The Biochemical Basis for the Mechanics of Cytokinesis

项目摘要 细胞质分裂，即将一个母细胞分裂成两个子细胞，是一个必不可少的生命过程。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。我们一直在努力理解 细胞如何利用蛋白质产生相关的细胞物理特性，从而驱动胞质收缩。 我们还对开发小分子抑制剂以帮助基因功能鉴定和 途径解剖，但最终目标是这些小分子抑制剂中的一些将用于临床 申请。在这项提案中，我们将建立在我们在第一个周期启动的分析框架的基础上 但我们也将在几个方面扩大我们的努力。在目标1中，我们将测量肌球蛋白-II的寿命 和不同突变背景中的各种肌动蛋白交联物，其中机制已知，以便 评估机械应变对交联剂寿命的影响。我们将测试我们对 通过测量分裂突变体的皮质力学来控制胞质分裂的力学和动力学 我们对其机理有具体预测的菌株。我们还将开始研究较低的等级 通过重组交联的肌动蛋白网络，对其施加机械应变，从而提高细胞骨架的功能水平， 并研究交联剂和网络的行为。我们将使用FRAP或单粒子 分析以评估交联剂寿命的应变相关性；基于我们的预测，我们有几个预测 活体研究。在目标2中，我们将利用我们的观察结果，即种族是产生阻力的原因 在胞质分裂过程中的压力，以及我们发现的仅限于胞质分裂的机械感觉系统。 在这个目标中，我们将确定种族效应器来充实这一途径。我们将研究已确定的14-3-3 作为诺康唑的抑制剂。小种和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，进而调节全球肌动蛋白交联物以 控制胞质收缩的动力学和力学。在目标3中，我们将扩展我们的分子研究 通过鉴定REMI突变体中受影响的基因，我们已经恢复了。

Project Summary 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.