MAMMALIAN KINETOCHORE CONTROL OF MICROTUBULE DYNAMICS

描述（由申请人提供）：这个项目的长期目标是定义一个高分辨率的结构图，解释哺乳动物动着点如何协调和控制微管动力学。哺乳动物着丝点是一种独特的结构，其功能是将染色体连接到纺锤体微管上，产生染色体排列所需的运动，并在染色体排列完成之前阻止细胞分裂。这一功能对于遗传密码的可靠传递至关重要，因此对人类健康和所有生物的生存至关重要。在这个应用中，我们建议使用高通量电子断层扫描来研究着丝点功能和微管正端发生的组装和拆卸之间的动态转变之间的关键联系。我们的方法利用了伴随动态变化的微管正端的显著结构变化。在Specific Aim 1中，我们通过优化标本制备以获得最大对比度来建立高通量数据处理；采用最新的自动层析数据采集技术；开发降噪和自动特征提取算法；并制定了分类分析方案。在具体目标2中，我们将使用这种高通量分析来创建有丝分裂不同阶段的着丝点微管加端结构的大型数据库。该数据库的分类分析将用于检验当前关于着丝点在不同方向运动过程中与微管正端相互作用的假设和模型。在特异性目标3中，我们将使用相同的方法来测试假设，即选择抗有丝分裂药物治疗会改变正端构象，去除着丝点蛋白CENP-E会改变正端构象和微管附着的模式。拟议的研究将推进我们对着丝点功能机制的认识，从而提高我们理解和治疗由有丝分裂和减数分裂功能障碍引起的人类疾病的能力，包括出生缺陷、流产和多种形式的癌症。此外，Specific Aim 1的技术发展将使其他使用电子断层扫描的研究人员受益。

DESCRIPTION (provided by applicant):The long-range goal of this project is to define a high-resolution structural map that explains how mammalian kinetochores coordinate and control microtubule dynamics. The mammalian kinetochore is a distinctive structure that functions to attach chromosomes to spindle microtubules, produce motion required for chromosome alignment, and prevent cell division before proper alignment is achieved. This function is crucial for reliable transmission of genetic code, and thereby vital to human health and the survival of all organisms. In this application, we propose to use high-throughput electron tomography to investigate the critical link between the kinetochore function and the dynamic transitions between assembly and disassembly that take place at microtubule plus-ends. Our approach takes advantage of the striking structural changes of microtubule plus-ends that accompany changes in dynamic state. In Specific Aim 1 we establish high-throughput data processing by optimizing specimen preparation for maximize contrast; implementing the latest advances in automated tomographic data collection; developing algorithms for noise reduction and automated feature extraction; and developing a scheme for classification analysis. In Specific aim 2 we will use this high-throughput analysis to create a large database of kinetochore microtubule plus-end structures from different stages of mitosis. Classification analysis of this database will be used to test current hypotheses and models concerning kinetochore interactions with microtubule plus-ends during different directions of kinetochore movement. In Specific Aim 3 we will use the same approach to test the hypotheses that treatment with select anti-mitotic drugs alters the plus-end conformations and that removal of kinetochore protein CENP-E alters plus-end conformation and the pattern of microtubule attachments. The proposed studies will advance our knowledge of kinetochore functional mechanisms and thereby enhance our ability to understand and treat human disorders arising from mitotic and meiotic malfunction, including birth defects, miscarriages, and many forms of cancer. In addition, technical development in Specific Aim 1 will benefit other investigators using electron tomography.