The degree of assembly into filaments and the length distribution of the arginylated actin filaments. (A) Phalloidin Alexa Fluor-488–labeled Ac- and R-actin filaments were loaded on the surfaced coated with NM myosin II in the absence of ATP. (B) The number of the landed filaments per movie. (C) The length distributions of the landed filaments. (D) Schematic of the image analysis pipeline to detect and analyze detachment/ wobbling of actin filaments in the surface motility assay. The pixels that showed a large difference from the previous time frame were detected by dividing the (i+1)-th images with the ith images after smoothing with Gaussian blurring. The maximum projections through the time frames are shown in a pseudocolor, as shown in Fig. 3D. For quantification of the detachment/wobbling events, a threshold was calculated based on the mean and the SD of the background areas in each movie, and the spots above this threshold value in the (i+1)-th/ith ratio movie were detected. (E) Actin-gliding data obtained from analysis of motility assay performed with tissue isolated full-length cardiac myosin.