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: 10291393
• 负责人: Daniel Bernstein
• 金额: $ 2.09万
• 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/10291393
• 关键词: Actins; Address; Adult; Affect; Autophagocytosis; Award; Biological; Biological Models; Biomechanics; Calcium; Calcium Signaling; Cardiac Myocytes; Cardiac development; Cell Line; Cell physiology; Cells; Cellular Stress Response; Cellular Structures; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Development; Down-Regulation; Embryonic Development; Engineering; Ensure; Functional disorder; Future; Generations; Genes; Goals; Grant; Growth; Heart; Heart Abnormalities; Hereditary Disease; Homeostasis; Human; Hypertrophic Cardiomyopathy; Hypertrophy; Impairment; Individual; Kinetics; Knowledge; Lead; Length; Life; Link; Maintenance; Measures; Mechanics; Messenger RNA; Mission; Mitochondria; Molecular; Molecular Motors; Motor; Muscle; Muscle Cells; Muscle Fibers; Mutation; Myofibrils; Myosin ATPase; National Institute of General Medical Sciences; Organ; Organelles; Output; Parents; Pathway interactions; Patients; Phenotype; Physiological; Pilot Projects; Point Mutation; Positioning Attribute; Production; Proteins; Quality Control; Reactive Oxygen Species; Regulation; Reporter; Research; Research Personnel; Sarcomeres; Series; Skeletal Muscle; Structure; Subgroup; System; Techniques; Testing; Thick Filament; Thin Filament; Time; TimeLine; Tissues; Traction Force Microscopy; Translating; Western Blotting; Width; Work; alpha Actinin; beta-Myosin; body system; cell type; clinical phenotype; connectin; disease phenotype; disease-causing mutation; heart function; high throughput screening; human disease; induced pluripotent stem cell; inhibition of autophagy; innovation; lens; link protein; mechanical force; mechanical properties; model development; multidisciplinary; mutant; nebulin; new therapeutic target; parent project; programs; protein folding; protein structure function; proteostasis; prototype; response; single molecule; single-cell RNA sequencing; trafficking

The overarching goal of the parent 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.

母项目的首要目标是使用肌球蛋白作为模型系统，以解决 一个基本的生物学问题，即组织组织和功能的改变是如何发生的， 在分子水平上的功能的微妙变化。肌球蛋白产生的力不仅是 骨骼肌和心脏的生理功能，但也为适当的发展和维护 这些组织在胚胎发育过程中和以后。我们的团队旨在开发一个详细的机制， 了解肌球蛋白产生的力如何调节肌肉组织发育， 体内平衡我们通过一个透镜来研究这个一般性问题，即： 单个肌球蛋白分子的活性可以驱动组织水平组织和功能的显著变化， 例如在遗传性疾病的情况下。在目标1中，我们将确定肌球蛋白的结构变化 影响肌球蛋白-肌动蛋白相互作用的化学机械特性， 马达蛋白这一目标将利用我们团队开发的创新技术来量化生物力学 在单分子水平上肌球蛋白突变引起的变化以及 肌节水平的结构和功能。在目标2和3中，我们将确定肌球蛋白动力学的变化， 力的产生影响细胞和组织的生长、成熟和功能，使用心肌细胞， 骨骼肌细胞作为模型系统。这些目标将利用CRISPR编辑来引入肌球蛋白突变 在同基因hiPSC衍生的心肌和骨骼肌细胞中。我们就能比较生物力学 在单个分子水平上的改变与亚细胞器（肌原纤维）、细胞和微 组织中我们希望回答基本的机械问题，如蛋白质结构的改变， 功能影响细胞和组织功能，改变力和可塑性，并提供了解 细胞如何适应不断变化的机械力。然后，我们将利用我们的hiPSC 用于高通量筛选的平台，以开发针对肌球蛋白表型亚组的新型疗法 突变。我们的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，探索与我们的研究相关的创新研究问题。 研究计划。对NIGMS使命至关重要的是，我们团队的多学科综合方法， 从单个分子到亚细胞结构到整个细胞再到工程微组织的规模， 作为未来研究团队使用hiPSC探索其他生物蛋白质的原型 组装，使用人类致病突变作为扰动来定义它们的分子和 器官系统的功能机制。