Regulation of Cdc42-Dependent Signaling Pathways Throughout the Cell Cycle

这个项目的长期目标是了解在真核细胞周期中控制细胞形态发生的信号转导机制。正常的形态发生，即细胞复杂的三维组织被发展和维持的过程，对于细胞分化和繁殖的保真度是必不可少的。本项目的目标是破译酿酒酵母中控制细胞极性过程的信号转导机制。在酵母细胞周期中，响应不同信号的极化生长可以导致几种不同的形态结构的产生，如芽、交配突起和假菌丝。酵母细胞极性结构中的两个蛋白质组分：Ras相关的GTP酶CDc42p及其鸟嘌呤核苷酸交换因子(Gef)的cdc24p已在本实验室中被鉴定。这些成分是导致细胞周期中细胞极性产生的信号转导途径的组成部分。这些成分在其他真核生物中具有保守的对应物，这表明可能存在控制细胞极性的共同信号转导机制。这个项目的具体目的是为了了解CDC42P GTP酶的调节以及它如何与不同的细胞周期调节过程相结合。这些实验将检验的假设是：1)。细胞极性信号的启动依赖于CDC24P的局部靶向，而不是CDC42P的定位；以及2)。在细胞周期中，CDC42p被激活和去激活。CDC24P在细胞周期的特定时间定位于核和极化生长的位置。然而，最近的数据表明，在整个细胞周期中，CDC42p定位于细胞的整个外围，并在激活后的极化生长部位富集。为了更好地了解这一关键的调控过程，将使用绿色荧光蛋白(GFP)标记的野生型和突变蛋白来确定CDC24P和CDC42P的靶向机制。酿酒酵母CDC42p被CDc24p环境基金激活，并通过Bem3p、Rga1p和Rga2p GTP酶激活蛋白(GAP)的作用失活。本实验室最近的研究表明，在细胞周期的多个阶段，CDC24P激活了CDC42P，很可能是在G1期萌芽和M期细胞质分裂之前。如果是这样的话，那么在其间的时间范围内，由于其间隙的作用，很可能会使Cdc42p失活。将使用GFP标记的蛋白质来研究单个GAP的细胞周期特异性定位。这些问题的答案不仅关系到对细胞生物学中信号转导机制的基本理解，而且也关系到对酵母和其他真核生物细胞形态发生过程的理解。

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.