Spatial and temporal regulation of polarity establishment in budding yeast

 描述（由申请人提供）：细胞极性的建立对于各种细胞过程（包括增殖和细胞运动）至关重要，如在酵母生长期间发育胚胎、伤口愈合和芽形成中所见。因此，细胞极性的丧失涉及几种疾病过程，例如上皮肿瘤。Cdc 42 GT3是酵母和动物细胞极性的中心调节因子，也是转移的关键调节因子。虽然细胞极化发生沿着一个单一的轴，通常是由空间线索，大量的最近的研究集中在极性的建立在缺乏空间线索的机制。因此，如何确定细胞极化的特定方向以及如何以空间和时间方式调节该过程在很大程度上是未知的。拟议的工作利用易处理的酵母系统来阐明空间线索导向的细胞极化的机制。芽殖酵母的细胞以两种空间编程模式进行极化生长。我们最近发现Cdc 42在G1期以两个时间步骤被激活。第一步依赖于Bud 3，一种在单倍体中标记细胞分裂位点的皮质蛋白，而随后的激活依赖于Cdc 24，迄今为止Cdc 24是Cdc 42的唯一GDP-GTP交换因子（GEF）。这一发现使我们假设Cdc 42在G1期的顺序激活是在单倍体芽殖酵母中适当识别和读出细胞极性线索的基础。目的1旨在研究空间地标和Rsr 1 GTdR模块如何调节Cdc 42在G1早期的激活。我们的遗传和生化数据表明，Cdc 42在建立一个适当的芽网站意想不到的作用。因此，目的2寻求Cdc 42及其调节剂在适当的芽位点建立细胞极性的作用机制。最后，目的3探索细胞类型特异性出芽模式的分子基础，并研究决定极性轴方向的皮质标记物的组装。这项研究计划将采用生物化学方法和定量显微镜，以及计算建模。本研究旨在确定Cdc 42的顺序激活如何控制空间地标的识别和极性建立的执行。鉴于Cdc 42在从酵母到人类的细胞极性事件中的普遍重要性，这项工作的发现将与高等真核生物（包括哺乳动物）中的空间线索导向细胞极化有关。

 DESCRIPTION (provided by applicant): Establishment of cell polarity is critical for a variety of cellular processes including proliferation and cell movement, as seen in developing embryos, wound healing, and bud formation during yeast growth. Loss of cell polarity is, therefore, implicated in several disease processes, for example, in epithelial tumors. The Cdc42 GTPase is a central regulator of cell polarity in both yeast and animals and is also a critical regulator f metastasis. Although cell polarization occurs along a single axis that is generally determined by a spatial cue, a large number of recent studies have focused on the mechanisms of polarity establishment in the absence of spatial cues. Thus, how a specific direction of cell polarization is determined and how the process is regulated in a spatial and temporal manner are largely unknown. The proposed work capitalizes on the tractable yeast system to elucidate the mechanisms underlying spatial cue-directed cell polarization. Cells of the budding yeast undergo polarized growth in two spatially programmed patterns. We recently discovered that Cdc42 is activated in two temporal steps in the G1 phase. The first step depends on Bud3, a cortical protein marking the cell division site in haploids, while subsequent activation depends on Cdc24, which has heretofore been the sole GDP-GTP exchange factor (GEF) for Cdc42. This finding led us to hypothesize that sequential activation of Cdc42 in the G1 phase is fundamental to appropriate recognition and readout of cell polarity cues in haploid budding yeast. Aim 1 seeks to investigate how spatial landmarks and the Rsr1 GTPase module regulate activation of Cdc42 in early G1. Our genetic and biochemical data suggest an unexpected role of Cdc42 in establishment of a proper bud site. Thus, Aim 2 seeks the mechanism of action of Cdc42 and its regulators in establishment of cell polarity at a proper bud site. Finally, Aim 3 explores the molecular basis of the cell-type-specific budding pattern and investigates the assembly of the cortical markers that determines the orientation of the polarity axis. This research plan will employ biochemical methods and quantitative microscopy, as well as computational modeling. This study is expected to establish how sequential activation of Cdc42 governs recognition of spatial landmarks and execution of polarity establishment. Given the ubiquitous importance of Cdc42 in cell polarity events from yeast to humans, findings from this work will be relevant to spatial cue-directed cell polarization in higher eukaryotes, including mammals.