Extended Data Fig. 8: Loss of MROH1 affects acidity and proteolytic activity of lysosomes.
From: The HEAT repeat protein HPO-27 is a lysosome fission factor
a-a”, Super-resolution images of a WT COS7 cell stably expressing GFP-MROH1 and containing chased fluorescent Dextran A555. The boxed region is magnified in the zoom images. Arrows and arrowheads indicate association of MROH1 with vesicular and tubular lysosomes labeled by Dextran. b, CRISPR-mediated generation of the MROH1 knockout COS7 cell line, and verification by sequencing. The knockout clone contains a 28-bp deletion, which causes a frameshift and results in a premature stop codon. c, qPCR analysis of MROH1 expression in the indicated cells. Three independent experiments were performed. Data are shown as mean±SD. d-o, Confocal fluorescence images of WT (d-f), MROH1 KO (g-i), siControl (j-l), and siMROH1 (m-o) COS7 cells stained by Lysotracker Red (d, g, j, m), Magic Red (e, h, k, n), or DQ-BSA (f, i, l, o). Cell boundaries are outlined by dashed circles. Quantifications are shown in p (n = 20 cells in each of 4 biological repeats), q (n = 20 cells in each of 3 biological repeats). Data are shown as mean±SD. r-s”’, Confocal fluorescence images of Lysotracker Blue-stained COS7 cells co-expressing GFP-MROH1 and CHERRY-RAB7A(WT) (r-r”’) or CHERRY-RAB7A(T22N) (s-s”’). Arrowheads indicate enrichment of MROH1 on RAB7-positive vesicles. In c, p, q, Student’s two-tailed unpaired t-test was performed to compare MROH1 mutant or siRNA datasets with WT or siControl. P values are indicated. Scale bars: represent 5 μm.
