The Biochemical Basis for the Mechanics of Cytokinesis

描述（由申请人提供）：细胞分裂是一个母细胞分裂成两个子细胞，是一个重要的生命过程。细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使用蛋白质来产生驱动细胞分裂收缩的相关细胞物理特性。我们也对开发小分子抑制剂来帮助基因功能鉴定和途径解剖感兴趣，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。在这项建议中，我们将以我们在赠款的第一个周期中发起的分析框架为基础，但我们也将在几个方面扩大我们的努力。在目标1中，我们将测量不同突变背景下肌凝蛋白- ii和各种肌动蛋白交联剂的寿命，其中力学已知，以评估机械应变对交联剂寿命的影响。我们将通过测量分裂突变株的皮质力学来测试我们对细胞分裂力学和动力学的分子控制的理解，我们对这些力学有具体的预测。我们还将通过重构交联肌动蛋白网络，对其施加机械应变，并研究交联剂和网络的行为，开始研究较低层次的细胞骨架功能。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；根据我们的体内研究，我们有几个预测。在目标2中，我们将借鉴我们的观察，RacE负责在细胞质分裂过程中产生抗性应力，并限制我们发现的机械感觉系统进行细胞质分裂。在本研究中，我们将确定RacE效应物来充实这一途径。我们将研究14-3-3，它被确定为诺可达唑的抑制因子。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，而14-3-3反过来调节全球肌动蛋白交联剂来控制细胞分裂收缩的动力学和机制。在Aim 3中，我们将通过识别我们已经恢复的REMI突变体中的受影响基因来扩展我们的分子调查。

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.