MICROFILAMENTS IN THE YEAST SACCHAROMYCES CEREVISIAE

存活能力是活细胞的基本属性， 无论是上皮细胞、神经细胞、迁移细胞，还是单纯的生长细胞， 必须选择细胞分裂的轴。为了开始理解 细胞极性的结构基础，我们采用芽殖酵母，因为 精湛的分子和经典遗传学，生物化学和细胞生物学， 在这个有机体中。此外，酵母用于 它的分泌途径，以及它如何分离细胞器 在细胞分裂过程中，与脊椎动物 细胞在目前的阶段，我们已经表明，酵母有一个特殊的组织 驱动含原肌球蛋白的肌动蛋白索组装的中心。这些 电缆是机械基板的极化运动， 由酵母MYO 2编码的非常规肌球蛋白-V。我们已经证明，Myo 2 p结合 通过它的尾巴到分泌囊泡，并将它们运送到肌动蛋白 两极化增长的电缆。我们还发现， 具有细胞分裂轴的细胞核是通过 Myo 2 p货物结构域与Kar 9 p一起将其运送到芽中。我们提出 三个具体目标建立在这些研究的基础上。首先，我们建议确定 组件参与组装和调节极化肌动蛋白电缆。我们 重点放在酵母formins，Bnilp和Bnrlp，因为这些似乎是关键 支架蛋白参与了电缆的形成过程。通过 遗传分析，我们提出的方法，以确定参与肌动蛋白的成分， 电缆组装，然后，基于这些研究，提出方法， 在体外重建这个过程。第二，我们建议审查其他 Myo 2 p可能会分离器官，并且鉴定分子的方法 让Myo 2 p能够运输特定的货物第三，由于Myo 2 p是最好的 作为肌球蛋白-V超家族的一员，我们建议分析 其货物结合域的结构，通过解剖特异性活性 从基因上以及在原子水平上确定其结构。这些 研究将为了解酵母如何建立一个 极化的细胞骨架，并反过来使用它来靶向极化生长， 在细胞周期中分离细胞器。因为分子的同系物 酵母中这些过程的关键与小鼠的缺陷有关 在人类疾病中，这项工作应该具有普遍意义。

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.