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

父项目的总体目标是使用Myosin作为模型系统，在其中解决 基本的生物学问题，即组织组织和功能的改变是如何经常引起的 在分子水平上的功能的微妙变化。肌球蛋白产生的力量不仅是为了 骨骼肌和心脏的生理功能，也是为了适当的发育和维持 这些组织在胚胎发育期间和以后。我们团队的目标是开发一种详细的机械 了解肌球蛋白如何产生力量来调节肌肉组织的发育和 动态平衡。我们通过询问看似很小的变化是如何 单个肌球蛋白分子的活性可以推动组织水平组织和功能的戏剧性变化， 例如，在遗传性疾病的背景下。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性质 马达蛋白质。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 肌球蛋白突变在单分子水平上的变化及其相应后果 肌节水平的结构和功能。在目标2和目标3中，我们将确定肌球蛋白动力学和 力量产生影响细胞和组织的生长、成熟和功能，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编辑来引入肌球蛋白突变 在同基因的hipsc来源的心肌和骨骼肌细胞中。然后我们将能够比较生物力学 单个分子水平上的变化与亚细胞细胞器(肌原纤维)、细胞和微结构的变化 纸巾。我们希望回答一些基本的机械问题，比如蛋白质结构的改变和 功能影响细胞和组织的功能，改变力量和可塑性，并提供了解的窗口 细胞如何适应变化的机械力的变化。然后，我们将能够利用我们的HiPSC 用于高通量筛选的平台，以开发针对肌球蛋白表型亚群的新疗法 突变。我们研究计划的另一个主要目标是支持早期调查人员(ESI)。我们会 支持来自ESI调查人员的试点研究，探索与我们的 研究计划。对于NIGMS任务至关重要，我们团队的多学科集成方法跨越 从单个分子到亚细胞结构再到整个细胞再到工程微组织的规模，将 作为团队进行未来研究的原型，使用HiPSCs探索其他生物蛋白质 组件，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。

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.