Signaling mechanisms in cell polarity in yeast

描述(申请人提供)：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建立和维持细胞极性的分子机制。更具体地说，我感兴趣的是了解CDC42P，一种进化上保守的小GTP酶，如何通过独立的途径控制肌动蛋白细胞骨架和间隔蛋白的组织。此前，我们已经证明了酵母中一对高度同源的蛋白质Msb3p和Msb4p是CDC42突变的剂量依赖性抑制因子，这两种蛋白也通过作为Rab GTPase Sec4p的GTP酶激活蛋白(GAP)来调节分泌，从而定义了CDC42p介导的信号通路和Sec4p介导的分泌之间的分子联系。最近，我们发现Msb3p和Msb4p直接与CDC42p-GDP结合，还与Spa2p结合，Spa2p是“极化体”的支架蛋白，还包括Forin Bnilp和肌动蛋白结合蛋白Bud6p，从而定义了从CDc42p到肌动蛋白组织的具体途径。目前，我们正试图确定极化体功能的详细生化机制，其组成部分在进化上是保守的。我们最近的另一项工作导致了一个关于细胞周期开始时隔质环形成的两步模型：将隔质招募到萌芽部位，并将招募的隔质组装成皮质环。CDC42P在这两个步骤中都起到了作用。在这个框架下，我们试图定义Axl2p和Rgalp在Septin组织中的作用。Axl2p是一种I型膜，Rgalp是一种CDC42P间隙。最后，我们分离到PXL1作为几个CDC42-TS等位基因的多拷贝抑制因子。Pxllp在氨基酸序列和结构组织上显示出与巴西林的广泛相似性，巴西林是一种对发育和细胞运动至关重要的局灶性黏附蛋白。我们正在使用Pxllp作为一个模型来理解这个蛋白质家族是如何通过它们的C-末端LIM结构域来定位于离散的细胞位置的，这是帕克西林生物学中的一个关键缺口。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的信号转导机制将对基础生物学和临床科学具有深远的意义。

DESCRIPTION (provided by applicant): My long-term objective is to use the genetically tractable eukaryote Saccharomyces cerevisiae to determine the molecular mechanisms underlying the establishment and maintenance of cell polarity. More specifically y, I am interested in understanding how Cdc42p, an evolutionarily conserved small GTPase, controls the organization of the actin cytoskeleton and of the septins through independent pathways. Previously, we have shown that Msb3p and Msb4p, a pair of highly homologous proteins in yeast, function as dosage-dependent suppressors of cdc42 mutations and that both proteins also regulate secretion by functioning as GTPase-activating proteins (GAPs) for the Rab GTPase Sec4p; thus, defining a molecular linkage between Cdc42p-mediated signaling pathway and Sec4p-mediated secretion. Recently, we found that Msb3p and Msb4p bind directly to Cdc42p-GDP and also to Spa2p, a scaffold protein of the "polarisome" that also includes the formin Bnilp and the actin-binding protein Bud6p; thus, defining a concrete pathway from Cdc42p to actin organization. Currently, we are trying to determine the detailed biochemical mechanisms underlying the function of the polarisome, the components of which are evolutionarily conserved. Another line of our recent work has led to a two-step model on the septin-ring formation at the beginning of the cell cycle: the recruitment of septins to the incipient bud site and the assembly of the recruited septins into a cortical ring. Cdc42p plays a role in both steps. With this framework, we are trying to define the role of Axl2p, a type I membrane, and Rgalp, a Cdc42p GAP, in septin organization. Finally, we have isolated PXL1 as a multicopy suppressor of several cdc42-Ts alleles. Pxllp shows extensive similarity in amino-acid sequence and structural organization with paxillin, a focal adhesion protein that is essential for development and cell motility. We are using Pxllp as a model to understand how this family of proteins is targeted to discrete cellular locations through their C-terminal LIM domains, a crucial gap in paxillin biology. 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 will have profound implications in basic biology and clinical sciences.