Saccharomyces cerevisiae microtubule cytoskeleton

描述（由申请人提供）：酵母有丝分裂纺锤体可能是第一个完整分子细节描述的细胞器，提供了一个机会，以了解其功能和调节，其水平是任何其他生物体都无法达到的。这里提出的研究对实现这一目标至关重要。我们发现的Dam 1复合体由7个与纺锤体和动粒相关的亚基组成。- 该复合物在纺锤体完整性和动粒功能中具有重要作用。这项研究的目的是确定Dam 1复合物对纺锤体完整性和动粒功能的精确机制，以及其活性如何在体内调节。由于Daml复合物与内部动粒组分和微管相互作用，并且对于建立新的染色体附着是必不可少的，因此它可以将染色体和纺锤体微管聚集在一起。为了更深入地了解这一重要功能，将进行详细的表型研究的突变体的Dam 1 i复杂的，和组装的动粒组件的相互依赖性。相互作用的合作伙伴将被确定遗传发展更好地了解其功能的生化背景。将确定复合物对微管的生化活性。为了阐明单个Dam 1 i亚基的作用，并为理解翻译后修饰的作用提供基础，将绘制复合物中每个蛋白质的相互作用结构域并测试其体内重要性。Dam 1复合物的三个组分在体内被磷酸化，并且它们的磷酸化位点已经被绘制。这些磷酸盐中的一些或全部似乎是由保守的有丝分裂激酶Iplip和Mps 1 p添加的;这些激酶提供了必需的有丝分裂和有丝分裂检查点功能。每个已确定的磷酸化位点的突变将提供一个独特的机会，以发展新的见解的机制，精心策划的复杂事件的有丝分裂。由于减数分裂I具有不同于减数分裂II和有丝分裂的性质，因此将在有效且同步进行减数分裂的菌株中研究Dam 1亚基的突变体。比较这些突变如何影响有丝分裂和减数分裂II与减数分裂I，有望提供对减数分裂I独特性质的更深入理解。

DESCRIPTION (provided by applicant): The yeast mitotic spindle will likely be the first such organelle described in complete molecular detail, providing an opportunity to develop an understanding of its function and regulation at a level not achievable for any other organism. The studies proposed here are critical for attaining this goal. The Dam1 complex that we discovered, consists of 7 subunits that associate with the spindle and kinetochores. - The complex has essential roles in spindle integrity and kinetochore function. The goal of the proposed research is to determine the precise mechanisms by which the Dam1 complex contributes to spindle integrity and kinetochore function, and how its activities are regulated in vivo. Because the Daml complex interacts both with inner kinetochore components and with microtubules, and appears essential for establishment of new chromosome attachments, it may bring together chromosomes and spindle microtubules. To develop a deeper understanding of this important function, detailed phenotypic studies of mutants of the Dam1i complex, and of interdependencies in assembly of kinetochore components, will be performed. Interacting partners will be identified genetically to develop a better understanding of the biochemical context within which it functions. Biochemical activities of the complex on microtubules will be identified. To elucidate the roles of individual Dam1i subunits and to provide a foundation for understanding effects of post-translational modifications, interaction domains of each protein in the complex will be mapped and tested for in vivo importance. Three components of the Dam1 complex are phosphorylated in vivo, and their phosphorylation sites have been mapped. Some or all of these phosphates appear to be added by the conserved mitotic kinases Iplip and Mps1p; kinases that provide essential mitotic and mitotic checkpoint functions. Mutagenesis of each identified phosphorylation site will provide a unique opportunity to develop novel insights into mechanisms for orchestrating the complex events of mitosis. Because meiosis I has properties distinct from meiosis II and mitosis, mutants of Dam1 subunits will be studied in a strain that efficiently and synchronously undergoes meiosis. Comparison of how these mutations affect mitosis and meiosis II vs. meiosis I promises to provide a deeper understanding of the unique properties of meiosis I.