Molecular Mechanisms of Cytoskeletal Mechanosensory Systems

项目总结 细胞执行不同的过程，如细胞分裂、生长、运动、粘连的形成和组织 形态发生，在广泛的机械环境下。这些过程的核心是机械 力量，可能来自细胞外部或内部产生，并与信令集成 引导细胞过程的途径。细胞的大分子细胞骨架机制，包括肌动蛋白- 根据肌球蛋白II马达和肌动蛋白交联蛋白，组装，功能，然后拆卸响应 这些力量和信号通路。这种动态力响应型组件提供了对 机械，导致自然的正反馈和负反馈，并进一步允许机械输入 转换为信令输出。通过使用网柄菌细胞，我们发现这些成分中的许多都是 以机械反应收缩试剂盒(CKS)的形式预先组装在细胞质中，这允许高度 对强制输入的有效响应。CKs包括肌球蛋白II、皮质素I、IQGAP1、IQGAP2以及其他几种 我们所知道的蛋白质。对于此应用程序，大量已发布和未发布的数据激发了问题 有待回答，我们的工作从Dictyostelials延伸到人类蛋白质和模型系统。我们开始的时候 利用我们的一套实验和建模平台，包括一个名为 SpringSaLaD，它允许分子激励的、基于粒子的、随机的生化模拟 流程。利用SpringSaLaD，我们正在通过绘制活体测量的数据来模拟Cks的形成 浓度、扩散常数和体内“表观”KDS。从这个模型中，我们已经列出了一个初步列表 关于CKs特征的预测，我们将在Dictyostelial.我们还将探索 在有机械力和无机械力的情况下装配和拆卸CKS。对于装配，我们将确定 CKS和非肌肉肌球蛋白II双极粗丝(BTF)的力依赖组装的分子基础， 使用干涉散射质谱计。对于拆卸，我们将使用磁镊子来测量 BTF内的顺应性，然后确定这种顺应性如何限制肌球蛋白重的活性 链激酶(MHCKC用于Dictyostelials，PKcheeta用于NMIIB)。我们还发现， 网鞭毛菌肌球蛋白II和人NMIIB的机械敏感积累(机械积累) 最佳装配率为20%。此外，NMIIB的设定点是特定于细胞类型和细胞周期阶段的。我们会 使用我们建立的框架来确定设定点定位对细胞行为的影响， 包括NMIIB动态、细胞分裂和基因表达。我们将把这些信息合并到我们的 肌球蛋白II机械累积的计算模型，扩展模型以包括 公鸡们。总而言之，这项跨越分子到细胞尺度与物理理论相结合的研究成果 发展，将破译依赖于力的细胞骨架组装的关键原理和机制以及 对细胞行为的影响。

PROJECT SUMMARY Cells perform diverse processes, such as cell division, growth, motility, formation of adhesions, and tissue morphogenesis, under a wide range of mechanical environments. Central to these processes are mechanical forces, which may come from outside the cell or be generated internally and which are integrated with signaling pathways to guide the cellular process. The cell's macromolecular cytoskeletal machinery, including the actin- based myosin II motors and actin crosslinking proteins, assemble, function and then disassemble in response to these forces and signaling pathways. This dynamic force-responsive assembly provides self-tuning of the machinery, leading to natural positive and negative feedback and further allows mechanical inputs to be converted into signaling outputs. Using Dictyostelium cells, we discovered that many of these components are pre-assembled in the cytoplasm in the form of mechanoresponsive Contractility Kits (CKs), which allow for highly efficient responses to force inputs. The CKs include myosin II, cortexillin I, IQGAP1, IQGAP2, plus several other proteins that we know of. For this application, substantial published and unpublished data motivate the questions to be answered, and our work extends from Dictyostelium to human proteins and model systems. We begin by leveraging our suite of experimental and modeling platforms, including a new modeling framework called SpringSaLaD, which allows for molecularly motivated, particle-based, stochastic simulations of biochemical processes. Using SpringSaLaD, we are modeling the formation of CKs by drawing upon measured in vivo concentrations, diffusion constants, and in vivo “apparent” KDs. From this model, we have made an initial list of predictions about the features of the CKs, which we will test in Dictyostelium. We will also explore the kinetics of assembly and disassembly of the CKs with and without mechanical force. For assembly, we will determine the molecular basis for force-dependent assembly of the CKs and nonmuscle myosin II bipolar thick filament (BTF), using interference scattering mass spectrometry. For disassembly, we will use magnetic tweezers to measure the compliance within the BTF and then determine how this compliance restricts the activity of the myosin heavy chain kinase (MHCKC for Dictyostelium and PKCzeta for NMIIB). We have also found that the setpoint of mechanosensitive accumulation (mechanoaccumulation) by Dictyostelium myosin II and human NMIIB has an optimum of 20% assembly fraction. Further, NMIIB's setpoint is cell type- and cell cycle stage-specific. We will use the framework we have established to determine the consequences of setpoint positioning on cell behavior, including NMIIB dynamics, cell division, and gene expression. We will incorporate this information into our computational models for myosin II mechanoaccumulation, expanding the models to include the components of the CKs. In sum, this research effort, which spans molecular to cellular scales combined with physical theory development, will decipher key principles and mechanisms of force-dependent cytoskeletal assembly and the impact on cell behavior.