LRRK2-induced Ca ²⁺ entry defects are reversed by targeting lysosomal TPC2. (a) Schematic depicting permeabilization of lysosomes by LLOMe. (b) Epifluorescence micrographs of the indicated cell line loaded with rhodamine B–dextran and treated with vehicle (DMSO) or LLOMe (1 mM) for 1 h. Scale bar: 100 µm. (c) Schematic depicting fusion of lysosomes by vacuolin-1. (d) Transmitted light micrographs of the indicated cell line treated with vehicle (DMSO) or vacuolin-1 (1 µM) overnight. Arrows highlight the presence of vacuoles. Scale bar: 100 µm. (e) Effect of LLOMe and vacuolin-1 on depolarization-evoked Ca²⁺ signals in the indicated cell type (mean ± SEM, n = 3 independent biological replicates). (f) Effect of thapsigargin on depolarization-evoked Ca²⁺ signals in cells expressing LRRK2 G2019S (mean ± SEM, n = 3 independent biological replicates). (g) Summary data (mean ± SEM, n = 3 independent biological replicates) from e and f quantifying the peak change in the Ca²⁺ signals from the indicated line and treatment. Each point represents the mean response from a cell population. *P < 0.05 (one-way ANOVA, Tukey’s test). (h) Schematic of TPC2 depicting blockade of NAADP activation by PF-543 and the channel pore by Tet or mutation of Leu265. (i) Effect of PF-5483 and Tet on depolarization-evoked Ca²⁺ signals in the indicated cell type (mean ± SEM, n = 3–4 independent biological replicates). (j) Confocal micrographs of cells expressing TPC2^PD-GFP or LAMP1-GFP. Scale bar: 10 µm. (k) Effect of TPC2^PD and LAMP 1 on depolarization-evoked Ca²⁺ signals in the indicated cell type (mean ± SEM, n = 3 independent biological replicates). (l) Summary data (mean ± SEM, n = 3–4 independent biological replicates) from i and k quantifying the peak change in the Ca²⁺ signals and area under the curve from the indicated line and treatment. Each point represents the mean response from a cell population. *P < 0.05, **P < 0.01 (one-way ANOVA, Tukey’s test). Tet, tetrandrine.