Figure 8.

M6PR and LRP1 mediate PGRN–PSAP lysosomal trafficking. (A) Both PSAP and PGRN are mislocalized in M6PR-deficient fibroblasts. Fibroblasts infected with lentiviruses harboring guide RNA against M6PR and Cas9 were selected with puromycin and fixed and stained with goat anti–mouse PSAP or sheep anti–PGRN antibodies as indicated together with rat anti-LAMP1 and rabbit anti-M6PR antibodies. Two neighboring cells with (a) or without (b) M6PR expression are shown. (B and C) Quantification of PSAP (B) and PGRN (C) localization within LAMP1-positive vesicles in control and M6PR^−/− fibroblasts as shown in A using ImageJ. Data are given as mean ± SEM from three independent experiments: **, P < 0.01; Student’s t test. (D and E) M6PR is required for PSAP-mediated PGRN lysosomal targeting from the extracellular space. Fibroblasts infected with lentiviruses harboring guide RNA against M6PR and Cas9 were selected with puromycin for a week and treated with recombinant human his-PSAP and human FLAG-PGRN-his proteins (5 µg/ml) in serum-free medium for 12 h, and then stained with hPGRN, LAMP1, and M6PR antibodies. Two neighboring cells with (a) or without (b) M6PR expression are shown. Intensities of endocytosed PGRN were quantified using ImageJ. Data are presented as mean ± SEM from three independent experiments: ***, P < 0.001, Student’s t test. (F and G) LRP1 is also critical for PGRN–PSAP uptake in fibroblasts. Fibroblasts infected with lentiviruses harboring guide RNA against LRP1 and Cas9 were selected with puromycin for 1 wk and treated as in D. Data are presented as mean ± SEM from three independent experiments: ***, P < 0.001, Student’s t test. Bars: (main) 20 µm; (inset) 5 μm.