Motor protein dynamics and mitotic mechanisms

描述（由申请人提供）：我们的基因组的稳定繁殖需要通过基于微管的双极纺锤体将DMA均等分配到子细胞中。细胞分裂的错误与发育缺陷和疾病有关。我们的长期目标是确定哺乳动物有丝分裂过程中双极纺锤体组装的分子基础。我们提出了一个跨学科的方法来研究如何微管运输和聚合有助于双极纺锤体组装，专注于驱动蛋白-5和Polo样激酶（Plks），进化保守的蛋白质，其功能丧失导致单极纺锤体。有丝分裂纺锤体组装需要驱动蛋白-5依赖的微管交联和滑动。单分子研究已经表征了驱动蛋白-5在单个微管上的运动性。然而，驱动蛋白-5交联两个微管时的运动特性仍然未知。此外，对于微管交联所必需的驱动蛋白-5的同源四聚体结构的贡献仍然知之甚少。最近，我们和其他人已经表征了Plks的化学抑制剂。当与高分辨率显微镜相结合时，这些抑制剂可以成为检查细胞分裂动力学的强大工具。类似于Plk 1敲除，Plk受体处理的细胞形成缺乏适当中心体功能的单极纺锤体。在驱动蛋白-5抑制后观察到的中心体分离失败不太可能是缺乏Plk功能的细胞中单极纺锤体的原因，因为中心体可以通过与正常中期细胞中的距离相当的距离分离。我们还发现，纺锤体崩溃后Plk抑制与动粒纤维缩短和持久的非动粒纤维的外观。这些发现表明，微管聚合的Plk依赖性调节是双极纺锤体形成所必需的.在本项目中，我们将：（1）检查单个Eg 5，脊椎动物驱动蛋白-5，分子在两个微管之间的运动。(2)确定Eg 5的结构组织如何影响其运动特性和功能。(3)研究Polo样激酶在调节纺锤体微管动力学中的作用。(4)鉴定在分裂细胞中调节微管动力学的Polo样激酶底物。Eg 5和Polo样激酶的化学抑制剂目前正作为抗癌剂进行临床试验。我们的研究将深入了解这些蛋白质的细胞分裂功能，并影响靶向药物的开发。

DESCRIPTION (provided by applicant): The stable propagation of our genomes requires equal partitioning of DMA into daughter cells by a microtubule-based bipolar spindle. Errors in cell division are associated with developmental defects and disease. Our long-term goal is to determine the molecular basis for bipolar spindle assembly during mammalian mitosis. We propose an interdisciplinary approach to examine how microtubule transport and polymerization contribute to bipolar spindle assembly, focusing on kinesin-5 and Polo-like kinases (Plks), evolutionarily conserved proteins whose loss-of-function leads to monopolar spindles. Mitotic spindle assembly requires kinesin-5 dependent crosslinking and sliding of microtubules. Single molecule studies have characterized kinesin-5 motility on individual microtubules. However, the motile properties of kinesin-5 while crosslinking two microtubules remain unknown. In addition, the contribution of kinesin-5's homo- tetrameric structure, which is essential for microtubule crosslinking, remains poorly understood. Recently, we and others have characterized chemical inhibitors for Plks. When combined with high-resolution microscopy, these inhibitors can be powerful tools to examine cell division dynamics. Similar to Plk1 knockdown, Plk inhibitor-treated cells form monopolar spindles that lack proper centrosome function. Failure in centrosome separation, as observed upon kinesin-5 inhibition, is not likely to be the cause of monopolar spindles in cells lacking Plk function as centrosomes can be separated by distances comparable to that in normal metaphase cells. We have also found that spindle collapse upon Plk inhibition is associated with kinetochore fiber shortening and the appearance of persistent non-kinetochore fibers. These findings suggest that Plk- dependent regulation of microtubule polymerization is needed for bipolar spindle formation. In this project we will: (1) Examine the motility of single Eg5, vertebrate kinesin-5, molecules between two microtubules. (2) Determine how Eg5's structural organization influences its motile properties and function. (3) Examine the role of Polo-like kinases in regulating spindle microtubule dynamics. (4) Characterize Polo-like kinase substrates that regulate microtubule dynamics in dividing cells. Chemical inhibitors of Eg5 and Polo-like kinases are currently in clinical trials as anti-cancer agents. Our studies will provide insight into the cell division functions of these proteins and also impact the development of drugs that target them.