The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps

Abstract

Membrane rupture of lysosomes results in leakage of their contents, which is harmful to cells. Recent studies have reported that several systems contribute to the repair or elimination of damaged lysosomes. Lysophagy is a type of selective autophagy that plays a crucial role in the lysosomal damage response. Because multiple pathways are involved in this response, an assay that specifically evaluates lysophagy is needed. Here, we developed the TMEM192-mKeima probe to evaluate lysophagy. By comparing the use of this probe with the conventional galectin-3 assay, we showed that this probe is more specific to lysophagy. Using TMEM192-mKeima, we showed that TFEB and p62 are important for the lysosomal damage response but not for lysophagy, although they have previously been considered to be involved in lysophagy. We further investigated the initial steps in lysophagy and identified UBE2L3, UBE2N, TRIM10, 16, and 27 as factors involved in it. Our results demonstrate that the TMEM192-mKeima probe is a useful tool for investigating lysophagy.

Figure 1.

Establishment of a lysophagy flux assay using TMEM192-mKeima. (A) Schematic diagram of the lysophagy assay using TMEM192-mKeima. (B) HeLa cells stably expressing TMEM192-mKeima were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM containing Hoechst without LLOMe. After incubation for 10 h, cells were observed under a fluorescence microscope. Scale bars, 10 µm. (C) Quantification of the number of acidic TMEM192-Keima puncta per cell for Fig. S2 A. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). (D) HeLa cells stably expressing TMEM192-mKeima were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM containing Hoechst without LLOMe. Cells were observed using a 100× oil immersion objective under a fluorescence microscope. Scale bars, 10 µm. (E) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against FIP200 or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 10 h and observed by fluorescence microscopy. Scale bars, 10 µm. (F) Quantification of the number of acidic mKeima puncta per cell in cells for Fig. 1 E. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test). (G) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against FIP200, ATG14, ATG7, ATG12, ATG16, or Stx17, or treated with control siRNA. After 72 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 10 h and observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows the number of acidic TMEM192-mKeima puncta per cell in these HeLa cells. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05; **P < 0.01 (one-way ANOVA followed by Dunnett’s test). (H) U2OS cells stably expressing TMEM192-mKeima were treated with siRNA against FIP200 or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 6 h and observed by fluorescence microscopy. Scale bars, 10 µm. (I) Quantification of the number of acidic mKeima puncta per cell in cells for Fig. 1 H. More than 30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test).

Figure S1.

TMEM192-mKeima localizes to the lysosome even under conditions with lysosomal damage. HeLa cells expressing TMEM192-mKeima were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured for 20 h in DMEM. Cells were immunostained with LAMP1 antibody and then analyzed by fluorescence microscopy. Scale bars, 10 µm.

Figure S2.

The conversion of mKeima increases in a time- and concentration-dependent manner with LLOMe. (A) HeLa cells stably expressing TMEM192-mKeima were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM containing Hoechst without LLOMe. After incubation for 1, 3, 6, or 10 h, cells were observed under a fluorescence microscope. Scale bars, 10 µm. (B) HeLa cells stably expressing TMEM192-mKeima were incubated for 1 h in the presence of 125, 250, 500, or 1,000 µM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 20 h and then analyzed by fluorescence microscopy. The graph shows the number of acidic TMEM192-mKeima puncta per cell. Error bars indicate means ± SEM in at least three independent experiments (n = 3). >30 cells were analyzed per condition in each experiment.

Figure S3.

Conversion to acidic mKeima depends on the acidity of the lysosome and autophagy pathway. (A) HeLa cells stably expressing TMEM192-mKeima were incubated for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst and bafilomycin A1 for 20 h and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (B) WT, ATG7-KO, ATG9-KO, ATG14-KO, ATG16L1-KO, and STX17-KO HeLa cells expressing TMEM192-mKeima were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM containing Hoechst without LLOMe. After 21 h of incubation, cells were observed by fluorescence microscopy. The graph shows the number of acidic TMEM192-mKeima puncta per cell in WT and KO HeLa cells. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test).

Figure 2.

TMEM192-mKeima can assess lysophagic activity separately from ESCRT-mediated and TFEB-mediated lysosomal damage recovery. (A) HeLa cells stably expressing both TMEM192-mKeima and GFP-Gal3 were treated with siRNA against FIP200 or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM containing Hoechst without LLOMe. After incubation for 1, 3, 6, or 10 h, cells were observed under a fluorescence microscope. Scale bars, 10 µm. (B) The left graph shows the number of Gal-3 puncta per cell for Fig. 2 A. The right graph shows the number of acidic mKeima puncta per cell for Fig. 2 A. N indicates no drug treatment and L indicates 1 h of LLOMe treatment. More than 30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05; **P < 0.01 (unpaired two-tailed Student’s t test). (C) HeLa cells stably expressing GFP-Gal3 were treated with siRNA against Alix, TFEB, or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM without LLOMe. After 10 h, cells were observed by fluorescence microscopy. Scale bars, 10 µm. (D) Quantification of the percentage of Gal-3 puncta remaining after 10 h of incubation for Fig. 2 C. More than 80 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). **P < 0.01 (one-way ANOVA followed by Dunnett’s test). (E and F) Quantification of the number of acidic mKeima puncta per cell in cells for Fig. S4, A and B. More than 30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05, NS: not significant (one-way ANOVA followed by Dunnett’s test).

Figure S4.

Knockdown of ALIX or TFEB does not affect the number of acidic mKeima in the TEME192-mKeima assay but causes defects in the clearance of damaged lysosomes in the GFP-Gal3 assay. (A and B) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against FIP200, Alix, or TFEB, or treated with control siRNA. After siRNA treatment, cells were cultured for 5 h in the presence of 1 mM LLOMe and Hoechst. Cells were observed by fluorescence microscopy. Scale bars, 10 µm. (C) HeLa cells stably expressing GFP-Gal3 were treated with siRNA against FIP200, Alix, TFEB, or treated with control siRNA. After siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM without LLOMe. After 10 h, cells were observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows the percentage of Gal-3 puncta remaining after 10 h of incubation. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). **P < 0.01 (one-way ANOVA followed by Dunnett’s test).

Figure S5.

Autophagic adaptors localize to lysosomes under conditions with lysosomal damage. (A) HeLa cells were treated with or without 1 mM LLOMe for 3 h, immunostained with p62 and LAMP1 antibodies, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (B) HeLa cells stably expressing GFP-OPTN, GFP-NDP52, GFP-TA1BP1, or GFP-NBR1 were treated with or without 1 mM LLOMe for 3 h, immunostained with LAMP1 antibody, and then analyzed by fluorescence microscopy. Scale bars, 10 µm.

Figure 3.

OPTN, NDP52, and TAX1BP1 play crucial roles in lysophagy. (A) WT and Penta-KO (p62, OPTN, NDP52, TAX1BP1, and NBR1 KO) HeLa cells stably expressing TMEM192-mKeima were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 10 h and then analyzed by fluorescence microscopy. Scale bars, 10 µm. The graph shows the number of acidic TMEM192-mKeima puncta per cell in WT and Penta-KO HeLa cells. More than 30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). **P < 0.01 (unpaired two-tailed Student’s t test). (B) HeLa cells stably expressing TMEM192-mKeima were treated with two siRNAs against each of p62, NBR1, FIP200, or control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM containing Hoechst and observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows the number of acidic TMEM192-mKeima puncta per cell in each cell. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). **P < 0.01, NS: not significant (one-way ANOVA followed by Dunnett’s test). (C) HeLa cells stably expressing GFP-Gal3 were treated with siRNA against p62 or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM without LLOMe. After 10 h, cells were observed by fluorescence microscopy. Scale bars, 10 µm. Quantification of the percentage of Gal-3 puncta remaining after 10 h of incubation. >80 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05 (unpaired two-tailed Student’s t test). (D) Penta-KO cells stably expressing TMEM192-mKeima and each FLAG-adaptor (p62, OPTN, NDP52, TAX1BP1, and NBR1) were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 10 h and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (E) Quantification of the number of acidic TMEM192-mKeima puncta per cell for Fig. 3 D. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05; ***P < 0.001, NS: not significant (one-way ANOVA followed by Dunnett’s test). (F) HeLa cells stably expressing TMEM192-mKeima were treated with two siRNAs against each of OPTN, NDP52, TAX1BP1, or control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM containing Hoechst and observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows the number of acidic TMEM192-mKeima puncta per cell in each cell. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test).

Figure S6.

The recovery of lysosomal enzyme activity is suppressed in Penta-KO cells. WT or Penta-KO HeLa cells were cultured for 1 h in the presence of 1 mM LLOMe, followed by culture in DMEM medium containing Hoechst without LLOMe. After incubation for 4 h, cells were observed under a fluorescence microscope. Scale bars, 10 µm. The graph shows the percentage of Magic Red dots in cells after 4 h of incubation compared with untreated cells. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). **P < 0.01, NS: not significant (one-way ANOVA followed by Dunnett’s test).

Figure 4.

TBK1 is important for lysophagy. (A) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against TBK1 or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 1 h in the presence of 1 mM LLOMe. After LLOMe was removed, cells were cultured in DMEM containing Hoechst for 10 h and observed by fluorescence microscopy. Scale bars, 10 µm. (B) Quantification of the number of acidic TMEM192-Keima puncta per cell for Fig. 4 A. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test). (C) HeLa cells transiently expressing GFP-TBK1 were cultured for 3 h with or without 1 mM LLOMe, immunostained with LAMP1 antibody, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (D) HeLa cells were treated with or without 1 mM LLOMe and 10 µM BAPTA-AM for 1 h, immunostained with p-TBK1 and LAMP1 antibodies, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (E) Quantification of the number of p-TBK foci exhibiting a LAMP1 signal for Fig. 4 D. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). ***P < 0.001 (one-way ANOVA followed by Dunnett’s test). (F) HeLa cells were treated with LLOMe for the indicated times. Cell lysates were analyzed by immunoblotting using the indicated antibodies. Source data are available for this figure: SourceData F4.

Figure 5.

UBE2N and UBE2L3 are identified as factors required for lysophagy. (A) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against FIP200, UBE2L3, or UBE2N, or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM containing Hoechst and observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows the number of acidic TMEM192-mKeima puncta per cell in each cell. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05, ***P < 0.001 (one-way ANOVA followed by Dunnett’s test). (B) HeLa cells stably expressing GFP-UBE2L3 or GFP-UBE2N were treated with or without 1 mM LLOMe for 2 h, immunostained with LAMP1 antibody, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (C and D) HeLa cells were treated with siRNA against each of UBE2L3, UBE2N, or control siRNA. After 48 h of siRNA treatment, cells were treated with or without 1 mM LLOMe for 2 h, immunostained with LAMP1 and polyubiquitin chain (K48 or K63) antibody, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (E and F) Quantification of the number of K48 or K63 puncta per cell for Fig. 5, C and D. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05, NS: not significant (one-way ANOVA followed by Dunnett’s test).

Figure 6.

Small-scaled screening using TMEM192-mKeima system identified TRIM10 and TRIM27 as factors that function in lysophagy. (A) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against each TRIMs or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM containing Hoechst and observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows lysophagic activity with reference to the wild type. >30 cells were analyzed per condition in each experiment. For lysophagic activity in each siRNA-treated cell, a robust z-score was calculated for the overall lysophagic activity value. Red highlighting indicates those with significantly lower (less than −1.8) z-score values compared with other factors. (B) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against each of FIP200, TRIM16, TRIM10, TRIM27, or control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM-containing Hoechst and observed by fluorescence microscopy. The graph shows the number of acidic TMEM192-mKeima puncta per cell in each cell. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05; ***P < 0.001 (one-way ANOVA followed by Dunnett’s test). (C) HeLa cells were treated with siRNA against each of TRIM10, TRIM16, TRIM27, or control siRNA. After 48 h of siRNA treatment, cells were treated with or without 1 mM LLOMe for 2 h, immunostained with polyubiquitin chain (FK2) antibody, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (D) Quantification of the number of polyubiquitin chain (FK2) puncta per cell for Fig. 6 C. More than 30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). NS: not significant (one-way ANOVA followed by Dunnett’s test).