PLEKHM1 acts as a multivalent adaptor that promotes physical interaction between Rab7 and Arl8b. (a–c) Live-cell imaging was performed on cells expressing GFP-Rab7 and Arl8b-tomato along with either FLAG-PLEKHM1 or FLAG-NΔ300 PLEKHM1. The yellow arrowhead depicts kiss-and-run events in a and clustered enlarged endolysosomes in b, respectively. Rab7- and Arl8b-positive punctate structures that do not fuse in c are marked by white and yellow arrowheads. (d) HEK293T cell lysates expressing HA-Rab7 alone or coexpressed with either FLAG-PLEKHM1 (WT) or FLAG-PLEKHM1 (HRR→A) were immunoprecipitated (IP) with anti–HA antibody resin and immunoblotted (IB) using the indicated antibodies. (e) PC of Arl8 and Rab7 immunostained in HeLa cells transfected with indicated plasmids (n = 3; 30 cells analyzed per experiment). (f) Arl8b-HA was transfected in control- or PLEKHM1-siRNA–treated HEK293T cells. The lysates were immunoprecipitated with anti–HA antibody resin and immunoblotted using the indicated antibodies. (g) Lysates of WT- and PLEKHM1 KO-HeLa cells were immunoprecipitated with anti–Rab7 antibody resin and immunoblotted with the indicated antibodies. (h–j) Representative confocal images of HeLa cells treated with control siRNA or PLEKHM1 siRNAs and immunostained with anti–Arl8 and anti–Rab7 antibodies. Arrowheads mark colocalized pixels, and the nucleus was stained using DAPI. (k and l) Representative confocal micrographs of PLEKHM1 siRNA–treated HeLa cells expressing siRNA-resistant GFP-PLEKHM1 (WT) or GFP-PLEKHM1 (HRR→A) and immunostained for Arl8 and Rab7. In the insets, yellow arrowheads mark colocalized pixels. (m and n) PC and MC were calculated for Arl8 and Rab7 colocalization in indicated siRNA treatments of HeLa cells (n = 3; 30 cells analyzed per experiment). Data represent mean ± SEM (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test). Bars: (main) 10 µm; (insets) 2 µm.