Figure 5.

Rapalog-induced recruitment is not reversible by rapalog washout. (A) Frames from a time-lapse recording showing RFP-labeled peroxisomes in a KIF1A-FRB expressing cell. Rapalog was added at time 0:00 and washed out at time 30:00. (B) Time-coded color plot from the recording in A, in which blue and red mark distributions before (starting 15 min after Rapalog addition) and after washout, respectively. (C) Time traces of R_90% for four different cells (red). The black curve shows average ± SE; 0.1 μm rapalog was present from 0 to 30 min. (D) Recruitment of GFP-FRB (bottom) to RFP-FKBP labeled peroxisomes (top) using 1 mM rapalog present from 0 to 15 min. (E and F) Time trace of GFP intensity in regions with (black) and without (red) peroxisomes in the presence of 1 μM (E) and 10 nM (F) rapalog. Regions with peroxisomes were identified by thresholding the RFP time series. Traces were normalized by the average GFP intensity before rapalog addition in peroxisome-containing regions. Plots show average ± SE for 5 (E) and 4 (F) cells. (G) Ratio between the average GFP intensity in regions with and without peroxisomes calculated from E and F. Ratios start just above one because free cytoplasmic GFP levels are higher in the thicker perinuclear region that contains the peroxisomes. (H) Time traces of peroxisome-bound GFP intensities relative to the initial intensity of free GFP, obtained by subtracting a normalized time trace of cytoplasmic GFP intensity from the GFP intensity in peroxisome-containing regions.