Mechano-molecular regulation of kinetochore function

描述（由申请人提供）：染色体分离错误导致称为非整倍性的病理性细胞状况。非整倍体导致大多数妊娠早期流产、出生缺陷，并与肿瘤发生和转移有关。长期以来，人们一直认为细胞分裂的准确性取决于染色体的双向取向，即每个姐妹染色单体从相对的纺锤体两极附着到微管上。力和它产生的张力是染色体双向取向调节不可或缺的输入。事实上，正确的双向附着是通过跨动粒产生的张力来稳定的，动粒是一种蛋白质复合物，在细胞分裂期间在每个姐妹染色单体的着丝粒上组装，并将染色体连接到微管。尽管染色体双定向对基因组的完整性至关重要，但它并不是一个可靠的结果。事实上，错误的连接在细胞分裂过程中是常见的，它们必须被纠正以避免非整倍体。错误纠正需要选择性的不稳定的着丝粒微管（kt-MT）的相互作用不正确连接的染色体。目前对导致不正确的kt-MT附件不稳定的机制的了解还远远不够。长期目标是描述细胞分裂的基本分子特性，并在此过程中确定可以通过治疗来控制非整倍性的细胞过程。本提案的目的是通过将体外生物化学技术与D.黑腹组织培养细胞核心假设是错误纠正通过两条途径发生：基于着丝粒的系统和基于纺锤体的机制，每一个都受到在动粒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调节纠错途径以调节非整倍性的新疗法。中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 的张力依赖性的结构变化，称为动粒内拉伸，对kt-MT附着稳定性。目的2的目标是描述一种新的错误校正途径，该途径假设由基于极点的激酶梯度介导。目标3将解决由驱动蛋白-10产生极性喷射力的机械基础。电池的稳定的细胞系和成像技术已经开发和实施的程度，完成这项工作是可行的，并预计将大大推进错误校正的基本过程和力的贡献，其监管的理解。该方法是创新的，因为它将分子工程与高分辨率和超分辨率显微镜技术结合在一起，在体外和活细胞中定义关键细胞过程的分子基础。这项研究意义重大，因为它有望确定矫正机制的可利用接入点，这些接入点可以用于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.