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. 2009 Nov 1;20(21):4531–4540. doi: 10.1091/mbc.E08-10-0997

Figure 5.

Figure 5.

Analysis of Cav-1-GFP mobility by TIR-FM. (A) Comparison of epifluorescent (green) and TIR-FM (red) images of Cav-1-GFP in HLMVECs. (B) Time-series images obtained by TIR-FM were used to generate projections of the changes in Cav-1-GFP localization in or near the membrane over time. Cav-1-GFP membrane mobility includes lateral migration of vesicles, appearance and disappearance of Cav-1-GFP in the TIRF plane due to vesicle fission from and fusion with the plasma membrane, and separation of preexisting vesicle complexes. A single TIRF image is shown in a. Dynamic changes in the TIRF plane were estimated by subtracting each frame from the next using the Delta-F-up ImageJ module. The Z-projection over time of 29 resultant TIRF frames is presented in b. Mask of thresholded Z-projection is shown in c. Z-projection of 115 manually tracked vesicles is presented in d. Tracks observed by manual tracking were identical to those obtained with ImageJ software. (C) Three examples of Cav-1-GFP projection masks in control siRNA and FLN A siRNA-treated cells are shown. (D) Bar graph represents the total number of mobile Cav-1–GFP-labeled vesicle movements per unit area as a percentage of events registered in cells transfected with control siRNA (mean ± SEM). The overall mobility of Cav-1-GFP was reduced in FLN A siRNA-transfected cells by 41.4 ± 6.9% (p = 0.0052).