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

活细胞中的运动是蛋白质协调行为的结果。这个需要能量的过程是由细胞的分子燃料三磷酸腺苷(ATP)的分解推动的。肌肉细胞专门利用其精确尺寸和排列的细丝将这种化学能转化为运动和机械功，以最大限度地提高功率输出、速度、抗疲劳性和运动效率。在分子水平上，这种运动是由主要蛋白质之一肌球蛋白的构象周期性变化引起的。蛋白质构象变化的细节及其与ATP分解过程中的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在强制生产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上的特定区域，并利用能量转移过程来测量纳米范围内的距离。能量转移的过程意味着一个分子的荧光发射受到另一个分子的荧光机制的影响，这种影响是与距离有关的。肌节中细丝的规则排列允许在数十个功能相同的节段上进行空间平均，从而提高了信噪比，这是其他活细胞所不具备的。通过这种新的方法，我们计划研究肌球蛋白上特异附着的荧光分子在力量发展和肌肉长度扰动期间的距离变化。结果将是肌球蛋白分子这些部分的运动图，并确定这些运动的时间进程。

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.