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.

项目总结/摘要 该项目的总体目标是使用肌球蛋白作为模型系统，以解决基本的 组织结构和功能的改变如何从组织的细微变化中产生的生物学问题。 在分子水平上发挥作用。肌球蛋白产生的力不仅是肌球蛋白的生理功能所必需的， 骨骼肌和心脏，但也为这些组织的适当发展和维护， 胚胎发生及以后。我们的团队旨在对力如何作用于 肌球蛋白的产生起到调节肌肉组织发育和体内平衡的作用。我们研究这个一般 通过透镜的问题，问看似微小的变化，在个别肌球蛋白分子的活动， 可以推动组织水平的组织和功能发生巨大变化，例如在遗传性疾病的背景下， 疾病在目标1中，我们将确定肌球蛋白的结构变化如何影响化学机械性能 肌球蛋白-肌动蛋白相互作用的单个和小组件的运动蛋白。这一目标将利用 我们的团队开发的创新技术，用于量化肌球蛋白突变引起的生物力学变化 在单分子水平和相应的后果肌节水平的结构和功能。在 目标2和3，我们将确定肌球蛋白动力学和力产生的变化如何影响生长， 成熟和功能的细胞和组织，使用心肌细胞和骨骼肌细胞作为模型系统。 这些目标将利用CRISPR编辑在等基因hiPSC衍生的心脏和心肌细胞中引入肌球蛋白突变。 骨骼肌细胞然后我们就可以在单个分子水平上比较生物力学的变化 与亚细胞器（肌原纤维）、细胞和微组织中的那些。我们希望能回答基本的机械论 蛋白质结构和功能的改变如何影响细胞和组织功能，改变力 和可塑性，并提供了一个窗口，了解细胞如何适应变化的机械 力.然后，我们将利用我们的hiPSC平台进行高通量筛选，开发新颖的 针对肌球蛋白突变表型亚组的治疗。我们研究计划的另一个主要目标 支持早期研究者（ESI）。我们将支持ESI研究人员的试点研究， 与我们的研究计划相关的创新研究问题。对NIGMS的使命至关重要，我们的团队 多学科综合方法，从单个分子到亚细胞结构， 整个细胞到工程微组织，将作为一个原型，为团队进行未来的研究， hiPSC探索其他生物蛋白质组装，使用人类致病突变作为 扰动，以确定其跨器官系统的分子和功能机制。