Saccharomyces Cerevisiae Microtubule Cytoskeleton

酿酒酵母微管细胞骨架

• 批准号: 8115022
• 负责人: GEORJANA BARNES
• 金额: $ 34.59万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115022
• 关键词: Affect; Area; Bacteria; Binding; Biochemical; Biochemistry; Cells; Cellular biology; Chromosomal Instability; Complex; Congenital Abnormality; Consensus; Cytoskeleton; Detection; Dissection; Enzymes; Eukaryota; Foundations; Genes; Genetic; Genetic Screening; Goals; Human; In Vitro; Insecta; Investigation; Kinetochores; Location; Malignant Neoplasms; Microtubule Bundle; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Molecular Genetics; Monitor; Organism; Pathway interactions; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Prevention; Process; Property; Protein Kinase; Proteins; Regulation; Saccharomyces cerevisiae; Site; Structural Protein; Structure; Yeasts; aurora kinase; casein kinase II; centromere protein C; human disease; in vitro testing; in vivo; novel; novel strategies; public health relevance; reconstitution; success

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.

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分子更简单，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的研究，提供了一个在目前任何其他生物体中都无法达到的水平上对其功能和调节的理解的机会。拟议的研究对实现这一目标至关重要。动粒-微管连接的正确建立、功能和检查点监测是有丝分裂保真度的核心。尽管在识别参与这种物理连接的关键蛋白以及评估它们在体内和体外的活性方面取得了很大进展，但对于连接的调节机制，微管结合的外部动粒蛋白如何与中心动粒结合，或者当单独的亚复合体如Dam1和Ndc80在动粒中结合时产生的新特性，人们了解得很少。在对Dam1复合体的发现和分析以及对Ndc80复合体的结构研究的基础上，新的研究将确定它们的结合伙伴，确定Dam1和Ndc80复合体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，以及动粒复合体的集合如何与微管动态相互作用。以往的研究发现，极光激酶通过DAM1复合体的磷酸化来调节着丝粒的附着，鉴定了极光激酶的共同位点，鉴定了第四个酵母极光激酶复合体亚基，以及酪蛋白激酶2在动粒内部调节中的新意义，为揭示蛋白激酶如何调节关键的有丝分裂功能提供了坚实的基础。拟议的研究将确定极光激酶复合体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。对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决定该复合体的调控方式。最近发现酪蛋白激酶2是内部动粒中心蛋白NDC10和广泛保守的Mif2(CENP-C)连接蛋白的有丝分裂调节因子，并在体内获得了CK2和极光激酶双重调节这些蛋白的证据，将对这两种激酶如何调节动粒中心功能进行强大的体内和体外测试。由于酵母的独特优势，对有丝分裂纺锤体分解的全面分子解剖已经取得了很大进展，并将在此基础上充分阐明途径和机制。随着细胞的有丝分裂，复杂的有丝分裂纺锤体必须迅速解体，这包括拆除稳定的微管束、解离蛋白质亚复合体、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还未被探索，这是一个极其丰富和重要的研究领域。全面的遗传筛选和相互作用分析将确定基因和途径，重点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控的基本方面的了解，由于许多有丝分裂蛋白和机制在进化上是保守的，将为阐明人类的有丝分裂机制提供一个框架。染色体不稳定是导致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会为人类疾病的预防、检测和治疗提供新的策略。