Autolysosome homeostasis requires the MTMR8 PH-GRAM domain and catalytic cysteine residue. (A) Schematic of MTMR8 mutants used in (B, C, I, J, K, and O). An intact PH-GRAM domain and catalytic cysteine residue exist in WT MTMR8, MTMR8-C338S features a serine in place of the catalytic cysteine, and the PH-GRAM domain is truncated in MTMR8ΔPH. (B, C, and I) Representative maximum intensity projection of z-stack image from HeLa cells with the indicated genotypes stained with Hoechst to detect nuclei (blue) and antibodies to detect LAMP1 (green) and LC3B (magenta), indicative of lysosomes and autophagosomes, respectively. Autolysosomes contain both LAMP1 and LC3B. (D–H) Quantification of the size, number, and colocalization of both LAMP1 and LC3B puncta per cell as represented in B, C, and I. (J, K, and O) Representative maximum intensity projection of z-stack image from HeLa cells with the indicated genotypes stained with Hoechst to detect nuclei (blue) and antibodies to detect WIPI2 (magenta; J, K, and O) and LC3B (green; J′, K′, and O′), with merged channels shown in J″, K″, and O″ and dashed white boxes indicating the areas shown in zoom panels at the right. (L–N) Quantification of the number and size of WIPI2 puncta and colocalization between WIPI2 and LC3B as represented in J, K, and O. Data are presented as mean ± minimum to maximum (n ≥ 5 cells per 10 random images from three experimental replicates (D–H and L–N). Asterisks denote statistical significance (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001), using one-way ANOVA. Scale bars, 20 µm.