Determining the spindle dynamics regulatory network with an integrated approach

描述(由申请人提供)：对纺锤体微管(MT)动力学的严格调节对于有丝分裂的成功和精确度至关重要，但调节纺锤体微管动力学的机制仍不清楚。如果没有这些知识，就不可能完全了解有丝分裂过程中纺锤体的调节。近年来，一些MT调节蛋白已被鉴定，但对它们如何相互作用共同操纵纺锤体MT动力学知之甚少。沿着这个方向进行的第一个努力是最近发现了一个由五个调节蛋白(KLP59C、KLP67A、MAST、EB1和MSPs)组成的网络，它们在中期调控着动粒MT(KMT)+端动态。这个网络利用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，从而抵消负端的持续解聚，以维持中期KMT的稳定状态。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。这一应用的目的是确定只有少数MT调节蛋白的作用如何引起纺锤体MT+端从早中期到后期的广泛的动力学范围。中心假设是：控制其他有丝分裂阶段纺锤体MT动态的调控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。在强劲的初步数据指导下，这一假说将通过追求三个具体目标来验证：(1)确定国民党调控网络的变化，将加端动态从网络聚合(中期)转变为网络解聚(后期A)。(2)确定在前中期产生驱动染色体聚集的正端动态的KMT调控网络。(3)确定控制非动粒MT正端动力学的调控网络，以在前期和后期建立/维持两极纺锤体，并在后期B促进纺锤体的伸长。这些目标将通过补充计算机模拟、定制开发的自动图像跟踪方法、活细胞成像和基于RNAi的蛋白质敲除来实现。通过将假设的分子相互作用与细胞尺度的实验观测定量和严格地联系起来，模拟使我们能够区分实验本身由于缺乏必要的空间和时间分辨率而无法区分的替代分子机制。这一计划的创新既源于其假设的新颖性，也源于它所使用的广泛而独特的工具来检验这些假设。这项拟议的研究具有重要意义，因为它将提供对纺锤体机械的基本模块的系统水平的理解，这将填补目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体机制的理解--非整倍体是许多形式癌症的根本原因。 与公共卫生相关：拟议的研究是关于有丝分裂的一个重要且研究不足的领域，该领域可能适用于理解非整倍体的机制--非整倍体是许多形式癌症的根本原因。这项拟议的研究与公共卫生相关，因为预计要研究的基本机制将在整个门类中保守。因此，这些发现最终有望适用于人类的健康。

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.