，Cdc42p的活性如何受到其间隙RGa1p和Bem2p的时空调节，这是所有极化系统中的一个共同问题。已知有四个间隙(RGa1p、RGa2、Bem3p和Bem2p)作用于Cdc42p。在此之前，我们和其他人已经证明了三个间隙(Rga1p、RGa2和Bem3p)在细胞周期开始时参与隔膜素环的组装。最近，我们发现，在胞质分裂后的细胞中，Rga1p是唯一需要防止旧细胞分裂部位极化的基因。在这个提案中，我们将测试我们的新想法，即RGa1p和Bem2p在花蕾颈部冗余地发挥“把关人”的作用，将活性的CDc42p和Rho1p限制在单一的极化区域，即花蕾皮质。到目前为止，我们的研究表明，不同的间隙可以单独或共同作用于涉及CDC42P的特定细胞过程，并帮助建立了一个研究哺乳动物Rho GTP酶(人类基因组中~17个)的复杂调控的范例。在AIM2中，我们将提出实验来测试我们的整合模型，即CDC42p在极化肌动蛋白和Septin组织中的作用，这两个因素共同决定了细胞的整体形状，并决定了它们在基本过程中的不同功能，如极化胞吐和定向细胞迁移。我们的模型表明，CDC42p通过两条遗传上可分离但生物化学上相互作用的途径控制极化的肌动蛋白和间隔蛋白组织，其中一条途径涉及进化上保守的以Forin Bni1p、支架蛋白Spa2p以及Rab间隙Msb3p和Msb4p为中心的“极化体”，另一条途径涉及酵母特异的CDC42p效应器Gic1p和Gic2p。在这项提案中，我们将确定极化体途径如何通过肌动蛋白细胞骨架影响Septin组织，GIC途径如何通过Septins影响肌动蛋白组织，以及这两条途径如何通过基于Spa2p的相互作用在细胞压力下(如370C)相互作用。CDC42P的同系物参与多种细胞功能，如细胞极性、细胞迁移和细胞生长控制。哺乳动物中CDC42P活性的解除调控与严重的人类疾病有关，如癌症。因此，研究酵母中CDC42P的信号转导机制，将对基础生物学和临床科学具有深远的意义。 与公共健康相关：CDC42P是一种进化上保守的小GTP酶，在细胞极性、细胞迁移和细胞生长控制等多种细胞过程中发挥重要作用。哺乳动物中CDC42P活性的解除调控与严重的人类疾病有关，如癌症。因此，研究酵母中CDC42P的信号转导机制，将对基础生物学和临床科学具有深远的意义。

DESCRIPTION (provided by applicant): Cell polarity is essential for development and differentiation, and it plays vital roles in fundamental processes such as cell migration, nutrient transport across epithelia, and neuronal transmission. Defects in cell polarity are often associated with serious human diseases such as cancer. Our long-term objective is to use the genetically tractable eukaryote Saccharomyces cerevisiae to elucidate the principles of cell polarization. In this proposal, we will address two important questions regarding the central role of Cdc42p, an evolutionarily conserved small GTPase, in polarity development. In Aim1, we will determine how Cdc42p activity is spatiotemporally regulated by its GAPs, Rga1p and Bem2p, in the formation of a single polarization domain, a common issue among all polarization systems. There are four GAPs (Rga1p, Rga2, Bem3p, and Bem2p) known to act on Cdc42p. Previously, we and others have shown that three GAPs (Rga1p, Rga2, and Bem3p) share a role in septin ring assembly at the beginning of the cell cycle. Recently, we discovered that Rga1p is uniquely required for preventing polarization within old cell division sites in post-cytokinesis cells. In this proposal, we will test our novel idea that Rga1p and Bem2p function redundantly at the bud neck as "gatekeepers" to restrict active Cdc42p and Rho1p to a single polarization domain, the bud cortex. To date, our studies have indicated that different GAPs can act alone or together to regulate specific cellular processes involving Cdc42p, and have helped establish a paradigm for studying complex regulation of mammalian Rho GTPases (~17 in human genome) by their numerous GAPs (~68 Rho GAPs in human genome). In Aim2, we will propose experiments to test our integrative model for the role of Cdc42p in polarized actin and septin organization, which together determine the overall cell shape and dictate their diverse functions in fundamental processes such as polarized exocytosis and directed cell migration. Our model states that Cdc42p controls polarized actin and septin organization via two genetically separable, but biochemically cross-talking pathways, one involving the evolutionarily conserved "polarisome" that is centered on the formin Bni1p, scaffold protein Spa2p, and the Rab GAPs Msb3p and Msb4p, and the other involving the yeast-specific Cdc42p effectors Gic1p and Gic2p. In this proposal, we will determine how the polarisome pathway affects septin organization via the actin cytoskeleton, how the Gic pathway affects actin organization via the septins, and how these two pathways crosstalk under cellular stresses such as 370C via Spa2p-based interactions. Homologues of Cdc42p are involved in numerous cellular functions such as cell polarity, cell migration, and cell growth control. Deregulation of Cdc42p activity in mammals is associated with serious human diseases, such as cancer. Thus, studying the signaling mechanisms of Cdc42p in yeast will have profound implication in basic biology and clinical sciences. PUBLIC HEALTH RELEVANCE: Cdc42p, an evolutionarily conserved small GTPase, plays essential roles in diverse cellular processes such as cell polarity, cell migration, and cell growth control. Deregulation of Cdc42p activity in mammals is associated with serious human diseases, such as cancer. Thus, studying the signaling mechanisms of Cdc42p in yeast will have profound implication in basic biology and clinical sciences.