MICROFILAMENTS IN THE YEAST SACCHAROMYCES CEREVISIAE

极化能力是活细胞的基本属性， 无论它们是上皮细胞、神经细胞还是迁移细胞，或者仅仅是生长细胞 它们必须为细胞分裂挑选一个轴。要开始理解 细胞极性的结构基础，我们使用发芽酵母是因为 精湛的分子和经典遗传学、生物化学和细胞生物学 在这种有机体中可用。此外，酵母菌用来 极化它的分泌途径，可能还有它如何分离细胞器 在细胞分裂过程中，与脊椎动物使用的机制非常相似 细胞。目前，我们已经证明了酵母有一种特殊的组织 驱动含有原肌球蛋白的肌动蛋白电缆组装的中心。这些 电缆是极化运动的机械衬底， 酵母MYO2编码的非常规肌球蛋白-V。我们已经证明了Myo2p结合 通过它的尾巴来分泌小泡并将它们运送到肌动蛋白 用于极化生长的电缆。我们还发现，初始对齐 以细胞分裂为轴心的核是通过联会实现的 Myo2p货运域与Kar9p一起将其交付到萌芽状态。我们建议 建立在这些研究基础上的三个具体目标。首先，我们建议确定 组装和调节极化肌动蛋白电缆所涉及的组件。我们 重点关注酵母福尔马林、Bnilp和Bnrlp，因为这些似乎是关键 参与电缆形成过程的支架蛋白。穿过 基因分析我们提出了确定肌动蛋白相关成分的方法 电缆组装，然后在这些研究的基础上，提出 在体外重建这一过程。第二，我们建议研究其他 器官可能通过Myo2p和鉴定分子的方法来分离 允许Myo2p运输特定的货物。第三，因为Myo2p是最好的 了解肌球蛋白-V超家族成员，我们建议分析 通过解剖特定活性来确定其货物结合域的结构 从基因上以及在原子水平上决定其结构。这些 研究将为理解酵母如何建立一个 极化的细胞骨架，进而利用它来靶向极化的生长和 在细胞周期中分离细胞器。由于分子的同系物 酵母中这些过程的关键与小鼠的缺陷有关 在人类疾病方面，这项工作应该具有普遍的相关性。

The ability to polarize is a fundamental property of living cells, whether they are epithelial, nerve, or migrating cells, or simply growing cells that have to pick an axis for cell division. To begin to understand the structural basis for cell polarity, we employ the budding yeast because exquisite molecular and classical genetics, biochemistry and cell biology are available in this organism. Moreover, the basic principles that yeast uses to polarize its secretory pathway, and probably how it segregates its organelles during cell division, are very similar to the mechanisms employed by vertebrate cells. In the current period, we have shown that yeast has a special organizing center that drives the assembly of tropomyosin-containing actin cables. These cables are the mechanical substrates for polarized movement by the unconventional myosin-V encoded by yeast MYO2. We have shown that Myo2p binds through its tail to secretory vesicles and transports them down the actin cables for polarized growth. We have also found that the initial alignment of the nucleus with the axis of cell division is achieved through the association of the Myo2p cargo domain with Kar9p to deliver it into the bud. We propose three specific aims that build on these studies. First, we propose to identify components involved in assembling and regulating the polarized actin cables. We focus on the yeast formins, Bnilp and Bnrlp, as these appear to be key scaffolding proteins involved in the process of cable formation. Through genetic analysis we propose approaches to identify components involved in actin cable assembly, and then, based on these studies, propose approaches to reconstitute this process in vitro. Second, we propose to examine what other organdlies might be segregated by Myo2p, and approaches to identify molecules that allow Myo2p to transport specific cargoes. Third, since Myo2p is the best understood member of the myosin-V superfamily, we propose to analyze the structure of its cargo binding domain by both dissecting specific activities genetically as well as determining its structure at the atomic level. These studies will provide a foundation for understanding how yeast sets up a polarized cytoskeleton and in turn uses it to target polarized growth and segregate organelles during the cell cycle. Since homologues of molecules critical for these processes in yeast have been associated with defects in mice and in human diseases, this work should be of general relevance.