(A) Myo8A-GFP localizes to punctate structures throughout the cytosol as well as on the cell cortex. Images are maximum projections of z-stacks acquired with a spinning disc confocal. The punctate structures accumulate near the apex of the growing cell. Large globular structures are chloroplasts, which autofluorescence under these imaging conditions. Scale bar, 10 µm. (B) Images of Myo8A-GFP and Lifeact-mCherry in moss protonemata were simultaneously acquired with VAEM. In the merge Myo8A-GFP is green and Lifeact-mCherry is red. Scale bars, 2 µm. See also Video 1. Yellow box indicates the enlarged area shown in (C). Red line marks the trace for making the kymograph in (C). (C) An example of Myo8A-GFP particle moving along actin filaments. Six consecutive frames with 76 ms time interval are shown. Arrowhead indicates the starting position of a Myo8A-GFP particle, and arrows indicate the last position of that Myo8A-GFP. In the last frame, a new Myo8A-GFP particle binds to the same position indicated by the arrowhead. Linear movement of Myo8A-GFP is evident in kymograph. Scale bar, 1 µm. Scale bar in t, 1 s. (D) Moss protonemal cells expressing Myo8A-GFP, were treated with or without 25 µm Latrunculin B (LatB) and imaged with VAEM. In control samples, Myo8A-GFP linear trajectories are apparent in a frame average of 25 frames from approximately 2 s of real time, but absent in cells treated with LatB. Scale bars, 2 µm. See also Video 2. (E) Distribution of Myo8A-GFP velocities on actin filaments. Inset is a dot plot of the measured Myo8A-GFP velocities. Average velocity is 0.65 ± 0.57 µm/s; n = 249 events from seven cells.