RILPL1 promotes LRRK2 ⁺ lysosome clustering around the centrosome. (A) U2OS cells were treated with a nontargeting control (NTC) or siRILPL1 RNA. 24 h later, cells were transfected with HaloTag-LRRK2 for 36 h. Then, cells were treated with LLOME (2 h) and were fixed and stained with a pericentrin antibody. Volume view image of LRRK2⁺ lysosomes clustered around the centrosome (pericentrin). (B) The distance between LRRK2⁺ lysosomes and the centrosome was measured in cells treated with NTC or siRILPL1. Data are mean ± SEM (P < 0.0001, n = 221–222 lysosomes, from 7 to 9 cells). Unpaired t test with Welch’s correction. (C) The distance between LRRK2⁺ lysosomes and the center of the nucleus was measured in cells treated with NTC or siRILPL1. Data are mean ± SEM (P = 0.0206, n = 221–222 lysosomes, from 7 to 9 cells). Unpaired t test. (D and E) Time-lapse of a U2OS cell expressing HaloTag-LRRK2 and treated with LLOME. LRRK2 puncta recruitment and movement are followed at different time points up to 120 min. (D) is a control cell, and (E) is a cell previously transfected with a RILPL1 siRNA. (F) U2OS cells treated with a NTC and siRNA against DYNC1H1 were lysed and blotted against anti-DYNC1H1 and a-tubulin antibodies. (G) U2OS cells were incubated with NTC or siDYNC1H1 (24 h) and transfected with HaloTag-LRRK2, mBaoJin-RILPL1, and LAMP1-RFP (36 h). Cells were then imaged live after treatment with LLOME (2 h). Arrowheads indicate LRRK2⁺ lysosomes. Scale bar = 10 µm. Insets = 2 µm. Source data are available for this figure: SourceData F4.