Fig. 4: DCX-KO neurons show altered DCLK1 localization and lysosome motility.

A Compared mean intensity for MAP2, Tau, DCLK1, Kif1A, Kif3A, Kif5B, and Dynein in CTRL0 or DCXKO1 DIV3 immuno-stained neurons. The analysis pipeline is shown above the plot: a mask of the microtubules (MTs) was made, and the intensity of the marker was normalized to the intensity of the mask and to the control condition. Each transparent point represents a single image, while the opaque points show mean values, error bars represent the confidence intervals. Analyses are from n = 5, 5, 10, 5, 6,13, 6 CTRL0 images or 5, 5, 10, 5, 5, 13, 5 DCXKO1 images (MAP2, Tau, DCLK1, Kif1A, Kif3A, Kif5B, and Dynein respectively) from 2–4 experiments. Statistical analysis was performed using the Mann-Whitney non parametric U test: *p < 0.05. B Images representative of 3 experiments showing CTRL0 and DCXKO1 DIV3 neurons stained for DCLK1, Kif5B and Tubb3. C (Top) Representative still images chosen from 3 experiments showing CTRL0 and DCXKO1 straight neurite sections after incubation with the Lysotracker dye, and 2 min kymographs of those same sections. A = anterograde, and R = retrograde. (Bottom) Position plots for lysosomes detected using Trackmate during the 241 frames for CTRL0 and DCXKO1 neurons. The distribution of these positions was also plot and is represented on the right end side of the position plots. D Mean squared displacement (MSD) of lysosomes inside CTRL0 and DCXKO1 neurites. Data are presented as mean values ± SEM. E Proportion of tracked cargos that are either stationary (Stat.), Diffusive (Diff.) or Processive (Proc.). Data are presented as mean values ± SEM. Each point represents a single neuron. F Directional bias of lysosome cargos in CTRL0 and DCXKO1 neurons, where 0 corresponds to retrograde only and 1 corresponds to anterograde only. For D-F, n = 41 control and 30 DCXKO1 neurons from 3 independent experiments. Statistical analysis was performed using the Student’s t-test: ns nonsignificant, *p < 0.05. G Violin plots of the average velocity for anterograde and retrograde displacement of lysotracker cargos in CTRL0 and DCXKO1 DIV3 neurons. n = 1828 tracks for control and 1131 for DCXKO1 from 3 independent experiments. Statistical analysis was performed using the Student’s t-test: ns nonsignificant. p-values are detailed in Supplementary Table 2.