Identification and characterization of a novel mitotic kinesin Kif25

描述（由申请人提供）：细胞分裂过程中有丝分裂纺锤体的正确方向对组织中干细胞群的发育以及调节和维持至关重要。将纺锤体定位在适当的分裂平面是一个由膜锚定蛋白介导并受有丝分裂激酶调节的活跃过程。分子马达被认为是一种力的产生器，使主轴定向并固定在适当的分割平面上。我们已经发现，在人类细胞中，很少研究的运动蛋白家族马达Kif25的消耗会导致有丝分裂纺锤体的定向错误和纺锤体组装缺陷。我们已经确定Kif25是一个c端运动蛋白马达，尽管我们对这个马达的结构和功能知之甚少。目前的研究重点是验证Kif25通过从其中心体位置对纺锤体微管产生力来调节纺锤体组装和方向的假设。单分子研究将用于表征这种新型马达的微管依赖活性。这些实验将补充并为我们在活细胞中Kif25的研究提供信息，我们将使用活细胞和固定细胞成像的组合来评估蛋白质消耗和显性阴性结构，以及Kif25相互作用伙伴的鉴定和表征。总的来说，这一提议将充分表征Kif25的功能，并为有丝分裂细胞中控制纺锤体取向的机制提供重要的新成分。

DESCRIPTION (provided by applicant): Correct orientation of the mitotic spindle during cell division is critically important in development as well as in the regulation and maintenance of stem cell populations in tissues. Orienting the spindle in the appropriate plane of division is an active process mediated by membrane-anchored proteins and subject to regulation by mitotic kinases. Molecular motors are thought to act as force generators that both orient and anchor the spindle in the proper plane of division. We have found that depletion of a little studied kinesin family motor, Kif25, in human cells causes misorientation of the mitotic spindle and defects in spindle assembly. We have determined that Kif25 is a C-terminal kinesin motor although the structure and function of this motor are poorly understood. The focus of the current proposal is to test the hypothesis that Kif25 regulates both spindle assembly and orientation by producing force on spindle microtubules from its centrosomal location. Single molecule studies will be used to characterize the microtubule-dependent activity of this novel motor. These experiments will compliment and inform our investigations of Kif25 in live cells where we will use a combination of live and fixed cell imaging to evaluate protein depletions and dominant negative constructs, as well as the identification and characterization of Kif25 interacting partners. Overal this proposal will fully characterize Kif25 function and contribute an important new component to the machinery controlling spindle orientation in mitotic cells.