The Biochemical Basis for the Mechanics of Cytokinesis

项目摘要 胞质分裂，即母细胞分裂成两个子细胞，是生命的一个基本过程。胞质 成功对于单细胞的健康和保真度、多细胞发育和疾病预防至关重要。 在这个建议中，我们建立在我们的框架，破译细胞质分裂的分子基础上 力学和机械传感。在目的1中，我们使用网囊藻，研究了肌球蛋白II-皮质素I-IQGAP 2- 驱动蛋白-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 机械化学反馈系统，其整合来自有丝分裂纺锤体和机械应力的信号， 调节卵裂沟处的肌球蛋白II水平。使用光漂白后的荧光恢复，我们将 分析在施加和不施加机械应力的情况下以及在野生型和选择的情况下关键蛋白质的动力学 变种人由此，我们将破译蛋白质如何相互依赖以实现机械敏感性积累。 我们将使用下拉法，然后使用LC-MS来鉴定相互作用的蛋白质。相互作用蛋白质的列表将 然后与我们已经鉴定的遗传相互作用蛋白质列表进行比较。初步数据 鉴定参与翻译后修饰（PTM）的重要酶，如丙酰化， 乙酰化肌球蛋白II和其他蛋白质的乙酰化与哺乳动物的有丝分裂和细胞分裂有关。 心脏收缩系统功能。因此，我们有兴趣看看这些PTM是否有助于 机械感觉反馈系统和胞质分裂细胞形状改变。我们还将使用纯化的蛋白质， 确定IQGAP如何调节皮质素和可能的肌球蛋白II功能。在目标2中，我们将扩大 微管-RacE-14 - 3 - 3-Myosin II途径，我们发现。这条途径控制着全球/极地 皮质力学、皮质张力和胞质分裂形态改变。我们将利用遗传学和生物化学 鉴定racE相互作用物的方法。我们还将确定14 - 3 - 3控制 肌球蛋白II双极粗丝组装。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的保守性 14 - 3 - 3和另一种癌症相关蛋白之间的相互作用和可能的相似机制 S100A4/Mts1.总的来说，本更新申请中的拟议工作力求开发一个复杂的 理解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动力学和胞质分裂。

Project Summary Cytokinesis, the separation of a mother cell into two daughters, is an essential life process. Cytokinesis success is critical to the health and fidelity of 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 will 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.