Saccharomyces cerevisiae microtuble cytoskeleton

描述(由申请人提供)： 利用遗传学、生物化学、细胞生物学和超微结构的方法对酵母有丝分裂纺锤体进行了深入的研究，为理解其功能和调控提供了机会，目前在任何其他生物中都无法实现。拟议的研究对实现这一目标至关重要。DAM1复合体由10个蛋白质组成，在纺锤体的完整性和动粒-纺锤体的连接中起着重要的作用，也是发芽酵母极光蛋白激酶的重要靶点。DAM1复合体在微管上组装成环，优先与GTP-微管蛋白晶格结合，它稳定微管以防解体，并保持附着在解离的微管末端。这些特性非常适合于染色体和纺锤体微管之间的机械连接。利用最近开发的Dam1环移动和微管稳定的实时荧光分析，将测试蛋白质磷酸化和相互作用蛋白质对Dam1复合体生化活性的影响。通过将微珠连接到DAM1复合体上，微管拆解对环复合体产生的力将使用激光镊子进行测量。对已经相当先进的DAM1络合物涂层微管的冷冻-EM研究将用于螺旋重建，以获得可以对接到微管表面的已知结构上的复合体的3D结构。这一分析有望揭示该复合体作为动态微管结合界面功能的结构决定因素，并将扩展到翻译后修饰的Dam1复合体，以及具有相关相互作用蛋白的复合体。通过结合生物信息学、质谱学和化学遗传学来确定更多的Ipl1靶蛋白，将增加对Aurora Kinase Ipl1如何调节有丝分裂纺锤体功能的理解。分子遗传学将决定磷酸化如何影响目标蛋白质的功能。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了解，并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。

DESCRIPTION (provided by applicant): The yeast mitotic spindle has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to understand its function and regulation at a level not currently achievable in any other organism. The proposed studies are critical for attaining this goal. The 10- protein Dam1 complex provides critical functions in spindle integrity and kinetochore-spindle attachment, and it is a crucial target of the budding yeast Aurora kinase. The Dam1 complex assembles into rings on microtubules that associate preferentially with the GTP-tubulin lattice, it stabilizes microtubules against disassembly, and it remains attached to the disassembling microtubule ends. These properties are ideally suited for a mechanical linkage between chromosomes and spindle microtubules. Using a recently developed real-time fluorescence assay for Dam1 ring movement and microtubule stabilization, effects of protein phosphorylation and interacting proteins on biochemical activities of the Dam1 complex will be tested. By attaching microbeads to the Dam1 complex, forces generated on the ring complex by microtubule disassembly will be measured using laser tweezers. Cryo-EM studies of Dam1 complex-coated microtubules that are already quite advanced will be used for helical reconstructions to obtain a 3D structure of the complex that can be docked onto the known structure of the microtubule surface. This analysis promises to reveal structural determinants for the function of the complex as a dynamic microtubule-binding interface, and will be extended to post-translationally modified Dam1 complex, and to the complex with associated interacting proteins. Understanding of how the Aurora kinase Ipl1 regulates mitotic spindle functions will be increased by combining bioinformatics, mass spectrometry and chemical genetics to identify additional Ipl1 target proteins. Molecular genetics will determine how phosphorylation affects target protein functions. These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and 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.