For2A-GFP spots generate actin filaments at the cell cortex. (A and B) Simultaneous acquisition of For2A-GFP and Lifeact-mCherry at the cell cortex using VAEM demonstrates that For2A-GFP resides on the ends of newly generated actin filaments (A) and along preexisting filaments (B). See also Videos 9 and 10. In the merge images, For2A-GFP and Lifeact-mCherry are magenta and cyan, respectively. Numbers represent time in milliseconds (ms). Bar, 2 µm. Arrowheads in each frame of the merge indicate the For2A-GFP and the arrow in the last frame indicates the position of the For2A-GFP spot at the beginning of the time-lapse. (C) Quantification of the number of cortical For2A-GFP spots that moved in a linear trajectory and were found at the end of a new actin filament (blue), along a preexisting filament (green), or did not correlate with an actin filament (pink). 61 linear For2A-GFP trajectories were analyzed from 5 cells. (D) Quantification of the speed of For2A-GFP linear movements on new or preexisting filaments. (45 For2A-GFP spots from five cells). (E) Quantification of the change in Lifeact-mCherry fluorescence in the wake of a linearly moving cortical For2A-GFP spot on new or preexisting filaments (37 spots from five cells).