Figure 1. Starvation induces transport... | Rockefeller University Press

Figure 1.

$Starvation induces transport of SOD1 into lysosomes. (A) The schematic illustrates the detection of lysosomal targeting of substrate proteins using tandem mCherry and GFP reporter assays. Under neutral pH conditions, both mCherry and GFP signals are visible, while in the acidic environment of lysosomes, the GFP signal is quenched, leaving only the mCherry signal detectable. (B and C) Confocal images of N2a cells expressing mCherry-GFP-SOD1(G93A) under control (Ctrl) or starvation (Strv) conditions (B). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 21) (C). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (D and E) Confocal images of N2a cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (D). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 20) (E). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (F and G) Confocal images of 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (F). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 22; Strv, n = 23) (G). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (H and I) Confocal images of 293T cells expressing mCherry-GFP-SOD1 and LAMP1-BFP under starvation (Strv) conditions (H). (I) shows the relative fluorescence intensity plots along the dotted line in the inset in H. Bars: 5 μm; insets, 2 μm. (J) Schematic illustration of the Halo-tag assay to detect delivery of a substrate protein into lysosomes. Upon binding to the Halo ligand TMR, Halo cleaved from the substrate protein in lysosomes becomes resistant to lysosomal hydrolase digestion. The free Halo protein can be detected by in-gel fluorescence imaging. If the Halo-tagged candidate protein remains outside of the lysosomes, only the full-length band will be detected. (K) Halo-tag assays show strong free Halo bands in cells expressing Halo-LC3 or Halo-SOD1, compared with Halo-GFP, after starvation for 6 h. (L) Endogenous SOD1 is detected in purified lysosome fractions. Levels of SOD1 in lysosomes are increased after starvation (Strv), compared with control conditions (Ctrl). CTSL, cathepsin L. Source data are available for this figure: SourceData F1$

Starvation induces transport of SOD1 into lysosomes. (A) The schematic illustrates the detection of lysosomal targeting of substrate proteins using tandem mCherry and GFP reporter assays. Under neutral pH conditions, both mCherry and GFP signals are visible, while in the acidic environment of lysosomes, the GFP signal is quenched, leaving only the mCherry signal detectable. (B and C) Confocal images of N2a cells expressing mCherry-GFP-SOD1(G93A) under control (Ctrl) or starvation (Strv) conditions (B). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 21) (C). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (D and E) Confocal images of N2a cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (D). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 20) (E). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (F and G) Confocal images of 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (F). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 22; Strv, n = 23) (G). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (H and I) Confocal images of 293T cells expressing mCherry-GFP-SOD1 and LAMP1-BFP under starvation (Strv) conditions (H). (I) shows the relative fluorescence intensity plots along the dotted line in the inset in H. Bars: 5 μm; insets, 2 μm. (J) Schematic illustration of the Halo-tag assay to detect delivery of a substrate protein into lysosomes. Upon binding to the Halo ligand TMR, Halo cleaved from the substrate protein in lysosomes becomes resistant to lysosomal hydrolase digestion. The free Halo protein can be detected by in-gel fluorescence imaging. If the Halo-tagged candidate protein remains outside of the lysosomes, only the full-length band will be detected. (K) Halo-tag assays show strong free Halo bands in cells expressing Halo-LC3 or Halo-SOD1, compared with Halo-GFP, after starvation for 6 h. (L) Endogenous SOD1 is detected in purified lysosome fractions. Levels of SOD1 in lysosomes are increased after starvation (Strv), compared with control conditions (Ctrl). CTSL, cathepsin L. Source data are available for this figure: SourceData F1