Organization of the Mammilian Mitotic Spindle

描述（由申请人提供）：染色体分离对物种繁殖和细胞活力至关重要，它是由一个复杂的微管上层结构驱动的，称为纺锤体。越来越多的证据表明，纺锤体组织和染色体运动是由微管相关蛋白（运动蛋白和非运动蛋白、结构蛋白）的协同作用和微管固有的动态特性驱动的。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促进纺锤体形态发生和染色体运动。具体地说，我们将继续开发一种无细胞的纺锤杆组织分析，我们以前开发的。我们建议将其用作调节纺锤体形成的酶的纯化富集源，并作为生物物理分析的微管紫外光源。我们还建议使用活细胞成像来确定特定蛋白质和蛋白质复合物如何在有丝分裂中促进纺锤体形态发生和染色体运动。这些结合的方法将产生洞察纺锤体组装和染色体运动在哺乳动物细胞的分子机制。

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.