Saccharomyces Cerevisiae Microtubule Cytoskeleton

描述（由申请人提供）：酵母有丝分裂纺锤体比大型真核生物中的对应物简单，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，为了解其功能和调节提供了一个机会，目前在任何其他生物体中都无法实现。拟议的研究对实现这一目标至关重要。正确的建立，功能和检查点监测的着丝粒微管附件的核心有丝分裂的保真度。尽管有很大的进展，确定参与这种物理连接的关键蛋白质，并评估其在体内和体外的活动，很少有人了解有关机制调节附件，关于如何微管结合外动粒蛋白与中央动粒，或有关新兴的属性时，导致单独的亚复合物，如Dam 1和Ndc 80在动粒在一起。基于对Dam 1复合物的发现和分析，以及Ndc 80复合物的结构研究，新的研究将确定它们的结合伴侣，确定Dam 1和Ndc 80复合物的结构如何与功能相关，翻译后修饰和结合伴侣如何影响功能，以及动粒复合物的集合如何与微管动态相互作用。以前的发现，极光激酶调节动粒附着通过Dam 1复合物磷酸化，极光激酶共识网站的鉴定，第四酵母极光激酶复合物亚基的鉴定，和酪蛋白激酶2在内部动粒调节的新含义，提供了一个强大的基础研究，以揭示蛋白激酶如何调节关键的有丝分裂功能。拟议的研究将确定Aurora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。分子遗传学和生化分析的完全重建，四蛋白极光激酶复合物将确定如何调节这种复杂的。最近已经鉴定出酪蛋白激酶2作为内部动粒蛋白Ndc 10和广泛保守的Mif 2（CENP-C）连接蛋白的有丝分裂调节剂，并且获得了CK 2和Aurora激酶对这些蛋白质的双重调节的体内证据，将进行这两种激酶如何调节动粒功能的强有力的体内和体外测试。由于酵母的独特优势，有丝分裂纺锤体解体的完整分子解剖已经取得了很大进展，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，这涉及分解稳定的微管束，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是未开发的，这是一个非常肥沃和重要的调查领域。全面的遗传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。 公共卫生相关性：这些研究将增加对有丝分裂纺锤体功能和调节的基本方面的理解，并且由于许多有丝分裂蛋白和机制在进化上是保守的，因此将为阐明人类有丝分裂机制提供框架。染色体不稳定性是癌症和出生缺陷的关键因素。因此，了解纺锤体功能的原理可能为预防、检测和治疗人类疾病提供新的策略。

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.