Organization of the Mammilian Mitotic Spindle

描述（由申请人提供）：染色体分离对于物种繁殖和细胞活力至关重要，并由称为纺锤体的复杂微管超结构驱动。累积的证据表明，纺锤体组织和染色体运动是由微管相关蛋白（马达和非马达，结构蛋白）和微管的固有动力学特性的协同作用驱动的。我们的目标是使用体外生物化学技术来识别蛋白质的纺锤体组织，然后应用在体外和体内细胞生物学技术，以确定这些蛋白质如何有助于纺锤体形态发生和染色体运动。具体来说，我们将继续利用我们以前开发的无细胞检测纺锤极组织。我们建议使用它作为一个丰富的来源，用于纯化的酶，调节纺锤体的形成，并作为一个来源的微管星生物物理分析。我们还建议使用活细胞成像来确定特定的蛋白质和蛋白质复合物如何有助于纺锤体形态发生和有丝分裂中的染色体运动。这些结合的方法将产生深入了解哺乳动物细胞中的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）鉴定纺锤体组织蛋白NuMA上有丝分裂特异性磷酸化的特定位点，并使用体外和体内测定来确定这些修饰如何调节NuMA功能：2）使用色谱技术分离和鉴定在有丝分裂期间调节NuMA功能的酶; 3）使用活细胞成像来确定在两种KinI驱动蛋白不存在的情况下双极纺锤体是如何组织的; 4）将活细胞显微术与RNAi敲除结合联合收割机来确定动粒是如何精心制作纺锤体微管的; 5）采用光学捕获显微镜直接测量纺锤体两极微管负端的受力。

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.