Fig. 4.

Fig. 4.

Increased lysosomal exocytosis alters cathepsin activity in galns^m/m mutant larvae. (A) Schematic illustrating the hsp701:Lamp1-mCherry transgene and experimental workflow for analysis of Lamp1 abundance and subcellular location in developing larvae. (B,C) Western blots probed for mCherry in the whole animal indicate that the level of Lamp1-mCherry is increased 3 h post-heat shock (phs) but steadily declines until 24 h phs. (D,E) Lamp1-mCherry abundance at 0.5-10 h phs (D) and 0.5-24 h phs (E). The level present at each timepoint was normalized to the amount of Lamp1 present at 0.5 h phs. n=3 biological replicates of samples containing ten embryos per genotype per timepoint. Error bars=s.e.m. Two-tailed paired Student's t-test, **P<0.01, ****P<0.0001. (F) Confocal images of live 4 dpf galns^+/+ wild-type and galns^m/m mutant larvae expressing the fli1a:EGFP (green) and hsp701:Lamp1-mCherry transgenes (red). Analysis of Lamp1-mCherry 8 h phs identified several differences in Lamp1-mCherry abundance, character and location. Yellow arrows highlight cell surface signal in red-only panels in an ROI from example 1. Blue arrows highlight large swollen Lamp1-mCherry-positive lysosomes in an ROI from example 2. Scale bar: 10 µm. (G) Schematic illustrating scored parameters, including the number of cells exhibiting high versus moderate to no cell surface Lamp1-mCherry and measurement of fluorescent intensity within ROIs throughout cartilage (Klionsky et al., 2021). Graphs show that the abundance of cell surface-localized Lamp1-mCherry and overall Lamp1-mCherry signal are both increased in mutant larvae. n=15 larvae scored per genotype. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05, **P<0.01. (H) Schematic showing the general mechanism of labeling active cysteine cathepsin with the BMV109 activity-based probe. Created in BioRender by Flanagan-Steet, H. (2026). https://BioRender.com/ooh869k. This figure was sublicensed under CC-BY 4.0 terms. Confocal analysis of sections of larval cartilage stained with wheat germ agglutinin (WGA; blue) and the BMV109 activity-based probed (red) shows that cathepsin activity is only present within chondrocytes of 4 dpf galns^+/+ wild-type larvae. However, in galns^m/m mutants, activity is noted at the cell surface (indicative of exocytosis, white arrows) and outside the cell (see panel 1, in which the surface is denoted with a dashed line and extracellular activity with yellow arrows). Scale bar: 10 µm. (I) Schematic illustrating the procedure for labeling cathepsin activity in live larvae. ABP, activity-based probe. In-gel analyses of cathepsin activity in larval lysates show multiple alterations in Ctsk and Ctsl activity at 2-6 dpf. (J-L) Graphs show quantitation of individual cathepsins at 2-3 dpf (J), 3-4 dpf (K) and 5-6 dpf (L). n=3 biological replicates of samples containing ten embryos per genotype per labeling timepoint. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05, **P<0.01. (M,N) Quantitative RT-PCR (qRT-PCR) analysis showed increased expression of ctsk, ctsla and ctslb transcripts at 4 and 8 dpf in mutant larvae. n=5 biological replicates of samples containing ten embryos per genotype per labeling timepoint. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05, ***P<0.001. (O) Western blot of Ctsk showing that its inactive pro form is increased in mutant larvae, correlating with the noted reduction in Ctsk activity. Three different examples are shown. (P) Graphs show densitometry analysis of pro Ctsk. n=3 biological replicates of samples containing ten embryos per genotype per labeling timepoint. Error bars=s.e.m. Two-tailed paired Student's t-test, **P<0.01.