Multicolor TIRF microscope for studying mitotic spindle components at the single

描述(申请人提供)：染色体在细胞分裂过程中由微管分子机器，即有丝分裂纺锤体组织和分离。我们的总体目标是使用纯部件重建主轴功能，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行的。在此，我们请求支持一种能够同时记录单个蛋白质分子、多蛋白质复合体和动态微管细丝之间相互作用的超灵敏荧光显微镜。该仪器基于奥林巴斯IX51倒置显微镜，配备了全内反射荧光(TIRF)照明和三个ANDOR EMCCD摄像头，可以实现快速、多色的分子跟踪。它将由四名由NIH资助的研究人员使用，每个人都有互补的专业知识，相互密切合作。戴维斯实验室将研究由组成动点的六种蛋白质亚复合体的纯重组形式组装而成的超级复合体，以确定它们的相互作用如何使动点将染色体连接到纺锤体微管。Asbury和Biggins实验室将采取互补的方法，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-微管耦合的贡献。华德曼实验室将研究与动粒功能有关的微管调节酶的活性是如何通过与正端结合蛋白(+TIPS)和其他微管结合因子的相互作用来调节的。华德曼实验室还将观察单个微管解聚酶(MCAK)中的核苷酸周转，以测试它在从细丝分离之前是否通过酶作用移除多个微管蛋白亚基。这些项目将通过阐明染色体与纺锤体微管连接的潜在机制，纠正连接错误，以及染色体和纺锤体运动的调节，使我们更接近于完整地了解纺锤体的功能。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。

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.