Determining the spindle dynamics regulatory network with an integrated approach

描述（由申请人提供）：严格调节主轴微管（MT）动力学对有丝分裂的成功和保真度至关重要，但调节主轴微管动力学的机制尚不清楚。没有这些知识，一个完整的理解纺锤体调控在有丝分裂是不可能的。近年来，已经鉴定了许多MT调节蛋白，但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。沿着这一方向的第一个努力是最近鉴定的五种调节蛋白（KLP59C, KLP67A, Mast， EB1和Msps）网络，它们在中期控制着着丝粒MT (kMT) +端动力学。该网络利用MT聚合酶和解聚合酶（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，从而抵消负端的持续解聚合，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。本应用程序的目的是确定仅少数MT调节蛋白的作用如何在纺锤体MT +端从前期到后期产生广泛的动力学。中心假设是：控制其他有丝分裂阶段纺锤体MT动力学的调节网络可以通过改变中期网络各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，这一假设将通过追求三个具体目标来验证：(1)确定将正端动力学从净聚合（中期）转变为净解聚（后期A）的kMT调控网络的变化。(2)确定在中期前期产生驱动染色体聚集的正端动力学的kMT调控网络。(3)确定控制非着丝粒MT +端动态的调控网络，以在后期前期建立/维持双极纺锤体，并在后期b促进纺锤体延伸。这些目标将通过互补的计算机模拟、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。通过将假设的分子相互作用与细胞尺度的实验观察结果定量和严格地联系起来，模拟使我们能够区分单独实验无法由于缺乏必要的空间和时间分辨率而无法区分的其他分子机制。该计划的创新之处在于其假设的新颖性，以及用于检验这些假设的一系列广泛而独特的工具。提出的研究是重要的，因为它将提供主轴机械的一个基本模块的系统级的理解，这将填补在有丝分裂目前的知识严重的差距。此外，由此获得的知识将加深对非整倍体机制的理解——非整倍体是许多形式癌症的潜在原因。

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.