Figure 4.
TFEB promotes clearance of enlarged lysosomes and increases lysosomal motility in PD fibres.
A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible – overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.
B. FDB muscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to “exit” the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.
C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.