Regulation and Roles of Myosin V Interaction with Cargo

项目摘要/摘要 所有真核细胞的一个重要特性是细胞器的适当分布。细胞器的排列 对于每种细胞类型都是不同的。此外，作为分化的一部分，细胞器被移动到新的位置作为 细胞获得了新的功能。目前对运动机制的认识存在很大差距 在正确的时间将细胞器放到适当的位置。为了解决这些差距，这项建议的重点是 液泡/溶酶体的遗传。酵母中的液泡/溶酶体遗传提供了一个很好的模型，因为它 涉及细胞周期依赖性和肌球蛋白V分子马达(MyoV)的空间调节。中的缺陷 MyoV转运是一些人类疾病的基础，包括神经系统疾病，以及一些致命的 胃肠道。重要的是，人类MyoV和酵母MyoV高度相似。MyoV的一种主要模式 调节是通过控制它在细胞周期中附着和脱离液泡货物来实现的。 进步。鉴于人们对任何类型的分子马达是如何连接或脱离的知之甚少 在货物方面，拟议的研究将解决知识方面的一项重大差距。目标1将决定MyoV如何 货物从发动机中释放出来。初步数据表明，这种释放是通过分步后释放进行的。 连接MyoV与其货物的接头蛋白的翻译修饰。这些步骤受到高度监管。 监管所需的一些候选因素已经被发现。基因和基因的结合 生化分析将用于确定这些因子的具体功能。 对液泡遗传的研究还导致了一个意想不到的发现，即液泡/溶酶体是 对细胞周期进程是必不可少的，并与细胞周期蛋白依赖的激酶途径平行发挥作用。此外， 溶酶体在哺乳动物的细胞周期中可能也有类似的作用。对细胞器如何调节细胞的见解- Cycle可能为治疗癌症等疾病提供新的方法，在这些疾病中，细胞周期的正常控制是 迷路了。表征溶酶体在细胞周期进程中的作用，可能会发现新的靶点 以前从未被研究过的癌症。目标2试图获得对液泡是如何 对细胞周期有贡献。这一目标的一部分是基于一项新发现，即在压力之后， 以前未知的途径重新启动了暂停的细胞周期。酵母菌遗传学、化学抑制和体外试验 检测将被用来绘制重新启动细胞周期所需的特定路径。令人兴奋的是， 我们已经在酵母途径中发现的蛋白质在哺乳动物细胞中是保守的。因此，此外， 这项提议将测试哺乳动物溶酶体是否在细胞周期进程中发挥重要作用。

PROJECT SUMMARY/ABSTRACT A critical property of all eukaryotic cells is the proper distribution of organelles. The arrangement of organelles is distinct for each cell-type. Moreover, as part of differentiation, organelles are moved to new locations as a cell acquires new functions. There are large gaps in current knowledge of the mechanisms that move organelles to the proper place at the correct time. To address these gaps, this proposal is focused on the inheritance of the vacuole/lysosome. Vacuole/lysosome inheritance in yeast provides an excellent model as it involves cell cycle-dependent as well as spatial regulation of a myosin V molecular motor (MyoV). Defects in MyoV transport underlie some human diseases, including neurological diseases, and some fatal disorders of the gastrointestinal tract. Importantly human and yeast MyoV are highly similar. A major mode of MyoV regulation is via control of its attachment to and detachment from the vacuole cargo during cell cycle progression. Given that little is known about how any type of molecular motor attaches or detaches from cargoes, the proposed studies will address a critical gap in knowledge. Aim 1 will determine how a MyoV cargo is released from the motor. Preliminary data suggest that the release occurs via step-wise post- translational modification of an adaptor protein that links MyoV to its cargo. These steps are highly regulated. Some candidate factors required for the regulation have been uncovered. A combination of genetic and biochemical analyses will be used to determine the specific functions of these factors. Studies of vacuole inheritance also led to the unexpected discovery that the vacuole/lysosome is essential for cell-cycle progression, and acts in parallel with the cyclin-dependent kinase pathway. Moreover, lysosomes may have a similar role in the mammalian cell-cycle. Insights into how organelles regulate the cell- cycle may provide new approaches to treat diseases such as cancer, where normal control of the cell-cycle is lost. Characterization of a role for the lysosome in cell-cycle progression, will likely uncover new targets for cancer that have not been previously explored. Aim 2 seeks to gain mechanistic insights into how the vacuole contributes to the cell-cycle. A portion of the Aim is based on a new discovery that following stress, a previously unidentified pathway re-initiates a paused cell-cycle. Yeast genetics, chemical inhibition and in vitro assays will be used to map the specific pathways required for the re-initiation of the cell cycle. Excitingly, proteins that we already identified in the yeast pathway are conserved in mammalian cells. Thus, in addition, this proposal will test whether the mammalian lysosome plays an essential role in cell-cycle progression.