Figure 1. rnst-2 mutants accumulate enlarged lysosomes.

(A–I) Confocal fluorescence images of embryos at the 4-fold stage (4F, A–C), larvae 1 (L1, D–F) and adult hypodermis (G–I) in wild-type (WT, A, D, G), rnst-2(qx245) (B, E, H) and rnst-2(bp555) (C, F, I) expressing LAAT-1::GFP. (J–L”) Confocal fluorescence images of the hypodermis in wild-type (J–J″), rnst-2 (qx245) (K–K″) and rnst-2(bp555) (L–L″) adults expressing LAAT-1::GFP and stained by Lysotracker red. In (A–L’’), white arrowheads and arrows indicate globular and tubular lysosomes, respectively, and blue arrowheads indicate enlarged globular lysosomes. Scale bars: 5 µm. (M) Quantification of the average volume of lysosomes labeled by LAAT-1::GFP in 4-fold-stage embryos (4F), L1 larvae (L1) and adult hypodermis (day 3 of adulthood). (N) The percentage of LAAT-::GFP-positive lysosomes that were stained by lysotracker red was quantified in adult hypodermis. In (M, N), at least 10 worms were scored in each strain at each stage. Data are shown as mean ± SD. Two-way ANOVA with the Bonferroni post hoc test (M) or one-way ANOVA with Tukey’s post hoc test (N) was performed to compare mutant datasets with wild type. ***p<0.001, other points had p>0.05.

Figure 1—figure supplement 1. rnst-2 mutants accumulate enlarged lysosomes in multiple cell types.

(A–I) Confocal fluorescence images of embryos at the 4-fold stage (4F, (A–C), larvae 1 (L1, D–F) and adult hypodermis (G–I) in wild-type (WT, (A, D, G), rnst-2(qx245) (B, E, H) and rnst-2(bp555) (C, F, I) expressing NUC-1::CHERRY. (J–O) Confocal fluorescence images in sheath cells (J–L) and body wall muscle cells (M–O) in wild-type (J, M), rnst-2(qx245) (K, N) and rnst-2(bp555) (L, O) adults expressing LAAT-1::GFP driven by ced-1 (J–L) or myo-3 promoter (M–O). White arrowheads and arrows indicate globular and tubular lysosomes, and yellow arrowheads indicate enlarged lysosomes in rnst-2(qx245) and rnst-2(bp555). Scale bars: 5 µm. (P) Quantification of the volume of lysosomes labeled by NUC-1::CHERRY in 4-fold embryos (4F), L1 larvae (L1) and adult hypodermis (day 3 of adulthood). (Q) Quantification of the average volume of lysosomes labeled by NUC-1::CHERRY in wild type (WT) and rnst-2(qx245) at different stages. At least 10 worms were scored in each strain at each stage and data are shown as mean ± SEM. (R) Volume of lysotracker-stained- and LAAT-1::GFP-positive lysosomes was quantified in the adult hypodermis and compared. In (P, R), at least 10 worms were scored in each strain and data are shown as mean ± SD. Two-way ANOVA with the Bonferroni post hoc test (P) or one-way ANOVA with Tukey’s post hoc test (R) was performed to compare mutant datasets with wild type. ***p<0.001, other points had p>0.05.

Figure 1—figure supplement 2. Molecular cloning of rnst-2.

(A) Cloning of rnst-2. The top bar indicates the genetic position of rnst-2. The rnst-2 gene structure is shown with filled boxes representing exons and thin lines indicating introns. The arrow delineates the direction of transcription. The position of the mutation site in qx245 and bp555 is indicated. (B, F–K) Confocal fluorescence images of lysosomes in the indicated strains at the L1 stage expressing LAAT-1::GFP. White arrowheads and arrows indicate globular and tubular lysosomes, respectively, and yellow arrowheads indicate enlarged lysosomes. (C–E) Confocal fluorescence images of wild-type larvae expressing RNST-2::CHERRY (C), RNST-2(H118A)::CHERRY (D) and human RNASET2::CHERRY (E) driven by the rnst-2 promoter. Scale bars in (B–K): 5 µm. (L) Sequence alignment of C. elegans (c.e) RNST-2, Drosophila melanogaster (d.r) RNASET2 and human (h.s) RNASET2. Identical residues are shaded in black and similar ones are marked in white boxes. Blue boxes indicate two conserved catalytic active sites (CAS). Mutations identified in different rnst-2 alleles are in red.