Nano-Mechanics, Sarcomere, Myofibril and Protein Testing System

纳米力学、肌节、肌原纤维和蛋白质测试系统

• 批准号: RTI-2019-00173
• 负责人: Herzog, Walter
• 金额: $ 10.93万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=653497
• 关键词: Nano; Mechanics; Sarcomere; Myofibril; Protein

Actin and myosin are two proteins that make up molecular motors that drive many functions in animals including cell division, ATP production, hearing, and most importantly, contractions in muscles. We have studied the molecular mechanisms of muscle contractions for the past 15 years and realized that the textbook version of muscle contraction cannot explain many experimentally observed phenomena. Specifically, when an active muscle is stretched, its force, energetics and stiffness are not explained well by the molecular motor comprised of actin and myosin. In 2002, we discovered that structural proteins, thought to play only passive roles, contribute to active force production in muscles. We identified, through molecular, sub-cellular and cellular experiments, that the sarcomeric protein titin plays a crucial role in active force production. We have preliminary evidence, that titin binds calcium upon muscle activation, thereby increasing its stiffness and force, and that titin binds to actin, thus shortening its free spring length, thereby increasing its resistance to stretch. If our theories are correct, then the proximal segments of titin are bound to actin upon muscle activation, and thus cannot be stretched in an active muscle like they are in a passive muscle.***In order to test these hypotheses and provide fundamentally new insight into the molecular mechanisms of muscle contraction, we request monies for the purchase of a unique single sarcomere and myofibril testing system that allows for simultaneous force measurements and fluorescent microscopy to measure structural changes in sarcomeric proteins during muscle contraction with nanometre and millisecond resolution.***The requested system will be assembled in house using a unique configuration, and will vastly expand our current possibilities by allowing for simultaneous measurement of muscle energetics, muscle mechanics, and the structural changes in single proteins, in situ, on the sarcomere level.***Trainees will be exposed to a variety of micro-, nano- and pico-mechanical techniques in cellular and molecular biomechanics that will be valuable beyond skeletal muscle research. Trainees will learn animal handling skills, and the harvesting of skeletal muscle cells and myofibrils. They will also learn state-of-the-art fluorescent and atomic force microscopy, fluorescent spectroscopy, and single sarcomere and myofibril physiological and mechanical testing. A crucial set of experiments will involve the measurement of stretching, unfolding and refolding of sub-segments of titin's I-band domain. Such measurements have not been performed to date and could change the fundamental textbook notion of how muscles contract and how they produce force. ***In summary, the requested system is one of a kind, it will allow us to perform experiments that are currently not possible elsewhere, and will provide unique training opportunities for students in biomechanics and biomedical engineering.*****

肌动蛋白和肌球蛋白是构成分子马达的两种蛋白质，这些分子马达驱动动物的许多功能，包括细胞分裂，ATP生产，听力，最重要的是肌肉收缩。在过去的15年里，我们研究了肌肉收缩的分子机制，并意识到肌肉收缩的教科书版本无法解释许多实验观察到的现象。具体来说，当一个活跃的肌肉被拉伸时，它的力，能量和刚度不能很好地解释由肌动蛋白和肌球蛋白组成的分子马达。在2002年，我们发现，结构蛋白，被认为只发挥被动作用，有助于在肌肉中产生积极的力量。我们通过分子、亚细胞和细胞实验确定肌节蛋白肌联蛋白在主动力产生中起着至关重要的作用。我们有初步的证据表明，肌联蛋白在肌肉激活时与钙结合，从而增加其刚度和力量，肌联蛋白与肌动蛋白结合，从而缩短其自由弹簧长度，从而增加其拉伸阻力。如果我们的理论是正确的，那么肌联蛋白的近端片段在肌肉激活时与肌动蛋白结合，因此在主动肌肉中不能像在被动肌肉中那样被拉伸。为了检验这些假设，并提供肌肉收缩的分子机制的全新见解，我们要求资金购买一个独特的单一肌节和肌原纤维测试系统，该系统允许同时进行力测量和荧光显微镜检查，以纳米和毫秒分辨率测量肌肉收缩期间肌节蛋白的结构变化。所要求的系统将使用独特的配置在内部组装，并将通过允许在肌节水平上原位同时测量肌肉能量学，肌肉力学和单个蛋白质的结构变化来极大地扩展我们目前的可能性。学员将接触到各种微，纳米和皮机械技术在细胞和分子生物力学，这将是有价值的骨骼肌研究之外。学员将学习动物处理技能，以及骨骼肌细胞和肌原纤维的收获。他们还将学习最先进的荧光和原子力显微镜，荧光光谱，以及单个肌节和肌原纤维生理和机械测试。一组关键的实验将涉及测量的拉伸，展开和重折叠的亚段肌联蛋白的I带域。迄今为止还没有进行过这样的测量，这可能会改变教科书中关于肌肉如何收缩以及它们如何产生力量的基本概念。* 总之，所要求的系统是独一无二的，它将使我们能够进行目前在其他地方不可能进行的实验，并将为生物力学和生物医学工程的学生提供独特的培训机会。