Fig. 5. Expression of FLAG-MCLT reduces the velocity of SGs. (A,B) PC12 cells, either single-transfected with hCgB-GFP(S65T) (control) or double-transfected with hCgB-GFP(S65T) and FLAG (FLAG) or FLAG-MCLT (FLAG-MCLT), respectively, were incubated for 2 hours at 20°C and then at 37°C as indicated. Cells were imaged at 37°C and the movements of SGs were tracked automatically. For each time point and condition at least 20 cells from four independent experiments were analysed. (A) Mean velocities of all SGs per cell are plotted as a function of chase time. Prior to plotting, the system-inherent error of the automated tracking algorithm used was subtracted from all values (see Materials and Methods). Error bars, s.e.m. (B) Frequency distributions of all velocity steps (n) recorded from frame to frame for each condition over the observation time indicated in A. Open arrowhead, maximal number of steps measured for control and FLAG-transfected cells; filled arrowhead, maximal number of steps measured for FLAG-MCLT-transfected cells. (C) PC12 cells, co-transfected with pcDNA3/PTS1-GFP and FLAG or FLAG-MCLT, respectively, were incubated for 2 hours at 20°C and then for 90 minutes at 37°C. Cells were imaged at 37°C and the movements of peroxisomes were tracked automatically. The frequency distributions of all velocity steps (n) are shown for each condition (C).