Dyn2 depletion leads to decreased PM duration and increased mobility of caveolae in Cav1-mCh FlpIn cells. (A) Immunogold staining of Dyn2 on PM lawns prepared from 3T3-L1 cells. Arrows indicate caveolae and a CCP. Panels to the right show caveolae at higher magnification. Bars, 100 nm main figure, 50 nm small panels. (B) Representative first frame image from TIRF movie of Cav1-mCh cell. Scale bar, 10 μm. Dashed white square shows the region of the cell that was tracked over 5 min. Magnifications show last frame of the movie. Scale bar, 2 μm. Red dots illustrate mCh structures recognized by the tracking program as detailed described in the Materials and methods section. The color-coded trajectories illustrate the duration and length, whereby the speed is calculated, as well as the displacement length (shortest length between the first and last position) of tracked caveolae structures for 5 min. 1 in red represents caveolae with long duration time, short displacement length, and low speed. 2 in cyan represents caveolae with intermediate duration time, long displacement length, and high speed. 3 in blue represents caveolae with short duration time, short displacement length, and high speed. (C–H) Cav1-mCh cells were transfected with siRNA for 72 h before experiments were performed. (C) Immunoblots of cells treated with siRNAs as indicated. (D) Number of caveolae counted in the basal PM in cells imaged on TIRF. Mean ± SEM from at least 17 cells per condition. (E) Color-coded trajectories of caveolae in Cav1-mCh cells treated with siRNA as indicated. Cells were imaged by TIRF over 5 min. Scale bars, 10 μm. (F–H) Quantification of Cav1-mCh track duration time (F), speed (G), and displacement length (H). Numbers were related to ctrl-treated cells. Analyses were performed using Imaris software and track mean ± SEM from at least eight cells per condition are shown. Significance was assessed using t test, *P ≤ 0.05, **P ≤ 0.01. Source data are available for this figure: SourceData F1.