Loss of cbs-1 disrupts lysosomal acidification, maturation and cargo degradation. (A) Assessing lysosome acidity with the mKate2::GFP::LGG-2 fusion protein. Left: images of mKate2::GFP::LGG-2-labeled lysosomes in the hypodermis and muscle in WT and yq399 animals (2 days after L4 stage). Bars, 5 μm. Top right: schematic illustration of the lysosome acidification assay with the mKate2::GFP::LGG-2 reporter. Bottom right: quantification of the ratio (GFP/[GFP+mKate2]) of fluorescence intensities of lysosomes as shown in the left images. Hyp, hypodermis; Mus, muscle. ≥30 lysosomes in the indicated tissues from 3 animals of each genotype were analyzed. (B) Assessing lysosomal acidity using the NUC-1::pHTomato fusion protein (Phsp-16nuc-1::pHTomato). Left: NUC-1::pHTomato-labeled lysosomes in the hypodermis in WT, yq399, and yq357 animals (2 days after L4). NUC-1::pHTomato images were captured 30 min after heat shock of the animals (33°C for 30 min). Bars, 5 μm. Right: quantification of lysosomal pHTomato fluorescence intensity (arbitrary units, A.U.). ≥30 lysosomes from 5 animals were examined for each genotype. (C) Representative images (left) and quantification (right) of LysoTracker Red staining in the hypodermis in WT and cbs-1(yq357) animals (2 days after L4). Bars, 5 μm. A.U., arbitrary units. ≥30 lysosomes from 5 animals were examined for each genotype. (D) Pulse-chase analysis of lysosomal maturation. Left: images of lysosomal NUC-1::sfGFP::mCh in the hypodermis of WT and yq399 animals (2 days after L4). Bar, 5 μm. Right: quantification of florescence intensity ratio (GFP/[GFP+mCh]). Data were analyzed at the indicated time points following heat shock. ≥20 lysosomes from 3 animals were quantified for each genotype. (E) Relative activities of lysosomal cathepsin B/L, CTSB, and CTSD in animals with the indicated genotypes. Cathepsin activities were normalized to WT levels. Data are from three independent experiments. (F) Western blotting of CPL-1 in WT and cbs-1(yq357) animals (2 days after L4). α-Tubulin was used as loading control. Fold changes in mature-CPL-1 (mature-CPL-1/total CPL-1/tubulin) are indicated at the bottom. (G) Representative images of lysosomes in the hypodermis co-expressing GFP::LGG-1 and NUC-1::mCh (top) and the muscle co-expressing mCh::LGG-1 and LAAT-1::GFP (bottom) in WT and yq399 animals (2 days after L4). Bars, 5 μm. (H) Representative images of lysosomes in the hypodermis in WT and yq399 animals (2 days after L4) co-expressing SQST-1::GFP and NUC-1::mCh. Bars, 5 μm. (I) TEM analysis of lysosomes. Left: representative images of lysosomes in the hypodermis (Hyp), intestine (Int), and muscle (Mus) of WT and yq399 animals (2 days after L4 stage). Yellow, orange, and magenta arrowheads indicate normal lysosomes, damaged lysosomes, and enlarged lysosomes with cargo accumulation, respectively. Bars, 1 μm. Right: quantification of lysosome diameter. ≥30 lysosomes from each tissue of ≥3 animals were analyzed for each genotype. For all quantifications, data are presented as mean ± SEM. Statistical comparisons were performed using one-way ANOVA with Holm–Sidak’s multiple comparisons test (B and E) or the two-sided Student’s t test (A, C, D, and I). **P < 0.01, ***P < 0.001, and ****P < 0.0001. Source data are available for this figure: SourceData F2.