Control of Microtubule Dynamics for Spindle Assembly and Chromosome Segregation

描述(申请人提供)：有丝分裂的主要目标是在两个子细胞之间准确地分配遗传物质。减数分裂或有丝分裂的缺陷会导致非整倍体，这是导致出生缺陷的重要原因，也是肿瘤发生的标志。正确的纺锤体功能依赖于微管(MT)动力学的精确空间和时间控制以及马达蛋白质的力的整合。调节的MT动力学缺陷导致纺锤体多极化、动粒-MT连接不当、有丝分裂进程延迟和染色体分离不当。尽管为主轴生成了一个广泛的部件列表，但一个主要的悬而未决的问题是了解MT动力学和马达蛋白活性是如何在空间和时间上调节的，以确保正确的主轴结构和功能。 这在一定程度上是由于缺乏适当的工具，可以用来将关键的调控生化事件与这些事件在纺锤体中的空间控制相联系。我们最近实施的新的基于FRET的生物传感器与薄膜和超分辨率显微镜相结合，现在使我们能够解决这个重要的问题。在这个方案中，我们将：1)通过重构MT滑动，并使用新型分子间FRET报告的薄膜成像，定义MT滑动是如何在空间上被调制以控制纺锤体组织的。这些实验将建立一个范例，说明运动活动的空间控制如何有助于纺锤体的整体组织，并将定义 XCTK2有助于极聚焦，可作为中心体扩增肿瘤治疗开发的靶点。2)通过测试MCAK的CT如何调节MCAK的构象和活性，确定Aurora B激酶如何调节MCAK中的蛋白质构象、靶向和有丝分裂运动的功能。我们将使用非洲爪哇纺锤体组装分析和各种MCAK CT突变体以及我们的MCAK生物传感器的荧光寿命成像来确定这种构象调控发生的地点和时间。这项工作将定义控制MCAK的空间控制机制，也将作为通过激酶网络调节蛋白质活性的模型。3)通过检验非着丝点MT的过度聚合增加错误附着的动点并降低有丝分裂的保真度的模型，定义空间上不同MT群体的整合如何有助于有丝分裂的保真度。动蛋白敲除后染色体-MT相互作用的3D-SIM超分辨率成像将使我们能够可视化这些蛋白质的丢失是如何导致染色体定向错误的。使用单分子成像和Palm/Storm的体外重建分析将定义不同的MT动力学调节器如何协同控制加端动力学。总之，这些研究将弥合体外生化活动如何影响体内纺锤体结构和功能之间的知识差距。

DESCRIPTION (provided by applicant): The major goal of mitosis is to distribute the genetic material accurately between the two daughter cells. Defects in meiosis or mitosis lead to aneuploidy, which is a significant cause of birth defects and is a hallmark of tumorigenesis. Proper spindle function relies on precise spatial and temporal control of microtubule (MT) dynamics and the integration of forces of motor proteins. Defects in regulated MT dynamics lead to spindle multi-polarity, improper kinetochore-MT attachments, delayed mitotic progression, and improper chromosome segregation. Despite the generation of an extensive parts list for the spindle, a major unanswered question is to understand how MT dynamics and motor protein activity are spatially and temporally regulated to ensure proper spindle architecture and function. This has been due, in part, to a lack of appropriate tools that could be used to relate key regulatory biochemical events to where those events are controlled spatially in the spindle. Our recent implementation of new FRET-based biosensors combined with FLIM and super resolution microscopy is now enabling us to address this important question. In this proposal we will: 1) Define how MT sliding is spatially modulated to control spindle organization by reconstituting MT sliding, and using FLIM imaging of novel intermolecular FRET reporters. These experiments will establish a paradigm for how the spatial control of motor activity contributes to the global organization of the spindle and will also define the critical features of XCTK2 that contribute to pole focusing and could be targeted for therapeutic development for treatment of tumors with centrosome amplification. 2) Define how Aurora B kinase modulates protein conformation, targeting, and function of the mitotic kinesin MCAK by testing how the CT of MCAK modulates conformation and activity. We will use Xenopus spindle assembly assays with various MCAK CT mutants and Fluorescence Lifetime Imaging of our MCAK biosensors to define where and when this conformational regulation occurs. This work will define spatial control mechanisms for the control of MCAK that will also serve as a model for regulation of protein activity by kinase networks. 3) Define how the integration of spatially distinct MT populations contributes to mitotic fidelity by testing the model that excess polymerization of non-kinetochore MTs increases mal-attached kinetochores and reduces mitotic fidelity. 3D-SIM super-resolution imaging of chromosome- MT interactions after kinesin knockdown will allow us to visualize how loss of these proteins lead to mal- oriented chromosomes. In vitro reconstitution assays using single molecule imaging and PALM/STORM will define how different MT dynamics regulators cooperatively control plus-end dynamics. Together these studies will bridge the gap in knowledge between how biochemical activities in vitro impact spindle structure and function in vivo.