Novel approaches to elucidate the molecular basis of muscle contraction: FRET-FLIM imaging applied to single muscle fibres

活细胞中的运动是由蛋白质的协调行为引起的。这个需要能量的过程是由ATP（三磷酸腺苷）的分解驱动的，ATP是细胞的分子燃料。肌肉细胞专门将这种化学能转化为运动和机械功，其精确尺寸和排列的细丝可最大限度地提高动力输出，速度，抗疲劳性和运动效率。在分子水平上，运动是由主要蛋白质之一肌球蛋白的构象的周期性变化引起的。蛋白质构象变化的细节及其与ATP分解过程中步骤的联系尚不清楚。在这个项目中，我们打算开发先进的荧光显微镜技术来测量力产生过程中蛋白质构象的变化。为此，我们打算将荧光分子连接到肌球蛋白分子的特定区域，并利用能量转移过程来测量nm范围内的距离。能量转移的过程意味着一个分子的荧光发射以距离特异性方式受到另一个分子的荧光机制的影响。肌节中纤维的规则排列允许在数十个功能等同的节段上进行空间平均，从而提高了其他活细胞中无法获得的信噪比。有了这种新的方法，我们计划调查的荧光分子之间的距离的变化，特别是附着在肌球蛋白在力的发展和肌肉长度的扰动。结果将是肌球蛋白分子这些部分的运动图，以及这些运动的时间过程的确定。

Movement in living cells results from the coordinated behaviour of proteins. This energy-requiring process is driven by the breakdown of ATP (adenosine triphosphate), the cells' molecular fuel. Muscle cells specialise in converting this chemical energy into movement and mechanical work with their precisely dimensioned and aligned filaments to maximise power output, speed, fatigue resistance and efficiency of movement. At the molecular level, the movement is brought about by a cyclical change in conformation in one of the main protein, myosin. The details of the changes in protein conformation and their link to the steps in the ATP breakdown process are not known. In this project, we intend to develop advanced fluorescence microscopy techniques to measure changes in protein conformation during force production. To do this, we intend to attach fluorescent molecules to specific regions on the myosin molecule and make use of an energy transfer process to measure distances in the nm range. The process of energy transfer implies that the fluorescence emission of one molecule is affected by the fluorescence mechanism of the other, in a distance-specific manner. The regular arrangement of filaments into sarcomeres allows spatial averaging over dozens of functionally equivalent segments, thus giving rise to an improvement in the signal/noise ratio that is not available in other living cells. With this novel method, we plan to investigate changes in the distance between the fluorescent molecules specifically attached on myosin during force development and during perturbations of the muscle length. The result will be a map of the movement of these parts of the myosin molecule, and the determination of the time-course of these movements.