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.

项目概要 细胞执行多种过程，例如细胞分裂、生长、运动、粘附形成和组织 在各种机械环境下，形态发生是这些过程的核心。 力，可能来自细胞外部或内部产生，并且与信号传导集成 指导细胞过程的途径。 基于肌球蛋白 II 马达和肌动蛋白交联蛋白，组装、发挥作用，然后响应分解 这些力和信号通路提供了自我调节的动态力响应组件。 机械，导致自然的正反馈和负反馈，并进一步允许机械输入 使用盘基网柄菌细胞，我们发现其中许多成分都转化为信号输出。 以机械响应收缩套件 (CK) 的形式预先组装在细胞质中，可实现高度 对力输入的有效反应 CK 包括肌球蛋白 II、皮质西林 I、IQGAP1、IQGAP2 以及其他几种。 我们所知道的蛋白质，大量已发表和未发表的数据引发了这些问题。 我们的工作从盘基网柄菌扩展到人类蛋白质和模型系统。 利用我们的实验和建模平台套件，包括一个名为 SpringSaLaD，允许进行分子驱动的、基于粒子的、随机的生物化学模拟 使用 SpringSaLaD，我们通过体内测量来模拟 CK 的形成。 浓度、扩散常数和体内“表观”KD 根据该模型，我们列出了初始列表。 关于 CK 特征的预测，我们将在盘基网柄菌中进行测试，我们还将探索 CK 的动力学。 使用和不使用机械力进行 CK 的组装和拆卸 对于组装，我们将确定以下条件。 CK 和非肌肉肌球蛋白 II 双极粗丝 (BTF) 力依赖性组装的分子基础， 使用干涉散射质谱法进行拆卸，我们将使用磁力镊子进行测量。 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性 链激酶（盘基网柄菌属的 MHCKC 和 NMIIB 的 PKCzeta）我们还发现了设定点。 盘基网柄菌肌球蛋白 II 和人 NMIIB 的机械敏感性积累（机械积累）具有 此外，NMIIB 的设定点是特定于细胞类型和细胞周期阶段的。 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 动力学、细胞分裂和基因表达，我们会将这些信息纳入我们的研究中。 肌球蛋白 II 机械积累的计算模型，扩展模型以包括以下组件 总之，这项研究工作涵盖分子到细胞尺度并与物理理论相结合。 开发，将破译力依赖性细胞骨架组装的关键原理和机制以及 对细胞行为的影响。