Figure 4.

E193K variant affects vesicle trafficking. (A) Schematic representation of LRRK2 wild type, E193K variant and N-terminus domain. The distinct LRRK2 domains are indicated. (B) Western-blotting analysis of WT and E193K N2A clones expressing sypHy reporter together with empty vector or RFP-LRRK2 N-terminus constructs. Arrowhead indicates the specific RFP positive band detected by the anti-RFP antibody. (C) Vesicle density in the TIRFM zone after NH₄Cl treatment. The cells were incubated with the membrane permeant NH₄Cl solution for 5 minutes, to label all sypHy positive clusters. Then, cells were fixed and imaged by epifluorescence or TIRFM to visualize vesicles docked to the plasma membrane. Each spot corresponds to a sypHy positive cluster. Scale bar = 10 μm. (D) The graph reports the number of sypHy positive clusters visualized in the same cell under epifluorescence or TIRFM. Data are normalized for the cell area and are expressed as mean ± SE; n = 15 cells for construct. ***p < 0.001, Student’s T-test. (E) Time course analysis of synaptic events occurring in N2A stable clones expressing LRRK2 wild type (WT) or E193K LRRK2 and transfected with the empty-vector or with the isolated N-terminal domain together with the sypHy reporter. Transfected cells were imaged by TIRFM 48 h 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 (F) and the resulting fluorescence changes (G) expressed as Area Under Curve (AUC) for each construct. Data are normalized for the cell area and are expressed as mean ± SE of up to 20 cells per construct, in three independent experiments. *p < 0.05, **p < 0.01 compared to LRRK2 wild type, ^###p < 0.001 compared to empty vector transfected clones.