Mechano-molecular regulation of kinetochore function

描述（由申请人提供）：染色体分离错误导致病理细胞状况称为非整倍体。非整倍体在妊娠早期导致大多数流产、出生缺陷，并与肿瘤发生和转移有关。人们早就认识到，细胞分裂的准确性取决于染色体的生物定向，即每个姐妹染色单体附着在来自相反纺锤极的微管上。力及其产生的张力是染色体双向调节的重要输入。事实上，适当的生物定向附着是通过着丝点产生的张力来稳定的。着丝点是一种蛋白质复合物，在细胞分裂时聚集在每个姐妹染色单体的着丝粒上，并将染色体连接到微管上。尽管染色体双向性对基因组完整性至关重要，但它并不是一个确定的结果。事实上，错误的附着体在细胞分裂过程中很常见，它们必须被纠正以避免非整倍体。错误纠正需要在不正确连接的染色体上选择性地破坏着丝点-微管（kt-MT）相互作用。目前的机制的知识负责稳定不正确的kt-MT附件是远远不够的。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体的细胞过程。本提案的目的是通过结合体外生化技术和活细胞检测在黑腹龙葵组织培养细胞中表征纠错的新方面。中心假设是，纠错通过两种途径发生：以着丝粒为基础的系统和以纺锤杆为基础的机制，每一种都受到在着丝点产生张力的力的影响。支持这项研究的基本原理是，确定错误纠正的机械分子基础将形成新疗法的发展，这些疗法可以调节错误纠正途径来调节非整倍体。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

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.