The Biochemical Basis for the Mechanics of Cytokinesis

描述（由申请人提供）：胞质分裂，即母细胞分离为两个子细胞，是一个重要的生命过程。胞质分裂失败导致四倍体，然后是非整倍体，这是肿瘤形成的早期事件。我们一直在努力了解细胞如何利用蛋白质产生相关的细胞物理特性，驱动胞质分裂收缩。我们也有兴趣开发小分子抑制剂，以帮助基因功能鉴定和通路解剖，但最终目标是这些小分子抑制剂中的一些将具有临床应用。在本提案中，我们将以我们在第一个赠款周期启动的分析框架为基础，但我们也将以几种方式扩大我们的努力。在目标1中，我们将测量肌球蛋白-II和各种肌动蛋白交联剂在不同突变背景中的寿命，其中力学是已知的，以评估机械应变对交联剂寿命的影响。我们将测试我们的理解胞质分裂力学和动力学的分子控制通过测量皮质力学分裂突变株，我们有具体的预测力学。我们也将开始研究较低层次的细胞骨架功能，通过重建交联肌动蛋白网络，对它们施加机械应变，并研究交联剂和网络的行为。我们将使用FRAP或单颗粒分析来评估交联剂寿命的应变依赖性;我们有几个预测基于我们的体内研究。在目标2中，我们将利用我们的观察，即RacE负责在胞质分裂期间产生电阻应力，并限制我们发现的胞质分裂的机械感觉系统。在这个目标中，我们将确定RacE效应子来充实这一途径。我们将研究14-3-3，它被鉴定为诺考达唑的抑制剂。RacE和14-3-3之间的遗传相互作用进一步指向微管调节RacE和/或14-3-3的途径，其反过来调节全局肌动蛋白交联剂以控制胞质分裂收缩的动力学和机制。在目标3中，我们将通过鉴定我们已经恢复的REMI突变体中受影响的基因来扩大我们的分子研究。 公共卫生相关性：胞质分裂对于正常细胞生长和疾病过程如癌症非常重要，有希望提供新的抗癌药物靶点的丰富来源。我们正在努力了解胞质分裂在基本水平上是如何工作的，以及细胞如何使用蛋白质来产生收缩性的物理特征。最终，通过对这一过程的严格理解和完整的分子处理，应该可以开发出更好的癌症疗法，这些疗法针对特定类型的癌细胞的特性。

DESCRIPTION (provided by applicant): Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis failure leads to tetraploidy then aneuploidy, an early event in tumor formation. We have been striving to understand how cells use proteins to generate the relevant cellular physical properties that drive cytokinesis contractility. We are also interested in developing small molecule inhibitors to aid in gene function identification and pathway dissection, but with the ultimate goal that some of these small molecule inhibitors will have clinical applications. In this proposal, we will build on the analytical framework that we initiated in the first cycle of the grant, but we will also expand our effort in several ways. In Aim 1, we will measure the lifetimes of myosin-II and various actin crosslinkers in different mutant backgrounds where the mechanics are known in order to assess the consequences of mechanical strain on crosslinker lifetimes. We will test our understanding of the molecular control of cytokinesis mechanics and dynamics by measuring cortical mechanics of dividing mutant strains where we have specific predictions of the mechanics. We will also begin studying lower hierarchical levels of cytoskeletal function by reconstituting crosslinked actin networks, applying mechanical strain to them, and studying the behavior of the crosslinkers and the network. We will use either FRAP or single particle analysis to assess the strain dependency of crosslinker lifetimes; we have several predictions based on our in vivo studies. In Aim 2, we will draw upon our observations that RacE is responsible for generating resistive stresses during cytokinesis and for restricting the mechanosensory system that we discovered to cytokinesis. In this Aim, we will identify RacE effectors to flesh out this pathway. We will study 14-3-3, which was identified as a suppressor of nocodazole. Genetic interactions between RacE and 14-3-3 further point towards a pathway in which microtubules regulate RacE and/or 14-3-3, which in turn regulate global actin crosslinkers to control the dynamics and mechanics of cytokinesis contractility. In Aim 3, we will expand our molecular inquiry by identifying the affected genes in the REMI mutants we have already recovered. PUBLIC HEALTH RELEVANCE: Of great importance for normal cell growth and disease processes such as cancer, cytokinesis has the promise of providing a rich source of new anti-cancer drug targets. We are striving to understand how cytokinesis works at a fundamental level and how the cell uses proteins to generate the physical features of contractility. Ultimately, with a rigorous understanding and a complete molecular handle on the process, it should be possible to develop better cancer therapies that are tailored to the properties of specific types of cancer cells.