Nano-Mechanics, Sarcomere, Myofibril and Protein Testing System

肌动蛋白和肌球蛋白是组成分子马达的两种蛋白质，它们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，并意识到教科书版本的肌肉收缩无法解释许多实验观察到的现象。具体地说，当一块活跃的肌肉被拉伸时，它的力量、能量和僵硬不能很好地用由肌动蛋白和肌球蛋白组成的分子马达来解释。2002年，我们发现被认为只起被动作用的结构蛋白有助于肌肉中主动力量的产生。我们通过分子、亚细胞和细胞实验证实，肌瘤蛋白Titin在主动力量的产生中起着至关重要的作用。我们有初步证据表明，Titin在肌肉激活时结合了钙，从而增加了肌肉的僵硬和力量，并且Titin与肌动蛋白结合，从而缩短了其自由弹簧长度，从而增加了其拉伸的抵抗力。如果我们的理论是正确的，那么肌动蛋白的近端片段在肌肉激活时与肌动蛋白结合在一起，因此不能像在被动肌肉中那样在主动肌肉中拉伸。*为了检验这些假设，并为肌肉收缩的分子机制提供根本上的新见解，我们申请资金购买一套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，以测量肌肉收缩过程中肌节蛋白的结构变化。*所需的系统将使用独特的配置在内部组装，并将通过允许同时测量肌肉能量来极大地扩展我们目前的可能性。肌肉力学，以及单一蛋白质在肌节水平上的结构变化。*学员将接触到细胞和分子生物力学中的各种微观、纳米和微力学技术，这些技术的价值将超出骨骼肌研究的范围。学员将学习处理动物的技能，以及骨骼肌细胞和肌原纤维的采集。他们还将学习最先进的荧光显微镜和原子力显微镜，荧光光谱学，以及单个肌节和肌原纤维的生理和机械测试。一系列关键的实验将包括测量Titin的I-Band结构域的亚段的伸展、展开和重新折叠。到目前为止，这种测量还没有进行过，可能会改变教科书上关于肌肉如何收缩和如何产生力量的基本概念。*总而言之，所要求的系统是独一无二的，它将允许我们进行目前在其他地方不可能进行的实验，并将为学生提供独特的生物力学和生物医学工程培训机会。*

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.*****