Molecular Mechanisms of Cytoskeletal Mechanosensory Systems

细胞骨架机械感觉系统的分子机制

• 批准号: 10605572
• 负责人: Pablo A. Iglesias
• 金额: $ 61.98万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10605572
• 关键词: Actins; Adhesions; Affect; Biochemical Process; Biological Models; Cell Cycle Stage; Cell Proliferation; Cell Shape; Cell division; Cell physiology; Cells; Complex; Computational Biology; Computer Models; Contractile System; Cytoplasm; Cytoskeletal Proteins; Cytoskeleton; Data; Development; Dictyostelium; Diffusion; Elasticity; Environment; Evolution; Feedback; Filament; Gene Expression; Goals; Growth; Heterogeneity; Human; Kinetics; Lead; Learning; Location; Magnetism; Maintenance; Mass Spectrum Analysis; Measures; Mechanical Stress; Mechanics; Mediating; Microscopy; Modeling; Molecular; Monitor; Morphogenesis; Motion; Motor; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; Nonmuscle Myosin Type IIA; Nonmuscle Myosin Type IIB; Normal Cell; Organism; Outcomes Research; Output; Phosphorylation; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Process; Property; Protein Isoforms; Proteins; Publishing; Regulatory Pathway; Relaxation; Research; Role; Science; Shapes; Signal Pathway; Signal Transduction; Site; System; Testing; Thick Filament; Time; Tissues; Variant; Work; cell behavior; cell motility; cell type; cellular engineering; cortexillin I; force sensor; in vivo; insight; mathematical model; mechanical force; mechanical stimulus; mechanotransduction; myosin-heavy-chain kinase; non-muscle myosin; particle; programs; protein crosslink; response; simulation; single molecule; theories; therapeutic development

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.

项目摘要 细胞执行不同的过程，例如细胞分裂、生长、运动、形成粘附和组织粘附。 形态发生，在广泛的机械环境下。这些过程的核心是机械的 力，它可能来自细胞外或内部产生，并与信号整合 引导细胞进程的途径。细胞的大分子细胞骨架机制，包括肌动蛋白- 基于肌球蛋白II马达和肌动蛋白交联蛋白，组装，功能，然后分解，以响应 这些力量和信号通路。这种动态力响应组件提供了对传感器的自调节。 机械，导致自然的正反馈和负反馈，并进一步允许机械输入， 转换成信号输出。使用网骨藻细胞，我们发现这些成分中有许多是 在细胞质中以机械响应收缩试剂盒（CK）的形式预组装，其允许高度收缩。 对外力输入的有效反应。CK包括肌球蛋白II、皮质素I、IQGAP 1、IQGAP 2以及几种其他CK。 我们所知道的蛋白质。对于这个应用程序，大量的已发表和未发表的数据激发了这些问题 我们的工作从网骨藻扩展到人类蛋白质和模型系统。我们开始 利用我们的一套实验和建模平台，包括一个新的建模框架， SpringSaLaD，它允许生物化学的分子动机，基于粒子的随机模拟 流程.使用SpringSaLaD，我们通过绘制体内测量的CKs形成模型， 浓度、扩散常数和体内“表观”KD。根据这个模型，我们初步列出了 关于CKs特征的预测，我们将在网骨藻中进行测试。我们还将探讨 在有和没有机械力的情况下组装和拆卸CK。对于组装，我们将确定 CK和非肌肉肌球蛋白II双极粗丝（BTF）的力依赖性组装的分子基础， 使用干涉散射质谱法。对于拆卸，我们将使用磁性镊子测量 BTF内的顺应性，然后确定这种顺应性如何限制肌球蛋白重链的活性， 链激酶（MHCKC用于Dictyosteoblastoma，PKCzeta用于NMIIB）。我们还发现的设定点 网骨藻肌球蛋白II和人NMIIB的机械敏感性积累（机械积累）具有 最佳组装分数为20%。此外，NMIIB的设定点是细胞类型和细胞周期阶段特异性的。我们将 使用我们已经建立的框架来确定设定点定位对小区行为的影响， 包括NMIIB动力学、细胞分裂和基因表达。我们将把这些信息纳入我们的 肌球蛋白II机械累积的计算模型，扩展模型以包括以下组分： 的CK。总之，这项研究工作，其中跨越分子到细胞尺度结合物理理论 开发，将破译力依赖性细胞骨架组装的关键原则和机制， 影响细胞行为。