Saccharomyces cerevisiae microtuble cytoskeleton

描述（由申请人提供）： 酵母有丝分裂纺锤体已被深入研究，使用遗传学，生物化学，细胞生物学和超微结构的方法，提供了一个机会，以了解其功能和调节的水平目前无法在任何其他生物体。拟议的研究对实现这一目标至关重要。10蛋白Dam 1复合物在纺锤体完整性和着丝粒-纺锤体附着中提供关键功能，并且它是芽殖酵母Aurora激酶的关键靶标。Dam 1复合物在微管上组装成优先与GTP-微管蛋白晶格相关的环，它稳定微管以防止拆卸，并且它保持附着在拆卸的微管末端。这些特性非常适合染色体和纺锤体微管之间的机械连接。使用最近开发的实时荧光检测Dam 1环运动和微管稳定，蛋白磷酸化和相互作用的蛋白质对Dam 1复合物的生化活性的影响将进行测试。通过将微珠连接到Dam 1复合物上，将使用激光镊子测量由微管分解在环复合物上产生的力。已经相当先进的Dam 1复合物涂层微管的Cryo-EM研究将用于螺旋重建，以获得可以对接到微管表面已知结构上的复合物的3D结构。该分析有望揭示作为动态微管结合界面的复合物的功能的结构决定因素，并将扩展到后修饰的Dam 1复合物，以及与相关相互作用蛋白质的复合物。极光激酶Ipl 1如何调节有丝分裂纺锤体功能的理解将通过生物信息学，质谱和化学遗传学相结合，以确定其他Ipl 1靶蛋白。分子遗传学将确定磷酸化如何影响靶蛋白功能。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理解，并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 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.