FIGURE 4:

PICK1 binds F-actin in vitro but not in cells. (A) SDS–PAGE analysis of the pellet (P) and supernatant (S) fractions of high-speed sedimentations performed at a fixed F-actin concentration (1 μM) and various PICK1 concentrations (1–20 μM). The graph on the right shows the fraction of F-actin–bound PICK1 as a function of PICK1 concentration, determined from densitometric analysis of the gels. The solid line represents the global fit of the data from three independent experiments. (B) Localization of GFP-PICK1_129-415 (green) and RFP-actin (red) in double-transfected HeLa cells. The boxed region is enlarged on the right. Note the lack of colocalization of F-actin–rich structures with PICK1-associated vesicles. Occasional overlaps of the two markers do not persist over time (see Supplemental Movie S6). (C) Latrunculin B inhibits the motility of PICK1_129-415-associated structures in B16F1 cells, shown before (control) or after treatment with 2 μM latrunculin B. First column, first frame of time-lapse sequence. Second column, maximum projection of frames 1–15 taken at 4-s intervals. Third column, overlay of the first two columns (see Supplemental Movie S7). Fourth column, zooms of the regions boxed in the third column. The plot on the right shows the quantification of vesicle speeds as described in Figure 3C. The difference between control and latrunculin B–treated cells is statistically significant (p < 0.0001, n = 56 tracks from five control cells; n = 88 tracks from five latrunculin B–treated cells).