Suzuki Madoka, Fujita Hideaki, Ishiwata Shin'ichi
Department of Physics, School of Science and Engineering, Waseda University, Tokyo, Japan.
Biophys J. 2005 Jul;89(1):321-8. doi: 10.1529/biophysj.104.054957. Epub 2005 Apr 22.
To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.
为了弥合肌肉收缩系统与体外运动测定之间的差距，我们设计了一种A带运动测定系统。用凝溶胶蛋白处理甘油化的骨骼肌肌原纤维，以选择性地去除细肌丝并暴露出单个A带。使被光镊捕获的单根带尾肌动蛋白丝与A带的内表面或外表面相互作用，并通过相差显微镜和荧光显微镜在生理离子条件下测量带尾丝的位移。我们观察到细丝有大幅度的来回位移，并伴随着所产生力的大幅变化。尽管存在这种大的张力波动，但我们发现，平均力与A带内部和外部的重叠部分成正比，分别距A带末端约150 nm和300 nm。与肌球蛋白分子密度的差异一致，A带内部重叠部分每单位长度的平均力（时间平均力/肌球蛋白头部约为1 pN）约为外部的两倍。因此，我们得出结论，本文所述的A带运动测定系统适用于研究单根肌动蛋白丝上的力产生及其在规则三维粗肌丝晶格内的滑动运动。
