FIGURE 5.

Depolymerization of Asp-11-mutant actins. A and B, latrunculin-induced depolymerization of D11Q (A) and D11N (B) actins. Solutions of polymers of WT, mutant, and a 1:1 mixture of both (concentration of total actin was 5 μm in all samples) were ultracentrifuged with or without preincubation with 60 μm latrunculin A for 10 min. The supernatant and pellet fractions were analyzed by SDS-PAGE, and the factions of actin in pellets were calculated by densitometry. Error bars indicate standard deviation of three independent measurements, and Student's t test demonstrated that the difference between WT and D11Q, WT and WT + D11Q, WT and D11N, and WT and WT + D11N are all significant, with p values < 0.00006. C, latrunculin-induced depolymerization of pyrene-labeled WT actin copolymerized with the same concentration of unlabeled WT (filled circles) or D11Q (open circles) actin, respectively, and assayed as in A. D, depolymerization of individual actin filaments. Alexa-Fluor 488-labeled WT actin copolymerized with the same concentration of unlabeled WT or D11Q actin was immobilized on a heavy meromyosin-coated surface and imaged immediately and 10 min after flushing with F-buffer. Bar, 20 μm.