Spatial and temporal regulation of polarity establishment in budding yeast

 描述(由申请人提供)：在酵母生长过程中，细胞极性的确定对包括增殖和细胞运动在内的各种细胞过程至关重要，如胚胎发育、伤口愈合和芽形成。因此，细胞极性的丧失与几种疾病过程有关，例如在上皮性肿瘤中。在酵母和动物中，CDC42 GTP酶都是细胞极性的中心调节器，也是转移的关键调节器。虽然细胞极化发生在通常由空间线索决定的单一轴上，但最近的大量研究集中在没有空间线索的情况下极性建立的机制。因此，细胞极化的特定方向是如何确定的，以及这个过程是如何以空间和时间的方式被调节的，在很大程度上是未知的。这项拟议的工作利用易处理的酵母系统来阐明空间线索引导的细胞极化的机制。发芽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，在G1期，CDC42在两个时间步骤中被激活。第一步依赖于Bud3，这是一种皮质蛋白，标志着单倍体中的细胞分裂位置，而随后的激活依赖于CDc24，到目前为止，它一直是唯一的CDc42的GDP-GTP交换因子(Gef)。这一发现导致我们假设，在单倍体发芽酵母中，CDC42在G1期的顺序激活是适当识别和读出细胞极性线索的基础。目的1研究空间标志物和Rsr1 GTPase模块如何调控G1期早期CDC42的激活。我们的遗传和生化数据表明，在建立一个合适的芽位方面，CDC42起到了意想不到的作用。因此，目标2寻求在适当的芽位置建立细胞极性的CDC42及其调节因子的作用机制。最后，目标3探索了细胞类型特定萌发模式的分子基础，并研究了决定极轴方向的皮质标记的组装。这项研究计划将使用生化方法和定量显微镜，以及计算模型。这项研究有望确定CDC42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CDC42在从酵母到人类的细胞极性事件中无处不在的重要性，这项工作的发现将与高等真核生物(包括哺乳动物)中空间线索导向的细胞极化相关。

 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.