Mechano-molecular regulation of kinetochore function

项目概要和摘要 染色体错误分离导致称为非整倍性的病理细胞状况。非整倍体原因 大多数流产发生在怀孕的前三个月，出生缺陷，并与肿瘤发生和转移有关， tasis。细胞分裂的准确性取决于染色体的双向取向， 姐妹染色单体从相对的纺锤体极附着到微管（MT）上。力和张力 生产是基因组高保真传输的组成部分。双向附件变得稳定， 跨动粒（KT）产生的张力-一种大型蛋白质复合物，具有两种基本功能， (1)染色体和纺锤体MT之间的联系，以及（2）纺锤体组装检查的调节中心- 延迟后期开始的点（SAC），直到染色体附着在纺锤体MT上并双向定向。内含子- 空间无序蛋白（IDP），其是不具有可再现折叠或三级结构的蛋白质， 在KT区和染色体表面有丰富的事实上，约50%的Dro- 预测sophila KT是本质上无序的，而IDP富集的隔室称为perichro- 有丝分裂染色体中，mosomal层占染色体质量的30%以上。本文研究了“非- 结构”-特别是内在无序蛋白（IDP）在细胞分裂中的作用。长期目标是 描述细胞分裂的基本分子特性，并在此过程中识别细胞过程 可以通过治疗来控制非整倍体。本提案的目的是在体外将联合收割机与双- 化学和生物物理测定与活细胞实验在D.黑胃和人组织培养 细胞来研究参与细胞分裂的保守IDP。中心假设是机械感应和 力转导IDP，定位于KT，着丝粒和染色质，利用力的产生， 动力学纺锤体MT调节纺锤体组装检查点（SAC）信号传导和染色体运动。的 支持这项研究的基本原理是基于这样一个事实，即感兴趣的国内流离失所者处于独特的地位， 经验MT依赖的力量。中心假设将通过三个具体目标进行检验。目标1将重点 调节检查点蛋白-KT相互作用，我们假设是机械性质。的目标 目的2是表征围绕KT组装的新型杯结构，该结构涂覆有SAC蛋白质，并且 我们假设是为国内流离失所者准备的。目标3将研究一个非常大的蛋白质的贡献，这是97%的差异， 命令，称为Ki-67细胞分裂。这些目标的实现预计将对基本的 力转导IDP的知识，以细胞分裂的保真度。这种方法是创新的，因为它对 基于细胞的实验，包括使用活细胞力传感器和单分子生物物理测定， 国内流离失所者。这项研究意义重大，因为它开辟了研究保守的国内流离失所者的新途径， 在治疗上用于调节SAC活性和靶向Ki-67 -一种长期用作增殖抑制剂的蛋白质， 它是癌症诊断中的一种标志物，但其在细胞分裂中的确切功能尚不清楚。

PROJECT SUMMARY & ABSTRACT Chromosome mis-segregation results 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 metas- tasis. The accuracy of cell division depends on chromosomes becoming bioriented, a configuration where each sister chromatid is attached to microtubules (MTs) from opposing spindle poles. Force and the tension that it produces are integral to high fidelity transmission of the genome. Bioriented attachments become stabilized by tension generated across the kinetochore (KT) – a large protein complex that fulfills two essential functions as (1) the link between chromosomes and spindle MTs and (2) the regulatory hub for a spindle assembly check- point (SAC) that delays anaphase onset until chromosomes are attached to spindle MTs and bioriented. Intrin- sically disordered proteins (IDPs), which are proteins that do not have reproducible folds or tertiary structures, are abundant at the KT and on the surface of chromosomes. In fact, ~50% of the molecular mass of the Dro- sophila KT is predicted to be intrinsically disordered while an IDP enriched compartment called the perichro- mosomal layer accounts for >30% of the mitotic chromosome mass. This proposal studies the function of “un- structure” – specifically the role of intrinsically disordered proteins (IDPs) in cell division. 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 combine in vitro bi- ochemical and biophysical assays with live-cell experimentation in D. melanogaster and human tissue culture cells to study conserved IDPs involved in cell division. The central hypothesis is that mechano-sensing and force-transducing IDPs, which localize to KTs, centromeres and chromatin, harness force-generation by dy- namic spindle MTs to regulate spindle assembly checkpoint (SAC) signaling and chromosome movement. The rationale underpinning the research is based on the fact that the IDPs of interest are uniquely positioned to ex- perience MT-dependent forces. The central hypothesis will be tested with three specific aims. Aim 1 will focus on regulation of a checkpoint protein-KT interaction that we hypothesize is mechanical in nature. The goal of aim 2 is to characterize a novel cup structure assembled around KTs that is coated with a SAC protein and that we hypothesize is enriched for IDPs. Aim 3 will study the contribution of a very large protein, which is 97% dis- ordered, called Ki-67 to cell division. Completion of these aims is expected to significantly impact basic knowledge of force-transducing IDPs to the fidelity of cell division. The approach is innovative because it pairs cell-based experiments including the use of live-cell force sensors with single molecule biophysical assays on IDPs. The research is significant because it opens new avenues of research into conserved IDPs that could be exploited therapeutically to modulate SAC activity and to target Ki-67 – a protein long-used as a proliferation marker in cancer diagnosis, but whose precise function in cell division remains unclear.