Path reconstruction as a tool for actin filament speed determination in the in vitro motility assay

The in vitro motility assay is used to measure speed of actin filaments moving over a glass surface coated with heavy meromyosin. In this paper a new method, the path reconstruction method, is presented to evaluate observed speeds. The method is compared with the commonly used centroid method, in which the centroids of the filaments are followed from frame to frame. Instead, in the path reconstruction method speed is evaluated from determination of perimeters of the filaments in each frame and by reconstruction of the traversed paths of the filaments over a number of frames. Biases in the determination of speed occurring in the centroid method due to curvature of paths and to video noise and Brownian motion are eliminated in the path reconstruction method, allowing measurement over a range of frame rates from 5 to 25 per second. The path reconstruction method leads to a clear separation of motile and nonmotile filaments provided that filaments are analyzed over at least 10 successive frames and allows easier separation of uniform and nonuniform sliding behavior.