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: 8532926
• 负责人: Ao Ma
• 金额: $ 27.95万
• 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/8532926
• 关键词: Anaphase; Aneuploidy; Area; Automobile Driving; Binding; Biological; Cells; Chromosomes; Complex; Computer Simulation; Custom; Data; Equilibrium; Event; Goals; Health; Human; Image; Kinetochores; Knowledge; Length; Life; Link; Malignant Neoplasms; Metaphase; Methods; Microtubule Polymerization; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Morphogenesis; Plus End of the Microtubule; Polymerase; Positioning Attribute; Prometaphase; Proteins; Public Health; Publishing; RNA Interference; Regulation; Research; Resolution; Staging; System; Testing; Up-Regulation; Update; Work; base; cellular imaging; depolymerization; genetic regulatory protein; in vivo; innovation; katanin; polymerization; public health relevance; research study; segregation; simulation; stem; success; tool

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，但是调节纺锤体MT动力学的机制仍然未知。没有这些知识，就不可能完全了解有丝分裂期间的主轴调节。近年来，已经确定了许多MT调节蛋白，但对它们如何相互作用以集体操纵纺锤体MT动力学知之甚少。沿该方向的第一个努力是最近确定了五个调节蛋白（KLP59C，KLP67A，MAST，EB1和MSP）的网络，该网络在中期期间控制了动型MT（KMT）Plus-End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 Plus-End的净聚合，从而抵消了减去末端的恒定去聚合以将中期KMT保持在稳态状态。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。该应用的目的是确定仅少数MT调节蛋白的作用如何产生主轴MT Plus末端的广泛动力学，从Prometathase通过后期进行。中心假设是：可以通过转移中期网络组件之间的平衡来实现控制纺锤体MT动力学的调节网络。在强大的初步数据的指导下，将通过追求三个特定目的来检验该假设：（1）确定KMT调节网络的变化，这些变化将加利端动力学从净聚合（et exphase）转化为净解聚化（过后A）。 （2）确定在Prometathase期间生成驱动染色体会议的加号动力学的KMT调节网络。 （3）确定管理非运动型MT Plus-End动力学的调节网络，以建立/维持双极轴，并在后期B中促进纺锤体伸长率。这些目标将使用互补的计算机模拟来实现，这是一种自动开发的自动图像跟踪方法，实时的电池成像和RNAI基于RNAI的protein protine sumpterins。通过桥接假设的分子相互作用与细胞尺度的实验性观察物进行定量和严格的实验性相互作用，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无法实验的替代分子机制。该计划的创新既源于其假设的新颖性，也源于其对其进行测试的广泛而独特的工具。拟议的研究很重要，因为它将提供对纺锤体机械基本模块的系统级别的理解，这将填补当前有丝分裂知识的严重空白。此外，因此获得的知识将加深对非整倍性机制的理解 - 多种形式的癌症的根本原因。 公共卫生相关性：拟议的研究是有丝分裂的重要且不足的有丝分裂区域，具有潜在的适用性，可用于理解非整倍性的机制 - 多种形式的癌症的根本原因。拟议的研究与公共卫生有关，因为预计将要研究的基本机制在整个门中保存。因此，最终期望这些发现适用于人类的健康。