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Multicolor TIRF microscope for studying mitotic spindle components at the single

多色 TIRF 显微镜用于研究单次有丝分裂纺锤体成分

基本信息

批准号：
7791455
负责人：
CHARLES ASBURY
金额：
$ 21.42万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-06 至 2011-05-05
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Chromosomes are organized and separated during cell division by a microtubule-based molecular machine, the mitotic spindle. Our overall goal is to reconstitute spindle functions using pure components and apply new tools for manipulating and tracking single molecules to uncover how this machine operates. Here we request support for an ultrasensitive fluorescence microscope capable of simultaneously recording interactions between individual protein molecules, multiprotein complexes, and dynamic microtubule filaments. The instrument is based on an Olympus IX51 inverted microscope equipped with total internal reflection fluorescence (TIRF) illumination and with three Andor emCCD cameras, enabling fast, multicolor molecular tracking. It will be used by four NIH-funded investigators, each with complementary expertise, working in close collaboration with one another. The Davis lab will study super-complexes assembled from pure, recombinant forms of the six protein subcomplexes that make up kinetochores to determine how their interactions allow kinetochores to link chromosomes to spindle microtubules. The Asbury and Biggins labs will take a complementary approach, studying whole kinetochores isolated from wild-type yeast and from mutants with targeted defects to assess quantitatively the contribution each kinetochore subcomplex makes to chromosome-microtubule coupling. The Wordeman lab will study how the activities of microtubule regulatory enzymes implicated in kinetochore function are modulated through interactions with plus end binding proteins (+TIPs) and other microtubule binding factors. The Wordeman lab will also observe nucleotide turnover in a single microtubule-depolymerizing enzyme (MCAK) to test whether it enzymatically removes multiple tubulin subunits before detaching from the filament. These projects will bring us closer to a complete understanding of spindle function by elucidating the mechanisms underlying attachment of chromosomes to spindle microtubules, correction of attachment errors, and regulation of chromosome and spindle movements. Ultimately, having a mechanistic understanding of the spindle promises to revolutionize the design of chemotherapeutic drugs that target spindle components.

描述（由申请方提供）：细胞分裂期间，染色体通过基于微管的分子机器（有丝分裂纺锤体）进行组织和分离。我们的总体目标是使用纯组分重建纺锤体功能，并应用新的工具来操纵和跟踪单分子，以揭示这台机器是如何运作的。在这里，我们要求支持一个超灵敏的荧光显微镜能够同时记录单个蛋白质分子，多蛋白复合物和动态微管丝之间的相互作用。该仪器基于Olympus IX51倒置显微镜，配备全内反射荧光（TIRF）照明和三个Andor emCCD相机，可实现快速，快速的分子跟踪。它将由四名NIH资助的研究人员使用，每个人都具有互补的专业知识，彼此密切合作。戴维斯实验室将研究由构成动粒的六种蛋白质亚复合物的纯重组形式组装的超级复合物，以确定它们的相互作用如何使动粒将染色体连接到纺锤体微管。阿斯伯里和比金斯实验室将采取一种互补的方法，研究从野生型酵母和具有靶向缺陷的突变体中分离出的整个动粒，以定量评估每个动粒亚复合体对染色体微管偶联的贡献。沃德曼实验室将研究如何通过与正末端结合蛋白（+TIPs）和其他微管结合因子的相互作用来调节与动粒功能有关的微管调节酶的活性。沃德曼实验室还将观察单个微管解聚酶（MCAK）中的核苷酸周转，以测试它是否在从细丝分离之前酶促去除多个微管蛋白亚基。这些项目将使我们更接近一个完整的理解纺锤体的功能，阐明的机制，染色体的附件纺锤体微管，附件错误的纠正，染色体和纺锤体运动的调节。最终，对纺锤体的机械理解有望彻底改变针对纺锤体组件的化疗药物的设计。