Figure 6.

Munc18‐1 KO neurons show reduced endosome‐to‐Golgi retrograde transport of Cholera Toxin B. (a) Typical example of WT and Munc18‐1 KO neurons at DIV3 stained for MAP2 (dendritic marker) and LAMP1 (lysosomes). Scale bar is 25 μm. (b) The relative LAMP1 area, measured as the LAMP1‐positive area over soma area, was not changed in Munc18‐1 KO neurons (ns p = .41, Mann–Whitney test). N: WT = 25/3. KO = 26/3. (c) No differences were observed in LAMP1 intensity between both conditions (ns p = .81, unpaired T test). N: WT = 25/3. KO = 26/3. (d) Typical example of WT and Munc18‐1 KO neurons at DIV 3 stained for MAP2 (dendritic marker) and EAA1 (early endosomes). Scale bar is 25 μm. (e) Number of EEA1‐positive puncta per μm² was unchanged in Munc18‐1 KO neurons (ns p = .34, unpaired T test). N: WT = 30/3, KO = 29/3. (f) The size of EEA1‐positive puncta was comparable between the two conditions (ns p = .42, Mann–Whitney test). N: WT = 30/3, KO = 29/3. (g) Cartoon representing the CTB assay. After a 15‐min incubation of CTB, uninternalized CTB is washed out. Internalized CTB is allowed to retrogradely traffic to the Golgi for 2 hr. (h) Typical examples of WT and Munc18‐1 KO neurons fixed after the CTB assay. Neurons were stained for MAP2 (dendritic marker) and GM130&TGN46 (cis‐Golgi and TGN markers). (i) CTB intensity in Golgi was ~45% lower in Munc18‐1 KO neurons (p < .0001, Mann–Whitney test). N: WT = 35/3, KO = 35/3. (j) Total CTB internalized in Munc18‐1 KO neurons was ~30% lower (p < .0001, Mann–Whitney test). N: WT = 35/3, KO = 35/3. (k) Relative CTB distribution in the Golgi, measured by CTB intensity in Golgi (I) divided by total CTB intensity (J), was 20% lower in Munc18‐1 KO neurons (p = .004, unpaired T test). N: WT = 35/3, KO = 35/3. Data are represented in Tukey boxplots. Columns and dots represent individual litters and neurons, respectively. N = number of neurons/number of independent cell cultures