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

使用体外单分子成像和肌原纤维了解肌肉中的双丝调节

• 批准号: BB/Y001621/1
• 负责人: Neil Kad
• 金额: $ 58.8万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY001621%2F1
• 关键词: Understanding; dual; filament; regulation; muscle

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.

肌肉对于人类的生存至关重要，可以实现运动和心脏收缩等过程。即使进行了数十年的研究，仍然需要了解既可以启用和控制收缩的基本分子机制。也许最大的开放问题是如何在收缩中调节力，我们在这里寻求使用敏感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：厚而薄的细丝。当运动蛋白从厚细丝中称为肌球蛋白的运动蛋白以ATP的形式使用以在细丝中拉动肌动蛋白时，就会发生收缩。这是由钙结合在细丝上的常规间隔对照蛋白的调节，这有助于调节肌球蛋白对肌动蛋白的访问。收缩发生的力取决于可用的肌醇数量。如果不受控制，肌肉将不断收缩并消耗所有生物体的可用能量。因此，调节肌肉收缩的时间和力对于存活至关重要。我们在此提案中的架构目标是在测试管（体外）和肌肉组织提取物（体内）中清楚地了解此过程。我们将使用尖端的单分子成像直接研究厚细丝和薄丝的调节，它们在称为“双丝细丝调节”的过程中共同控制收缩。要研究双丝细丝调节，我们将使用两个独特的测定法，我们的实验室拥有。首先，一种体外的“钢丝”测定法，涉及将显微镜盖在玻璃珠之间的覆盖面板上方的细丝悬浮。使用该测定法，我们可以通过以下荧光标记的肌球蛋白结合的位置直接观察激活及其放松。为了触发肌球蛋白结合，我们可以添加钙，但是由于它沿薄丝沿着任何对照蛋白结合，因此我们不能确定薄丝在哪里激活。这很重要，因为从钙结合点开始，激活沿着细丝散布，形成“合作单元”。因此，为了控制激活点，我们将在没有钙的情况下设计一种荧光控制蛋白的活性。随着肌球蛋白的结合，它报告了相对于激活点的薄丝打开的空间范围。这将使能够确切地测量合作单元的物理大小，这将是一个重要的突破。第二个（体内）测定是研究从肌肉组织中提取的肌原纤维中的调节。由于肌球蛋白在收缩过程中使用ATP，因此我们可以通过标记为ATP来检测到何处。使用此测定，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。我们还打算调整成像系统以进行一系列系统研究，这些研究将揭示薄细丝如何在肌膜纤维的密集，复杂的3维矩阵中进行通信。迄今为止，没有人进行过这样的直接测量，这对于理解双丝细丝调节的分子基础非常有价值。这些研究位于当前的肌肉生物学研究领域，对于理解严重疾病（例如心肌病和骨骼肌病）非常重要。