Determining the spindle dynamics regulatory network with an integrated approach

用综合方法确定主轴动态调节网络

• 批准号: 8700573
• 负责人: Ao Ma
• 金额: $ 24.28万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700573
• 关键词: Determining; spindle; dynamics; regulatory; network

DESCRIPTION (provided by applicant): Tight regulation of the spindle microtubule (MT) dynamics is vital for the success and fidelity of mitosis, but the mechanism for regulating spindle MT dynamics remains unknown. Without this knowledge, a complete understanding of spindle regulation during mitosis is impossible. In recent years, a number of MT regulatory proteins have been identified, but little is known of how they interact with each other to collectively manipulate spindle MT dynamics. The first endeavor along this direction is the recent identification of a network of five regulatory proteins (KLP59C, KLP67A, Mast, EB1 and Msps) that governs kinetochore MT (kMT) plus-end dynamics during metaphase. This network utilizes a complex balance between MT polymerases and depolymerases (instead of polymerases alone) to induce net polymerization at kMT plus-ends, which counteracts constant depolymerization at minus-ends to maintain the metaphase kMTs in a steady state. The long-term goal is to elucidate the molecular events that drive the assembly and function of the mitotic spindle. The objective of this application is to determine how the actions of only a handful of MT regulatory proteins give rise to the broad range of dynamics at spindle MT plus-ends from prometaphase through anaphase. The central hypothesis is: the regulatory networks controlling spindle MT dynamics at other mitotic stages can be attained by shifting the balance among the components of the metaphase network. Guided by strong preliminary data, this hypothesis will be tested through the pursuit of three specific aims: (1) Determine the changes to the kMT regulatory network that transform the plus-end dynamics from net polymerization (metaphase) to net depolymerization (anaphase A). (2) Determine the kMT regulatory network that generates the plus-end dynamics driving chromosome congression during prometaphase. (3) Determine the regulatory networks governing non-kinetochore MT plus-end dynamics to establish/maintain a bipolar spindle during pre- anaphase and to promote spindle elongation during anaphase B. These aims will be achieved using complementary computer simulation, a custom-developed automatic image tracking method, live cell imaging and RNAi-based protein knockdowns. By bridging hypothesized molecular interactions with cellular-scale experimental observables quantitatively and rigorously, simulations allow us to discriminate alternative molecular mechanisms that experiments alone cannot due to lack of necessary spatial and temporal resolution. The innovation of this plan stems from both the novelty of its hypotheses and the broad and unique array of tools it wields to test them. The proposed research is significant because it will provide a systems-level understanding of an essential module of the spindle machinery, which will fill a severe gap in the current knowledge of mitosis. Moreover, the knowledge thus gained will deepen the understanding of the mechanisms of aneuploidy--the underlying cause of many forms of cancers. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under-investigated area of mitosis that has potential applicability to understanding the mechanisms of aneuploidy--the underlying cause of many forms of cancers. The proposed research has relevance to public health, because the fundamental mechanisms to be investigated are expected to be conserved across the phyla. Thus, the findings are ultimately expected to be applicable to the health of human beings.

描述（由申请人提供）：纺锤体微管（MT）动力学的严格调节对于有丝分裂的成功和保真度至关重要，但调节纺锤体微管（MT）动力学的机制仍然未知。如果没有这些知识，就不可能完全了解有丝分裂期间的纺锤体调节。近年来，许多 MT 调节蛋白已被鉴定，但人们对它们如何相互作用以共同操纵纺锤体 MT 动力学知之甚少。沿着这个方向的第一个努力是最近鉴定了一个由五种调节蛋白（KLP59C、KLP67A、Mast、EB1 和 Msps）组成的网络，这些蛋白在中期控制着丝粒 MT (kMT) 的正端动力学。该网络利用 MT 聚合酶和解聚酶（而不是单独的聚合酶）之间的复杂平衡来诱导 kMT 正端的净聚合，这抵消了负端的持续解聚，以维持中期 kMT 处于稳定状态。长期目标是阐明驱动有丝分裂纺锤体组装和功能的分子事件。本应用的目的是确定少数 MT 调节蛋白的作用如何在纺锤体 MT 正端（从前期到后期）产生广泛的动态。中心假设是：在其他有丝分裂阶段控制纺锤体 MT 动态的调节网络可以通过改变中期网络组成部分之间的平衡来实现。在强有力的初步数据的指导下，该假设将通过追求三个具体目标进行检验：（1）确定 kMT 调节网络的变化，将正端动态从净聚合（中期）转变为净解聚（后期 A）。 (2) 确定 kMT 调控网络，该网络在前中期产生驱动染色体会聚的正端动力学。 (3) 确定控制非着丝粒 MT 加端动力学的调控网络，以在前期前期建立/维持双极纺锤体，并促进后期 B 期间纺锤体伸长。这些目标将通过使用互补的计算机模拟、定制开发的自动图像跟踪方法、活细胞成像和基于 RNAi 的蛋白质敲低来实现。通过定量和严格地将假设的分子相互作用与细胞规模的实验观测值联系起来，模拟使我们能够区分由于缺乏必要的空间和时间分辨率而仅靠实验无法区分的替代分子机制。该计划的创新源于其假设的新颖性以及它用来测试假设的广泛而独特的工具。这项研究意义重大，因为它将提供对主轴机械基本模块的系统级理解，这将填补当前有丝分裂知识的严重空白。此外，由此获得的知识将加深对非整倍性机制的理解——非整倍性是多种癌症的根本原因。 公共健康相关性：拟议的研究涉及有丝分裂的一个重要且尚未充分研究的领域，该领域对于理解非整倍性的机制（多种癌症的根本原因）具有潜在的适用性。拟议的研究与公共卫生相关，因为要研究的基本机制预计将在整个门中得到保留。因此，这些发现最终有望适用于人类的健康。