Saccharomyces Cerevisiae Microtubule Cytoskeleton

描述（由申请人提供）：酵母有丝分裂纺锤体比其在大型真核生物中的对应物更复杂，并且已经使用遗传学，生物化学，细胞生物学和超微结构方法进行了深入研究，提供了一个机会来发展对其功能和调控的理解，这是目前在任何其他生物中都无法实现的。拟议的研究对实现这一目标至关重要。着丝点-微管附着物的正确建立、功能和检查点监测是有丝分裂保真度的核心。尽管在确定参与这种物理附着的关键蛋白方面取得了很大进展，并评估了它们在体内和体外的活性，但对于调节附着的机制、微管结合的外着丝点蛋白如何与中心着丝点结合，以及当像Dam1和Ndc80这样的分离亚复合物在着丝点中结合时产生的突发性特性，人们知之甚少。基于Dam1复合物的发现和分析，以及Ndc80复合物的结构研究，新的研究将确定它们的结合伙伴，确定Dam1和Ndc80复合物的结构如何与功能相关，翻译后修饰和结合伙伴如何影响功能，以及着丝点复合物的集合如何与微管动态相互作用。先前的研究发现，极光激酶通过Dam1复合物磷酸化调控着丝粒的附着，极光激酶共识位点的鉴定，第四个酵母极光激酶复合物亚基的鉴定，以及酪蛋白激酶2在着丝粒内部调控中的新含义，为揭示蛋白激酶如何调节关键的有丝分裂功能的研究提供了坚实的基础。拟议的研究将确定极光激酶复合物如何靶向特定的细胞位置，将确定其在每个位置的靶标，并将确定磷酸化如何影响其靶标的活性。完全重组的四蛋白极光激酶复合体的分子遗传学和生化分析将确定该复合体是如何被调节的。最近发现酪蛋白激酶2是内着丝粒蛋白Ndc10和广泛保守的Mif2 （CENP-C）连接蛋白的有丝分裂调节剂，并获得CK2和极光激酶双重调节这些蛋白的体内证据，将对这两种激酶如何调节着丝粒功能进行强有力的体内和体外测试。由于酵母的独特优势，对有丝分裂纺锤体拆卸的完整分子解剖已经取得了强有力的进展，并将建立在充分阐明途径和机制的基础上。当细胞退出有丝分裂时，极其复杂的有丝分裂纺锤体必须迅速分解，这包括分解稳定的微管束、分解蛋白质亚复合物、逆转翻译后修饰和破坏蛋白质。由于主轴拆卸机制在很大程度上未被探索，这是一个非常肥沃和重要的研究领域。全面的基因筛选和相互作用分析将确定基因和途径，集中的表型和生化研究将揭示详细的机制。

DESCRIPTION (provided by applicant): The yeast mitotic spindle is less complex than its counterparts in larger eukaryotes and has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to develop an understanding of its function and regulation at a level that is not currently achievable in any other organism. The proposed studies are critical for attaining this goal. Correct establishment, function and checkpoint monitoring of kinetochore-microtubule attachments are central to mitotic fidelity. Despite much progress identifying key proteins involved in this physical attachment, and assessing their activities in vivo and in vitro, little is understood about mechanisms regulating attachments, about how microtubule-binding outer kinetochore proteins associate with the central kinetochore, or about emergent properties that result when separate subcomplexes like Dam1 and Ndc80 are together in kinetochores. Building upon discovery and analysis of the Dam1 complex, and structure studies of the Ndc80 complex, new studies will identify their binding partners, determine how Dam1 and Ndc80 complex structures relate to function, how post-translational modifications and binding partners affect function, and how ensembles of kinetochore complexes interact dynamically with microtubules. Previous discoveries that Aurora kinase regulates kinetochore attachment via Dam1 complex phosphorylation, identification of an Aurora kinase consensus site, identification of the fourth yeast Aurora kinase complex subunit, and novel implication of casein kinase 2 in inner kinetochore regulation, provide a robust foundation for studies to reveal how protein kinases regulate critical mitotic functions. Proposed studies will determine how the Aurora kinase complex is targeted to specific cellular locations, will identify its targets at each location, and will determine how phosphorylation affects activity of its targets. Molecular genetics and biochemical analysis of the fully reconstituted, four-protein Aurora kinase complex will determine how this complex is regulated. Having recently identified casein kinase 2 as a mitotic regulator of the inner kinetochore protein Ndc10 and the widely conserved Mif2 (CENP-C) linker protein, and obtained in vivo evidence for dual-regulation of these proteins by CK2 and Aurora kinase, powerful in vivo and in vitro tests of how these two kinases regulate kinetochore function will be conducted. Thanks to unique advantages of yeast, strong inroads into a full molecular dissection of mitotic spindle disassembly have already been made, and will be built upon to fully elucidate pathways and mechanisms. As cells exit mitosis, the enormously complex mitotic spindle must be disassembled rapidly, which involves taking apart stabilized microtubule bundles, disassembling protein subcomplexes, reversing post-translational modifications and destroying proteins. Because spindle disassembly mechanisms are largely unexplored, this is an extremely fertile and important area for investigation. Comprehensive genetic screens and interaction analyses will identify genes and pathways, and focused phenotypic and biochemical studies will reveal detailed mechanisms. PUBLIC HEALTH RELEVANCE: These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and since many mitotic proteins and mechanisms are evolutionarily conserved, will provide a framework for elucidating mitotic mechanisms in humans. Chromosome instability is a key contributing factor in cancer and birth defects. Therefore, understanding principles of spindle function may suggest novel strategies for prevention, detection and treatment of human diseases.