Figure 4

G2385R mutation affects vesicle trafficking. (A) Schematic representation of LRRK2 wild-type and G2385R variant. The distinct LRRK2 domains are indicated. (B) Western-blotting analysis of cells expressing sypHy reporter together with RFP-LRRK2 derived constructs. (C) Western-blotting analysis of cells expressing sypHy reporter together with vector coding for DsRed fluorescent protein and shRNA targeting hLRRK2 (siLRRK2) or a control sequence (siControl). (D) Time course analysis of synaptic events occurring in SH-5YSY cells transfected with wild-type LRRK2 (WT), G2385R-LRRK2, siControl or siLRRK2 under resting conditions. SH-5YSY cells were co-transfected with sypHy and the indicated constructs and imaged by TIRFM 48 hours later. Peaks of variable fluorescence intensity correspond to single fusion events. Fluorescence data are expressed as F/F0. The graphs show the total number of fusion events (E) and the resulting fluorescence changes (F) expressed as Area Under Curve (AUC) for each construct. Data are normalized for the cell area and are the mean ± SE of up to 20 cells per construct. *p < 0.05, ***p < 0.001 ANOVA. (G) Vesicle density in the TIRFM zone after NH₄Cl₂ treatment. The cells were incubated with the membrane permeant NH₄Cl₂ solution for 5 minutes, to label all sypHy positive cluster. Then, cells were fixed and imaged by TIRFM to visualize vesicles docked to the plasma membrane. Each spot corresponds to a sypHy positive clusters. Scale bar = 10 μm. (H) The graph reports the number of sypHy positive clusters present under the TIRF zone. Data are normalized for the cell area and are expressed as mean ± SE; n = 15 cells for construct. *p < 0.05, **p < 0.01 ANOVA.