FIGURE 6.

Regulation of FMNL1-mediated actin filament severing by the srGAP2 SH3 domain. A, shown are time-lapse images of polymerized actin filaments every 10 s in the presence of purified active FMNL1-C (amino acids 449–1094). Numbers in the upper right corner of each frame indicate time. Extensive severing is observed during the 1-min time-lapse period. Yellow arrowheads mark the locations of new filament severing events in each frame. B–G, shown are images of actin filaments (2 μm) either alone (B), with FMNL1-C (400 nm) (C), or with FMNL1-C and increasing concentrations of the purified srGAP2 SH3 domain (D–G). Scale bar represents 15 μm. H–J, quantification of filament lengths from three independent severing assays show the fraction of filaments longer than 9 μm (H) or shorter than 3 μm for each condition (I). J, shown is percent severing as calculated from H and I for increasing concentrations of the srGAP2 SH3 domain. Scale bars represent 15 μm. Error is ±S.E. K, polymerized actin filaments (250 nm, lane 1), stabilized by phalloidin, are pelleted by ultracentrifugation in the presence of FMNL1-C (100 nm) without and with srGAP2 SH3 (200 and 400 nm, lanes 4 and 5, respectively). In the absence of actin, FMNL1-C does not co-pellet with actin filaments (lane 2). With actin filaments, FMNL1-C does co-pellet (lane 3), and the srGAP2 SH3 domain does not disrupt this interaction (lane 4 and 5).