The Biochemical Basis for the Mechanics of Cytokinesis

项目总结 胞质分裂，即一个细胞分裂成两个子细胞，是一种优雅的细胞行为，突出了 许多细胞形状改变过程所需的生物力学系统。在这笔赠款的有效期内，我们有 演示了作用力产生、皮质张力、表面曲率和 粘弹性驱动胞质皱纹内移。我们确定了控制这些基因的关键分子途径 属性，并发现电路就像一个控制系统一样，配有反馈环路，允许 机械和化学信号调节收缩机械的累积。最后，我们有 将这些概念应用于其他系统，如成肌细胞融合、齿状突、肝细胞力学、胰腺 癌症，和肺部生物学，展示了使用模型生物(Dictyostelius)和模型的力量 过程(胞质分裂)作为更复杂系统的概念生成器。在这项提案中，我们继续建设 基于我们对细胞质分裂和机械敏感收缩网络的理解，我们开发了一种 构成这个网络的蛋白质相互作用的生物化学基础图(目标1)，并识别 五个不寻常嫌疑人在细胞形状控制中的功能作用(目标2)。在目标1中，我们将绘制生物化学图谱 通过使用几种最先进的蛋白质组学和遗传分析相结合的方式揭示相互作用 方法论。我们将使用荧光互相关光谱来测量细胞浓度， 复杂的大小(反映在扩散系数中)和生化相互作用的强度(在体内 KD‘)。使用单分子下拉，我们将测量每个组分的化学计量比。最后，使用 结构照明显微镜、晶格光片显微镜和共聚焦成像的结合，我们 将对该系统的亚蜂窝分布和架构形成更完整的图景。这些 然后，研究将为我们提供收缩网络的物理生化相互作用图。在目标2中，我们将 继续对与机械感觉收缩系统有关的五种意想不到的蛋白质进行功能研究 通过两种或两种以上的蛋白质组学和/或遗传学策略被揭示。这些蛋白质包括 腺核苷酸转位酶(Anca)、甲基丙二酸半醛脱氢酶(Mmsdh)、2 核糖核酸蛋白(RNP1A和RNP1B)和盘状物复合体。每种蛋白质都提供了一个独特的切入点 破译细胞形状控制的新机制。ANCA提供了进入以下接口的途径 细胞力学和新陈代谢。Mmsdh建议了一种可能的收缩调节模式，通过 丙酰化。据预测，RNP1在本质上是无序的，但在基因和基因上相互作用 生化上与收缩网络的主要节点之一。最后，碟状蛋白复合体是一种凝集素， 其可以提供将收缩网络连接到质膜的锚定复合体的一部分。 总体而言，这些研究将产生一个定量的接线图，并提供新的洞察机制 胞质分裂和控制细胞形状的机械感觉收缩系统通常会发生变化。

PROJECT SUMMARY Cytokinesis, the division of a cell into two daughter cells, serves as an elegant cell behavior that highlights the biomechanical systems required for many cell shape change processes. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive cytokinesis furrow ingression. We identified key molecular pathways that control these properties and found that the circuitry is wired like a control system complete with feedback loops that allows mechanical and chemical signals to tune the accumulation of the contractile machinery. Finally, we have applied these concepts to other systems, such as myoblast fusion, entosis, hepatocyte mechanics, pancreatic cancer, and lung biology, demonstrating the power of using a model organism (Dictyostelium) and a model process (cytokinesis) as a concept generator for more complex systems. In this proposal, we continue to build upon our understanding of cytokinesis and the mechanosensitive contractile network by developing a biochemically grounded map of the protein interactions that constitute this network (Aim 1) and discern the functional roles of five unusual suspects in cell shape control (Aim 2). In Aim 1, we will map the biochemical interactions revealed through a combination of proteomics and genetic analyses using several state-of-the-art methodologies. We will use fluorescence cross-correlation spectroscopy to measure cellular concentrations, complex sizes (reflected in diffusion coefficients), and the strengths of the biochemical interactions (`in vivo Kd'). Using Single Molecule Pulldown, we will measure the stoichiometry of each component. Finally, using a combination of Structured Illumination Microscopy, Lattice Light Sheet Microscopy, and confocal imaging, we will develop a more complete picture of the sub-cellular distribution and architecture of the system. These studies will then give us a physical biochemical interaction map of the contractile network. In Aim 2, we will pursue functional studies of five unexpected proteins implicated in the mechanosensory contractile system that have been revealed through two or more proteomics and/or genetics strategies. These proteins include adenine nucleotide translocase (AncA), methylmalonate semialdehyde dehydrogenase (Mmsdh), two ribonucleotide proteins (RNP1A and RNP1B), and discoidin complex. Each protein offers a unique entry point into deciphering new mechanisms of cell shape control. AncA provides an in-road into the interface between cell mechanics and metabolism. Mmsdh suggests a possible mode of regulation of contractility through propionylation. The RNP1s are predicted to be intrinsically disordered, but interact genetically and biochemically with one of the main nodes of the contractility network. Finally, the discoidin complex is a lectin, which may provide part of the anchoring complex that links the contractile network to the plasma membrane. Overall, the studies will yield a quantitative wiring diagram and provide novel insights into the mechanisms of cytokinesis and the mechanosensory contractile system that governs cell shape change more generally.