The Biochemical Basis for the Mechanics of Cytokinesis

描述(申请人提供)：细胞质分裂，即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。胞质分裂的成功对单细胞的健康和保真度、多细胞的发展和疾病预防至关重要。在这项提议中，我们建立在我们的框架之上，以破译细胞质分裂机制和机械传感的分子基础。在目标1中，我们研究了肌球蛋白II-皮质素I-IQGAP2-Kinesin-6途径(赤道机械敏感途径)。这个蛋白质网络的结构就像一个机械力化学反馈系统，它整合了来自有丝分裂纺锤体和机械应力的信号，以调节卵裂沟处的肌球蛋白II水平。利用光漂白后的荧光恢复，我们将分析在施加机械应力和不施加机械应力的情况下以及在野生型和筛选出的突变体中关键蛋白的动态。由此，我们将破译蛋白质是如何相互依赖进行机械敏感积累的。我们将使用下拉图谱和LC-MS鉴定相互作用的蛋白质。然后将相互作用蛋白质清单与我们已经鉴定的遗传相互作用蛋白质清单进行比较。初步数据确定了参与翻译后修饰(PTM)的重要酶，如丙酰化和乙酰化。肌球蛋白II和其他蛋白质的乙酰化与哺乳动物有丝分裂和心脏收缩系统功能有关。因此，我们有兴趣了解这些PTM是否有助于机械感觉反馈系统和胞质分裂细胞形状的改变。我们还将使用纯化的蛋白质来确定IQGAP如何调节皮质素和可能的肌球蛋白II的功能。在目标2中，我们将扩展我们发现的微管-RACE-14-3-3-肌球蛋白II途径。这一途径控制着全球/极地皮质的力学、皮质张力和胞质形态的变化。我们将利用遗传和生化方法来确定种族的相互作用因素。我们还将确定14-3-3控制肌球蛋白II双极粗丝组装的机制。然后，我们将确定人类14-3-3蛋白如何调节人类肌球蛋白II粗丝组装。初步数据表明，14-3-3-肌球蛋白II相互作用的保守性，以及14-3-3和另一种癌症相关蛋白S100A4/MTS1之间可能存在的类似机制。总体而言，在这一更新应用中拟议的工作致力于发展对促进和调节细胞形状变化的力传递以及控制皮质张力、肌球蛋白II动力学和细胞质分裂的途径的复杂理解。

DESCRIPTION (provided by applicant): Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis success is critical to the health and fidelityof single cells, multi-cellular development, and disease prevention. In this proposal, we build upon our framework for deciphering the molecular underpinnings of cytokinesis mechanics and mechanosensing. Using Dictyostelium, in Aim 1, we study the Myosin II-Cortexillin I-IQGAP2- Kinesin-6 pathway (the equatorial mechanosensitive pathway). This network of proteins is structured like a mechanochemical feedback system that integrates signals from the mitotic spindle and mechanical stress to tune the myosin II levels at the cleavage furrow. Using fluorescence recovery after photobleaching, we will analyze the dynamics of key proteins with and without applied mechanical stress and in wild type and selected mutants. From this, we will decipher how proteins depend on each other for mechanosensitive accumulation. We will use pull-downs followed by LC-MS to identify interacting proteins. The list of interacting proteins wil then be compared to the lists of genetic interacting proteins we have already identified. Preliminary data identify important enzymes involved in post-translational modifications (PTMs), such as propionylation and acetylation. Acetylation of myosin II and other proteins has been implicated in mammalian mitosis and in cardiac contractile system function. Thus, we are interested to see if these PTMs contribute to the mechanosensory feedback system and cytokinesis cell shape change. We will also use purified proteins to determine how IQGAPs modulate cortexillin and possibly myosin II function. In Aim 2, we will expand the Microtubule-RacE-14-3-3-Myosin II pathway, which we discovered. This pathway controls the global/polar cortex mechanics, cortical tension and cytokinesis shape change. We will draw upon genetic and biochemical methods to identify interactors of racE. We will also determine the mechanism by which 14-3-3 controls myosin II bipolar thick filament assembly. We will then determine how human 14-3-3 proteins modulate human myosin II thick filament assembly. Preliminary data points toward the conserved nature of 14-3-3-myosin II interactions and a possible similar mechanism shared between 14-3-3 and another cancer-related protein S100A4/Mts1. Overall, proposed work in this renewal application strives to develop a sophisticated understanding of the force transmission that promotes and regulates cell shape change and the pathways that control cortical tension, myosin II dynamics, and cytokinesis.