Saccharomyces cerevisiae microtubule cytoskeleton

描述(由申请人提供)：酵母有丝分裂纺锤体很可能是第一个完整的分子细节描述的细胞器，提供了一个机会来发展对其功能和调节的理解，这是任何其他生物体都无法达到的水平。这里提出的研究对于实现这一目标至关重要。我们发现的DAM1复合体由7个亚基组成，分别与纺锤体和着丝点相关。-该复合体在纺锤体完整性和动粒功能中具有重要作用。这项拟议研究的目的是确定DAM1复合体促进纺锤体完整性和动粒功能的确切机制，以及它的活动在体内是如何调节的。由于DAML复合体既与内部着丝粒成分相互作用，又与微管相互作用，似乎是建立新的染色体附着所必需的，它可能会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，将对Dam1i复合体的突变体以及动粒组件组装中的相互依赖进行详细的表型研究。将从基因上确定相互作用的伙伴，以更好地了解它在其中发挥作用的生化环境。将确定该络合物对微管的生化活性。为了阐明单个Dam1i亚基的作用，并为理解翻译后修饰的影响提供基础，将绘制复合体中每个蛋白质的相互作用结构域，并测试其在体内的重要性。DAM1复合体的三个组分在体内被磷酸化，并绘制了它们的磷酸化位点图。部分或全部这些磷酸盐似乎是由保守的有丝分裂激酶Iplip和Mps1p添加的；这些激酶提供基本的有丝分裂和有丝分裂检查点功能。每个已识别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，以协调复杂的有丝分裂事件的机制。由于减数分裂I具有与减数分裂II和有丝分裂不同的性质，因此将在一种高效和同步进行减数分裂的菌株中研究Dam1亚基的突变。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独特性质的更深层次的理解。

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.