Figure 2.
SOD1 is transported into lysosomes through autophagy. (A) Flow cytometry analysis of 293T cells expressing mCherry-GFP-SOD1 under control conditions (Ctrl), starvation conditions (Strv), starvation plus Baf treatment, or starvation plus wortmannin (Wort) treatment. (B) Schematic of CRISPR interference (CRISPRi) screening using the mCherry-GFP-SOD1 reporter to identify factors involved in SOD1 delivery to lysosomes. 293T cells stably expressing mCherry-GFP-SOD1 and dCas9 were transduced with a genome-wide lentiviral CRISPRi gRNA library. After puromycin selection, cells were starved in EBSS for 16 h. The top 40% and bottom 40% of mCherry/GFP ratios correspond to increased and inhibited SOD1 delivery to lysosomes, respectively. Cells were sorted by flow cytometry and processed for NGS to identify gRNAs. (C) Volcano plot of mCherry/GFP fluorescence intensity ratios in the cells in B. After NGS, the top hits in the screen (highlighted in magenta) were found to be sgRNAs targeting genes encoding components of the autophagy pathway. All other targeting sgRNAs are indicated in gray. (D) KEGG pathway analysis identifies autophagy as a major factor responsible for SOD1 transport into lysosomes. High-confidence factors were defined as having opposite phenotypes in the enhanced and inhibited sort gates and gene levels P < 0.05. Pathways with Benjamini–Hochberg false discovery rate (FDR) < 0.05 are shown. (E and F) Confocal images of 293T cells expressing mCherry-GFP-SOD1 and BFP-LC3 under starvation (Strv) conditions. Cells were treated with digitonin before fixation to remove cytosolic signals (E). (F) shows relative fluorescence intensity plots along the dotted line in the inset in E. Bars: 5 μm; insets, 2 μm. (G and H) Confocal images of 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl), starvation (Strv), or starvation plus wortmannin (Strv + Wort) conditions (G). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 19; Strv + Wort, n = 21) (H). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (I and J) Confocal images of FIP200 KO 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (I). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 21; Strv, n = 20) (J). Bars: 5 μm; insets, 2 μm.

SOD1 is transported into lysosomes through autophagy. (A) Flow cytometry analysis of 293T cells expressing mCherry-GFP-SOD1 under control conditions (Ctrl), starvation conditions (Strv), starvation plus Baf treatment, or starvation plus wortmannin (Wort) treatment. (B) Schematic of CRISPR interference (CRISPRi) screening using the mCherry-GFP-SOD1 reporter to identify factors involved in SOD1 delivery to lysosomes. 293T cells stably expressing mCherry-GFP-SOD1 and dCas9 were transduced with a genome-wide lentiviral CRISPRi gRNA library. After puromycin selection, cells were starved in EBSS for 16 h. The top 40% and bottom 40% of mCherry/GFP ratios correspond to increased and inhibited SOD1 delivery to lysosomes, respectively. Cells were sorted by flow cytometry and processed for NGS to identify gRNAs. (C) Volcano plot of mCherry/GFP fluorescence intensity ratios in the cells in B. After NGS, the top hits in the screen (highlighted in magenta) were found to be sgRNAs targeting genes encoding components of the autophagy pathway. All other targeting sgRNAs are indicated in gray. (D) KEGG pathway analysis identifies autophagy as a major factor responsible for SOD1 transport into lysosomes. High-confidence factors were defined as having opposite phenotypes in the enhanced and inhibited sort gates and gene levels P < 0.05. Pathways with Benjamini–Hochberg false discovery rate (FDR) < 0.05 are shown. (E and F) Confocal images of 293T cells expressing mCherry-GFP-SOD1 and BFP-LC3 under starvation (Strv) conditions. Cells were treated with digitonin before fixation to remove cytosolic signals (E). (F) shows relative fluorescence intensity plots along the dotted line in the inset in E. Bars: 5 μm; insets, 2 μm. (G and H) Confocal images of 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl), starvation (Strv), or starvation plus wortmannin (Strv + Wort) conditions (G). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 20; Strv, n = 19; Strv + Wort, n = 21) (H). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (I and J) Confocal images of FIP200 KO 293T cells expressing mCherry-GFP-SOD1 under control (Ctrl) or starvation (Strv) conditions (I). Quantification of mCherry-only puncta is presented as mean ± SEM (Ctrl, n = 21; Strv, n = 20) (J). Bars: 5 μm; insets, 2 μm.

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