Organization of the Mammilian Mitotic Spindle

描述(申请人提供)：染色体分离对于物种的繁殖和细胞的活力是必不可少的，并由一种称为纺锤体的复杂的基于微管的超结构驱动。越来越多的证据表明，纺锤体的组织和染色体运动是由微管相关蛋白(运动和非运动、结构蛋白)以及微管固有的动力学特性共同作用的。我们的目标是使用体外生化技术来鉴定纺锤体组织所必需的蛋白质，然后应用体外和体内细胞生物学技术来确定这些蛋白质如何参与纺锤体的形态发生和染色体运动。具体地说，我们将继续开发我们之前开发的纺锤体极组织的无细胞测试。我们建议将其用作纯化调节纺锤体形成的酶的富集源，并用作生物物理分析的微管酯的来源。我们还建议使用活细胞成像来定义特定的蛋白质和蛋白质复合体如何在有丝分裂中对纺锤体形态发生和染色体运动做出贡献。这些结合的方法将有助于深入了解哺乳动物细胞中纺锤体组装和染色体运动的分子机制。 本研究的具体目的是：1)确定纺锤体组织蛋白NUMA上有丝分裂特异性磷酸化的特定位点，并利用体外和体内实验确定这些修饰如何调控NUMA功能；2)使用层析技术分离和鉴定在有丝分裂过程中调控NUMA功能的酶；3)使用活细胞成像确定在没有两种Kini Kinesin蛋白的情况下双极纺锤体的组织方式；4)结合活细胞显微镜和RNAi敲击来确定kintochore如何细化纺锤体微管；以及5)使用光学捕获显微镜直接测量纺锤体极微管末端的力。

DESCRIPTION (provided by applicant): Chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based superstructure called the spindle. Cumulative evidence demonstrates that spindle organization and chromosome movement are driven by the concerted actions of microtubule-associated proteins (motor and non-motor, structural proteins) and the inherent dynamic properties of microtubules. Our goal is to use in vitro biochemical techniques to identify proteins essential for spindle organization and then to apply in vitro and in vivo cell biological techniques to determine how those proteins contribute to spindle morphogenesis and chromosome movement. Specifically, we will continue to exploit a cell free assay for spindle pole organization that we developed previously. We propose to use it as an enriched source for purification of enzymes that regulate spindle formation and as a source of microtubule asters for biophysical analyses. We also propose to use live cell imaging to define how specific proteins and protein complexes contribute to both spindle morphogenesis and chromosome movement in mitosis. These combined approaches will generate insight into the molecular mechanisms of spindle assembly and chromosome movement in mammalian cells. The specific aims of this research are to: 1) identify specific sites of mitosis-specific phosphorylation on the spindle organizing protein NuMA and use in vitro and in vivo assays to determine how those modifications regulate NuMA function; 2) use chromatographic techniques to isolate and identify enzymes the regulate NuMA function during mitosis; 3) use live cell imaging to determine how bipolar spindles organize in the absence of two KinI kinesin proteins; 4) combine live cell microscopy with RNAi knock down to determine how kinetochores elaborate spindle microtubules; and 5) use optical trapping microscopy to directly measure force on microtubule minus ends at spindle poles.