Understanding dual filament regulation in muscle using single molecule imaging in vitro and in myofibrils

肌肉对人类生存至关重要，使运动和心脏收缩等过程成为可能。即使经过几十年的研究，仍然需要了解有关使收缩和控制收缩的潜在分子机制。也许最大的开放性问题是如何在收缩中调节力，在这里，我们试图使用敏感的成像技术直接观察调节。肌肉高度组织成两种类型的蛋白质链：粗丝和细丝。当粗肌丝中的肌球蛋白利用ATP形式的能量拉动细肌丝中的肌动蛋白时，收缩就发生了。这是由钙离子与细丝上规则间隔的控制蛋白质结合来调节的，这有助于调节肌球蛋白与肌动蛋白的接触。收缩发生的力量取决于可用的肌球蛋白的数量。如果不加控制，肌肉会不断收缩，耗尽机体所有可用的能量。因此，调节肌肉收缩的时间和力量对生存至关重要。我们在这个建议中的首要目标是提供一个清晰的理解，这一过程在试管（体外）和肌肉组织提取物（体内）。我们将使用最先进的单分子成像技术直接研究粗丝和细丝的调节，它们共同控制收缩过程中称为“双丝调节”。为了研究双丝调节，我们将使用我们实验室拥有的两种独特的检测方法。首先，体外“钢丝”测定，其涉及将细丝悬挂在玻璃珠之间的显微镜盖玻片表面上方。使用这种检测，我们可以通过跟踪荧光标记的肌球蛋白结合的位置来直接观察激活和松弛。为了触发肌球蛋白的结合，我们可以加入钙，但由于钙与任何控制蛋白质沿着细丝结合，我们不能确定细丝在哪里被激活。这一点很重要，因为从钙结合的角度来看，活化沿着细丝扩散，形成一个“协作单元”。因此，为了控制激活点，我们将设计一种荧光控制蛋白，使其在没有钙的情况下具有活性。当肌球蛋白结合时，它报告相对于激活点的细丝被打开的空间范围。这将使精确测量的物理尺寸的合作单位，这将是一个重要的突破。第二个（体内）试验是研究调节肌原纤维，这是从肌肉组织中提取。由于肌球蛋白在收缩过程中使用ATP，我们可以通过跟踪标记的ATP来检测这种情况发生的位置。使用这种检测，我们将发现我们从体外研究中学到的规则如何在体内翻译。我们还打算调整我们的成像系统来进行一系列系统的研究，这些研究将揭示细丝和粗丝如何在肌原纤维的致密、复杂的三维基质中进行交流。迄今为止，还没有人进行过这样的直接测量，这对于理解双丝调节的分子基础非常有价值。这些研究处于当前肌肉生物学研究的前沿，对于理解心肌病和骨骼肌病等重要疾病非常重要。

Muscle is essential for human survival, enabling processes such as locomotion and heart contraction. Even with many decades of investigation, much still needs to be understood about the underlying molecular mechanisms that both enable and control contraction. Perhaps the biggest open question is how force is regulated in contraction, here we seek to directly observe regulation using sensitive imaging technologies. Muscle is highly organised into two types of protein strands: thick and thin filaments. Contraction occurs when motor proteins called myosin from the thick filament use energy in the form of ATP to pull on actin in the thin filament. This is regulated by calcium binding to regularly spaced control proteins on the thin filament, which helps regulate access of myosin to actin. The force at which contraction occurs depends on the number of myosins available. If left uncontrolled, muscle would constantly contract and use up all the organism's available energy. Therefore, regulating the timing and force of muscle contraction is crucial for survival. Our over-arching goal in this proposal is to provide a clear understanding of this process both in the test-tube (in vitro) and in muscle tissue extracts (in vivo). We will use cutting-edge single molecule imaging to study regulation directly on both thick and thin filaments, which work together to control contraction in a process known as 'dual filament regulation'.To study dual filament regulation, we will use two unique assays that our lab possesses. Firstly, an in vitro 'tightrope' assay, which involves suspending thin filaments above a microscope coverslip surface between glass beads. Using this assay, we can watch activation and its relaxation directly by following where fluorescently tagged myosins bind. To trigger myosin binding we can add calcium, but because it binds to any control protein along the thin filament, we cannot be sure where the thin filament is activated. This is important because from the point of calcium binding, activation spreads along the thin filament forming a 'cooperative unit'. Therefore, to control the point of activation we will engineer a fluorescent control protein to be active in the absence of calcium. As the myosin binds it reports on the spatial range over which the thin filament is turned on relative to the point of activation. This will enable a precise measurement of the physical size of the cooperative unit to be made, which would be an important breakthrough.The second (in vivo) assay is to study regulation in myofibrils, which are extracted from muscle tissue. Since myosin uses ATP during contraction we can detect where this occurs by following labelled ATP. Using this assay, we will discover how the rules we learned from the in vitro studies translate in vivo. We also intend to adapt our imaging system to perform a series of systematic studies that will reveal how the thin and thick filaments communicate in the dense, complex 3-dimensional matrix of a myofibril. No-one has made such direct measurements to date, which are highly valuable for understanding the molecular basis of dual filament regulation. These studies lie at the current frontier of muscle biology research and are important for understanding significant diseases such as cardiomyopathies and skeletal myopathies.