Fig. 4. Time-lapse TIRF microscopy of Twf1 severing actin filaments. (A-C) 1.5 µM monomeric actin (30% Alexa Fluor 488 labeled) was assembled into filaments in a flow cell, and then control buffer at pH 5.0 (A), 10 µM Lat-A at pH 5.0 (B), or 2 µM Twf1 at pH 5.0 (C) was applied at time zero. The time in seconds following addition of buffer/proteins is shown in the upper right corner of each panel. (D) Following filament severing by Twf1 in C, the immobilized filaments were incubated with fresh 1.5 µM actin monomers (30% Alexa Fluor 488 labeled) at time zero. Yellow arrowheads track the growth of barbed ends in consecutive frames (barbed end growth rate 8.9 µM^–1 second^–1, 10 filaments measured). Blue arrows mark the original positions of barbed ends (at time zero) for frame of reference. The field in D is the same field as that shown in C. Movies of these TIRF microscopy experiments are provided in supplemental materials (Movies 1-3, supplementary material).