Fig. 6.
Lysosomal proteolytic abnormalities in cells lacking atlastin-1.
A) Day 14 i3Ns from the lines indicated were fixed and immunolabelled for LAMP1 and cathepsin D, as well as cell mask whole-cell and DAPI nuclear labels. Cells were imaged with a Zeiss Axio Observer microscope with a tile imaging function and the images were deconvolved using Huygens software. B) The diameter of the largest LAMP1-positive vesicle per cell was measured in at least 100 cells per genotype and the percentage of neurons with at least one lysosome over 1.6 μm in diameter was calculated and plotted. LAMP1 vesicle size measurement was performed blind to genotype. N = 6 biological repeat experiments. C) and D) The number and area of cathepsin D-positive puncta were measured using an automated protocol developed in Image J software. Only signal within the cell area outlined by the cell mask was considered. Cell number was calculated based on the number of nuclei in the field. The percentage of cells with cathepsin D puncta >0.3 μm2 in area is plotted in C), while the total number of cathepsin D puncta per neuron is shown in D). On average 300 cells were analysed per genotype in each experiment, N = 6 biological repeats. Error bars show SEM. n.s., p > 0.05; *, p < 0.05. E) Day 21 neurons from the i3N lines indicated were incubated with DQ-BSA substrate for 5.5 h and with a live-imaging whole cell stain (Cell tracker) for 20 min. Living cells were then imaged with a Zeiss LSM 780 Confocal Microscope. F) Neuron somas were selected using the cell tracker channel. The mean DQ-BSA fluorescent intensity was measured and recorded for each cell. Mean results from each experiment are plotted as percentage relative to the Scr control. 5 independent experiments were performed and a minimum of 50 cells per genotype in each experiment was used for quantification. Error bars show mean +/− SEM. ***, p < 0.001. In B), C), D) and F) statistical comparisons were made using repeated measures one-way ANOVA with Dunnett's correction for multiple testing.