Regulation of polarized cell growth by GTPases

描述（由申请人提供）：极性建立和定向细胞分裂是许多生物发育的核心。芽殖酵母的细胞根据其细胞类型表现出两种不同的芽殖模式，反映了细胞极化的遗传程序。人们认为细胞对一种细胞类型特异性的皮层标记物有反应，这种标记物与质膜有关。所有细胞类型都使用一个共同的下游途径来建立极性，这涉及Ras和Rho家族gtpase Rsr1/Bud1和Cdc42。尽管目前对许多参与出芽的蛋白质有所了解，但极性是如何建立在一个空间定义的位置上的，这在很大程度上是未知的。这一建议侧重于一个大分子复合体是如何指定一个特定的芽位点组装，以及极性建立是如何在空间和时间的方式控制。根据遗传和生化数据，假设gtpase调节极性建立的各个步骤。为了验证这一假设，将采用分子遗传学、生物化学和细胞生物学方法进行多方向的研究。具体目的是了解：1)指定芽位的大分子复合物是如何组装的以及在每个细胞分裂周期中瞬时空间信息是如何遗传的；2) Rsr1的调控因子Bud2和Bud5如何与不同细胞类型的空间地标相互作用，以及相互作用是否调节它们的活性；3) Rsr1如何调控Cdc42的GEF Cdc24，以及Cdc42复合物在芽位点的组装。虽然极性的建立是一个复杂的问题，但酵母的简单遗传学为在分子水平上研究导致肌动蛋白细胞骨架极化组织的信号通路提供了独特的机会。哺乳动物细胞的发育也需要肌动蛋白细胞骨架的持续变化，以响应内部和外部信号，如伤口愈合或感染期间的趋化性。了解酵母中极性建立的分子机制有望为哺乳动物细胞中肌动蛋白细胞骨架组织提供分子基础。鉴于真核生物中gtpase及其调节因子的结构和功能守恒，所获得的知识也将使我们能够深入了解这些正常发育的关键调节因子的新机制，这有望与包括人类在内的大多数真核生物相关。

DESCRIPTION (provided by applicant): Polarity establishment and oriented cell divisions are central to the development of many organisms. Cells of the budding yeast S. cerevisiae exhibit two distinct patterns of budding depending on their cell type, reflecting genetic programming of cell polarization. It is believed that a cell responds to a cell-type-specific cortical marker, which is associated with the plasma membrane. All cell types use a common downstream pathway for polarity establishment, which involves the Ras and Rho family GTPases Rsr1/Bud1 and Cdc42. Despite current knowledge of many proteins involved in budding, how polarity is established toward a spatially defined site is largely unknown. This proposal focuses on how a macromolecular complex that specifies a specific bud site is assembled, and how polarity establishment is controlled in a spatial and temporal manner. Based on genetic and biochemical data, it is hypothesized that GTPases regulate various steps of polarity establishment. To test the hypothesis, multi-directional approaches will be undertaken using molecular genetic, biochemical, and cell biological methods. Specific aims are to understand: 1) How a macromolecular complex that specifies a bud site is assembled and how the transient spatial information is inherited in every cell division cycle; 2) how Bud2 and Bud5, regulators for Rsr1, interact with a distinct spatial landmark in each cell type, and whether the interactions regulate their activity; and 3) how Rsr1 regulates Cdc24, a GEF for Cdc42, and assembly of the Cdc42 complex at the bud site. Although polarity establishment is a complex problem, the facile genetics of yeast provides the unique opportunity to study a signaling pathway leading to polarized organization of the actin cytoskeleton at the molecular level. Development of mammalian cells also requires continual changes in the actin cytoskeleton in response to internal and external signals as seen during wound healing or chemotaxis during infection. Understanding the molecular mechanism underlying polarity establishment in yeast is expected to provide the molecular basis of actin cytoskeleton organization in mammalian cells. Given the structural and functional conservation of GTPases and their regulators in eukaryotes, knowledge gained will also allow an insight into novel mechanisms for these critical regulators of normal development, which is expected to be relevant to most eukaryotes including humans.