Rock R S, Rief M, Mehta A D, Spudich J A
Department of Biochemistry, Stanford University School of Medicine, Beckman Center B405, Stanford, California 94305-5307, USA.
Methods. 2000 Dec;22(4):373-81. doi: 10.1006/meth.2000.1089.
Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.
肌球蛋白V是一种基于肌动蛋白的马达蛋白，被认为参与囊泡运输。由于这种马达蛋白的特性可能与肌肉肌球蛋白II不同，我们结合使用滑动丝和光镊测定法来高分辨率观察单个分子，研究了肌球蛋白V驱动的运动。结果清楚地表明，脑肌球蛋白V是一种高效的持续性马达蛋白。在低肌球蛋白V密度下的体外运动测定显示出明显的单分子支持运动。在肌球蛋白V驱动运动的光镊测定中也观察到了持续性步移。这里描述了用于证明肌球蛋白V持续性的方法。这些方法包括密度依赖性测定，该测定消除了多个马达蛋白聚集或偶然共定位导致明显单分子运动性的可能性。此类测定将成为鉴定其他持续性肌球蛋白类别的有用工具。
