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 细胞执行多种过程，例如细胞分裂，生长，运动，粘附的形成和组织 形态发生，在广泛的机械环境下。这些过程的中心是机械的 可能来自单元外部或内部产生的力，并与信号集成 指导细胞过程的途径。细胞的大分子细胞骨架机械，包括肌动蛋白 基于肌球蛋白II电动机和肌动蛋白交联蛋白，组装，功能，然后拆卸 这些力和信号通路。这种动态的力响应组装提供了对 机械，导致自然的正和负反馈，并进一步允许机械输入为 转换为信号输出。使用dictyostelium细胞，我们发现其中许多组件是 以机械响应收缩式（CKS）的形式预组装在细胞质中，这允许高度 对强制输入的有效响应。 CK包括肌球蛋白II，Cortexillin I，IQGAP1，IQGAP2，以及其他几个 我们知道的蛋白质。对于此应用程序，大量发布和未发表的数据动机这些问题 要回答，我们的工作从dictyostelium延伸到人类蛋白质和模型系统。我们开始 利用我们的实验和建模平台套件，包括一个名为的新建模框架 Springsalad允许生化的分子动机，基于粒子的随机模拟 过程。使用Springsalad，我们通过在体内测量来对CK的形成进行建模 浓度，差异常数和体内“明显” KD。从这个模型中，我们列出了 关于CK的特征的预测，我们将在dictyostelium中对其进行测试。我们还将探讨 带有和没有机械力的CK的组装和拆卸。对于组装，我们将确定 CK和非肌动蛋白II双极厚细丝（BTF）的分子基础 使用干扰散射质谱。对于拆卸，我们将使用磁性镊子测量 BTF内的合规性，然后确定该合规性如何限制肌球蛋白重的活动 链激酶（用于NMIIB的MHCKC，用于dictyostelium和Pkczeta）。我们还发现 肌动蛋白II和人NMIIB的机械敏感积累（机械蓄积）具有 最佳组装分数。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单元行为的后果， 包括NMIIB动力学，细胞分裂和基因表达。我们将将这些信息纳入我们的 肌球蛋白II机械蓄能的计算模型，扩展了模型以包括 CKS。总而言之，这种研究工作跨越分子到细胞量表与物理理论结合 开发，将破译力依赖性细胞骨骼组件的关键原理和机制 对细胞行为的影响。