Fig. 4: MAD1-deficiency changes Golgi morphology and post-Golgi trafficking.

a Colocalization of endogenous MAD1 (blue) with GM130 (red) in neurons at DIV3. A saturated EGFP signal (green) was used as a morphological marker. Dashed lines demarcate the cell periphery. b, c The morphology of Golgi was assessed using z-stacking and z-projection of images immunostained with GM130 (red). EGFP (green) with a dashed line demarcates cell morphology. Representative images from the “Normal” and “Fragmented” groups (b, upper). Graph showing the percentage of transfected cells with each pattern of Golgi morphology (b, lower). Representative images of “Dendritic” or “Non-dendritic” Golgi position in DIV3 neurons (c, upper) and the percentage of transfected cells with each pattern of Golgi positioning (c, lower, shCTL, N = 3; shMAD1, N = 3; n > 30 cells for each N). d–g Neurons were transfected with VSVG + shCTL, VSVG + shMAD1, or VSVG + shMAD1 + c-MAD1. Neurons were co-immunostained with GM130 (blue) and VSVG (red) in a time-dependent manner (0, 20, and 40 min). EGFP signal shows the morphology of transfected cells (gray fill). Representative images of the soma region showing Golgi-accumulated VSVG at 0 min (d, left). Representative time-dependent images from each group showing whole neuronal morphology with enlarged neurites in the 20 μm range (20–40 μm from Golgi at 20 min, 30–50 μm from Golgi at 40 min). Arrowheads point to the Golgi (d). Statistical analysis of colocalization between GM130 and VSVG particles. Statistical significance (*) was assessed by comparing shCTL with shMAD1 at the same time point (e). A schematic diagram for the measurement of maximal VSVG moving distance from the Golgi (f, upper). Statistical analysis of maximal moving distance from Golgi at 20 min (f, lower) and 40 min (g, shCTL, 0 min, n = 22; 20 min, n = 23; 40 min, n = 23; shMAD1, 0 min, n = 17; 20 min, n = 27; 40 min, n = 29; shMAD1 + c-MAD1, 0 min, n = 14; 20 min, n = 23; 40 min, n = 27). Scale bars represent 10 μm (a–d). Statistical analysis was conducted by using one-way ANOVA followed by Turkey’s post-hoc test. Analysis with nested model was conducted (e–g; Supplementary Dataset). Data are presented as means ± SEM. Statistical significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 or n.s. (not significant).