From proteins to cells to tissues: A multi-scale assessment of biomechanical regulation by the myosin molecular motor

从蛋白质到细胞再到组织：肌球蛋白分子马达生物力学调节的多尺度评估

• 批准号: 10396504
• 负责人: Daniel Bernstein
• 金额: $ 207.59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396504
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actins; Address; Affect; Animal Model; Biological; Biological Assay; Biological Models; Biology; Biomechanics; Biophysical Process; Biophysics; Cardiac Myocytes; Cardiac Myosins; Cell fusion; Cell physiology; Cells; Cellular Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Computer Models; Data; Development; Disease; Embryonic Development; Engineering; Future; Gene Expression; Generations; Goals; Growth; Heart; Hereditary Disease; Homeostasis; Human; Human Engineering; Individual; Kinetics; Knowledge; Lead; Length; Link; Maintenance; Measurement; Mechanics; Methods; Microfilaments; Mission; Modeling; Molecular; Molecular Motors; Molecular Target; Motor; Muscle; Muscle Cells; Muscle Development; Muscle Fibers; Muscle function; Mutation; Myocardium; Myofibrils; Myosin ATPase; Myosin Heavy Chains; National Institute of General Medical Sciences; Organ; Organelles; Performance; Phenotype; Physiological; Pilot Projects; Point Mutation; Positioning Attribute; Production; Property; Proteins; Protocols documentation; Recombinants; Regulation; Research; Research Personnel; Sarcomeres; Shapes; Signal Pathway; Signal Transduction; Skeletal Muscle; Skin; Structure; Subgroup; Sum; Techniques; Testing; Thick Filament; Tissue constructs; Tissues; Translating; Work; Working stroke; biomechanical test; body system; cell motility; disease phenotype; disease-causing mutation; experimental study; high throughput screening; human disease; induced pluripotent stem cell; innovation; insight; lens; mechanical force; mechanical properties; multidisciplinary; new therapeutic target; optical traps; programs; protein structure; protein structure function; prototype; sensor; single molecule; skeletal; skeletal stem cell; stem cell model; success; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT The overarching goal of this project is to use myosin as a model system in which to address the fundamental biological question of how alterations in tissue organization and function can arise from often subtle changes in function at the molecular level. Force generation by myosin is required not only for the physiological functions of skeletal muscle and the heart, but also for the proper development and maintenance of these tissues during embryogenesis and beyond. Our team aims to develop a detailed mechanistic understanding of how force generation by myosin acts to regulate muscle tissue development and homeostasis. We examine this general question through the lens of asking how seemingly small changes in the activity of individual myosin molecules can drive dramatic changes in tissue-level organization and function, for example in the context of inherited disease. In Aim 1, we will determine how structural changes in myosin affect the chemo-mechanical properties of the myosin-actin interaction for individual and small assemblies of motor proteins. This aim will leverage innovative techniques developed by our team to quantify biomechanical changes induced by myosin mutations at the single molecule level and the corresponding consequences for sarcomere-level structure and function. In Aims 2 and 3, we will determine how changes in myosin kinetics and force production influence the growth, maturation, and function of cells and tissues, using cardiomyocytes and skeletal myocytes as model systems. These aims will leverage CRISPR-editing to introduce myosin mutations in isogenic hiPSC-derived cardiac and skeletal myocytes. We will then be able to compare biomechanical alterations at the individual molecule level with those in sub-cellular organelles (myofibrils), cells and micro-tissues. We expect to answer basic mechanistic questions as to how alterations in protein structure and function affect cell and tissue function, changing force and plasticity, and provide a window into understanding how cells adapt to alterations in changing mechanical forces. We will then be positioned to utilize our hiPSC platforms for high-throughput screens to develop novel therapies targeted to phenotypic subgroups of myosin mutations. Another major goal of our Research Program is to support Early Stage Investigators (ESI). We will support pilot studies from ESI investigators that explore innovative research questions relevant to our Research Program. Critical to the NIGMS mission, our team’s multi-disciplinary integrated approach, spanning the scale from individual molecules to sub-cellular structures to whole cells to engineered micro-tissues, will serve as a prototype for teams undertaking future studies using hiPSCs to explore other biological protein assemblies, using human disease-producing mutations as perturbations to define their molecular and functional mechanisms across organ systems.

项目摘要/摘要 该项目的总体目标是将肌球蛋白作为模型系统，以解决基本 关于组织组织和功能改变的生物学问题通常是由于经常因微妙的变化而引起的 分子水平的功能。肌球蛋白产生力不仅需要 骨骼肌和心脏，但也用于在 胚胎发生及其他。我们的团队旨在对强制的方式发展详细的机械理解 肌球蛋白产生的作用是调节肌肉组织发育和稳态。我们检查了这个将军 通过询问单个肌球蛋白分子活动的看似很小的变化的镜头的问题 可以推动组织级的组织和功能的巨大变化，例如在继承的背景下 疾病。在AIM 1中，我们将确定肌球蛋白的结构变化如何影响化学机械特性 肌球蛋白 - 肌动蛋白的相互作用，用于运动蛋白的个体和小组件。这个目标将利用 我们的团队开发的创新技术旨在量化肌球蛋白突变引起的生物力学变化 在单分子水平以及肌节级结构和功能的相应后果。在 目标2和3，我们将确定肌球蛋白动力学的变化和力产生如何影响增长， 使用心肌细胞和骨骼心肌细胞作为模型系统的成熟和细胞和组织的功能。 这些目的将利用CRISPR编辑来引入ISEGENIC HIPSC衍生心脏和 骨骼肌细胞。然后，我们将能够比较单个分子水平的生物力学改变 与细胞细胞器（肌原纤维）中的细胞和微观组织中的那些。我们希望回答基本机械 关于蛋白质结构和功能改变如何影响细胞和组织功能，力变化力的问题 和可塑性，并提供一个窗口，以了解细胞如何适应改变机械的改变 力量。然后，我们将定位利用我们的HIPSC平台进行高通量屏幕来开发新颖 针对肌球蛋白突变的表型亚组的疗法。我们研究计划的另一个主要目标 是支持早期研究人员（ESI）。我们将支持ESI研究人员的试点研究 创新的研究问题与我们的研究计划有关。对纽格姆的任务至关重要，我们的团队的 多学科的综合方法，涵盖了从单个分子到亚细胞结构的量表 整个细胞进行设计的微型组织，将作为使用未来研究的团队的原型 HIPSC使用产生人类疾病的突变探索其他生物蛋白组件 扰动以定义其跨器官系统的分子和功能机制。