Mechano-molecular regulation of kinetochore function

描述(申请人提供)：染色体分离错误导致一种称为非整倍体的病理细胞疾病。非整倍体导致了怀孕前三个月的大多数流产，出生缺陷，并与肿瘤的发生和转移有关。长期以来，人们一直认为，细胞分裂的准确性取决于染色体变得双向，即每个姐妹染色单体连接到来自相反纺锤体极的微管上。力及其产生的张力是调节染色体双向的不可或缺的输入。事实上，正确的双向连接是通过跨着丝粒产生的张力来稳定的--着丝粒是一种蛋白质复合体，在细胞分裂期间组装在每个姐妹染色单体的着丝粒上，并将染色体与微管连接起来。尽管染色体双向定位对基因组的完整性至关重要，但它并不是一个确定的结果。事实上，错误的附着在细胞分裂过程中很常见，必须加以纠正，以避免非整倍体。纠错需要在不正确连接的染色体上选择性地使着丝粒-微管(kt-MT)相互作用失稳。目前对导致不正确的kt-mt附着体失稳的机制的了解还远远不完整。长期目标是描述细胞分裂的基本分子特性，并在这样做的过程中，确定可以通过治疗来控制非整倍体的细胞过程。这项建议的目的是通过将体外生化技术与活细胞分析相结合来表征在黑腹果蝇组织培养细胞中纠错的新方面。中心假设是，纠错通过两条途径发生：基于着丝粒的系统和基于纺锤杆的机构，每一条途径都受到在动心产生张力的力的影响。支持这项研究的基本原理是，确定纠错的机械-分子基础将有助于开发新的疗法，通过调节纠错途径来调节非整倍体。核心假设将通过三个具体目标进行检验。目标1将侧重于功能贡献 依赖于张力的结构变化，称为动粒内拉伸，到kt-MT附着稳定性。目标2的目标是描述一种新的错误纠正途径，该途径假设是由基于极点的激酶梯度介导的。目标3将阐述运动蛋白-10产生极地弹射力的力学基础。已经开发和实施了一系列稳定的细胞系和成像技术，以至于完成这项工作既是可行的，也有望大大促进对纠错的基本过程以及力对其调节的贡献的理解。这种方法是创新，因为它将分子工程与体外和活细胞中的高分辨率和超分辨率显微镜技术相结合，以确定关键细胞过程的分子基础。这项研究意义重大，因为它有望确定可利用的纠正机制的接入点，这些接入点可以在治疗上有针对性地治疗和预防一系列人类疾病。

DESCRIPTION (provided by applicant): Errors in chromosome segregation result in a pathological cellular condition called aneuploidy. Aneuploidy causes a majority of miscarriages in the first trimester, birth defects and has been linked to tumorigenesis and metastasis. It has long been appreciated that the accuracy of cell division depends on chromosomes becoming bioriented, a configuration where each sister chromatid is attached to microtubules from opposing spindle poles. Force and the tension that it produces are integral inputs to the regulation of chromosome biorientation. In fact, properly bioriented attachments are stabilized by tension generated across the kinetochore - the protein complex that assembles during cell division on the centromeres of each sister chromatid and links chromosomes to microtubules. Despite its central importance to genomic integrity, chromosome biorientation is not an assured outcome. In fact, erroneous attachments are common during cell division and they must be corrected to avoid aneuploidy. Error correction requires the selective destabilization of kinetochore-microtubule (kt-MT) interactions on improperly attached chromosomes. Current knowledge of the mechanisms responsible for de- stabilizing incorrect kt-MT attachments is far from complete. The long-term goal is to describe the fundamental molecular properties of cell division and, in doing so, to identify cellular processes that can be targeted by therapies to control aneuploidy. The objective of this proposal is to characterize novel aspects of error correction by combining in vitro biochemical techniques with live-cell assays in D. melanogaster tissue culture cells. The central hypothesis is that error correction occurs via two pathways: a centromere-based system and a spindle pole-based mechanism, each of which is impacted by forces that produce tension at kinetochores. The rationale underpinning the research is that determining the mechano-molecular basis of error correction will in- form the development of novel therapies that modulate error correction pathways to regulate aneuploidy. The central hypothesis will be tested with three specific aims. Aim 1 will focus on the functional contribution of a tension-dependent structural change, called intrakinetochore stretch, to kt-MT attachment stability. The goal of aim 2 is to describe a novel error correction pathway that is hypothesized to be mediated by pole-based kinase gradients. Aim 3 will address the mechanical basis of polar ejection force generation by kinesin-10. A battery of stable cell lines and imaging techniques have been developed and implemented to an extent that completion of the work is both feasible and expected to significantly advance the understanding of the essential process of error correction and the contribution of force to its regulation. The approach is innovative because it unites molecular engineering with high- and super-resolution microscopy techniques both in vitro and in living cells to define the molecular foundations of a critical cellular proces. The research is significant because it is expected to identify exploitable access points to the correction machinery that could be therapeutically targeted to treat and prevent a range of human diseases.