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, 36 (8), 1100-1116

Development of LC3/GABARAP Sensors Containing a LIR and a Hydrophobic Domain to Monitor Autophagy

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Development of LC3/GABARAP Sensors Containing a LIR and a Hydrophobic Domain to Monitor Autophagy

You-Kyung Lee et al. EMBO J.

Abstract

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR(TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.

Keywords: LC3‐interacting region motif; autophagosome sensor; autophagy; hydrophobic domain.

Figures

Figure 1
Figure 1. Efficient localization of HyD‐LIRs‐GFP to mRFPLC3B‐positive autophagosomes

Schematic model of the development of new autophagosome sensors. LIR: LC3‐interacting region, HyD: hydrophobic domain.

Confocal images showing cellular localization of various LIR‐GFP constructs (HyD‐GFP, LIR(p62)‐GFP, HyD‐LIR(p62)‐GFP, LIR(Fy)‐GFP, or HyD‐LIR(Fy)‐GFP) together with mRFP‐LC3B in MEFs incubated with 100 nM rapamycin (Rapa) + 10 mM NH4Cl for 4 h. Scale bar: 10 μm.

The bar graphs illustrate the ratios of sensor‐positive mRFP‐LC3B spots (D) and ratios of autophagosomal/cytosol (A/C) fluorescence intensity (E) of each tested GFP sensor construct. The values are presented as the mean ± SEM. ***< 0.001, two‐tailed unpaired Student's t‐test. **P < 0.001, two‐tailed Mann–Whitney U‐test. The numbers on the bars indicate the number of cells used for the experiment.

Figure EV1
Figure EV1. Cellular localization of HyD‐LIR(X)‐GFP in MEFs expressing mRFPLC3 upon autophagy induction
Confocal images showing cellular localization of various HyD‐LIR(X)‐GFP incorporating LIR motifs of different LC3‐binding proteins together with mRFP‐LC3B in MEFs upon autophagy induction (100 nM rapamycin + 10 mM NH4Cl, 4 h). Scale bar, 10 μm. Each X (LC3‐binding proteins) indicates HyD‐LIR(X)‐GFP.
Figure 2
Figure 2. Cellular localization of HyD‐LIR(X)‐GFP to mRFPLC3(A, B, C)‐positive autophagosomes in MEFs upon autophagy induction

Confocal images showing cellular localization of various HyD‐LIR(X)‐GFP constructs together with either mRFP‐LC3B (A), mRFP‐LC3A (D), or mRFP‐LC3C (G) in MEFs upon autophagy induction (100 nM rapamycin + 10 mM NH4Cl, 4 h). X: LC3/GABARAP‐binding proteins (ULK2, ATG13, Bnip3, FUNDC1, ScAtg3, TBC1D25, FY (FYCO1), or TP (TP53INP2)). Scale bar, 10 μm. The bar graphs illustrate the ratios of sensor‐positive mRFP‐LC3B (B), mRFP‐LC3A (E), or mRFP‐LC3C (H) and A/C ratios of the fluorescent intensity (C, F, I) of each tested GFP sensor construct. *< 0.05 (comparison with all other groups by Kruskal–Wallis test followed by Dunn's multiple comparison test), **P < 0.01 (comparison with ULK2, Bnip3, and FUNDC1 by Kruskal–Wallis test followed by Dunn's multiple comparison test). The values are presented as the mean ± SEM. For the quantitative analysis of the A/C ratio, 20 randomly selected cells in each group were used.

Figure 3
Figure 3. Cellular localization of HyD‐LIR(X)‐GFP to mRFPGABARAP/GABARPL(1, 2)‐positive autophagosomes in MEFs upon autophagy induction

Confocal images showing cellular localization of various HyD‐LIR(X)‐GFP constructs together with either mRFP‐GABARAP (A), mRFP‐GABARAPL1 (D), or mRFP‐ GABARAPL2 (G) in MEFs upon autophagy induction (100 nM rapamycin + 10 mM NH4Cl, for 4 h). X: LC3/GABARAP‐binding proteins (ULK2, ATG13, Bnip3, FUNDC1, ScAtg3, TBC1D25, FY (Fyco1), or TP (TP53INP2)). Scale bar, 10 μm. The bar graphs illustrate the ratios of sensor‐positive mRFP‐GABARAP (B), mRFP‐GABARAPL1 (E), or mRFP‐GABARAPL2 (H) and A/C ratios of the fluorescence intensity (C, F, I) of each tested GFP sensor construct. ***< 0.001 (comparison with all other groups by Kruskal–Wallis test followed by Dunn's multiple comparison test). The values are presented as the mean ± SEM. For the quantitative analysis of the A/C ratio, 20 randomly selected cells in each group were used.

Figure EV2
Figure EV2. Efficient localization of HyD‐LIR(TP)‐GFP or HyD‐LIR(Fy)‐GFP to mRFPLC3/GABARAP‐positive or mRFPLC3A/B‐positive autophagosomes in HEK293T cells upon serum starvation and effects of HyD‐LIR(Fy)‐GFP and HyD‐LIR(TP)‐GFP expression on the autophagic flux

Confocal images showing cellular localization of HyD‐LIR(TP)‐GFP (A) or HyD‐LIR(Fy)‐GFP(B) into individual mRFP‐LC3/GABARAP‐positive autophagosome or mRFP‐LC3A/B‐positive autophagosome in HEK293T cells upon autophagy induction (serum starvation, 8 h).

HEK293T cells were transfected with a vector encoding GFP, GFP‐LC3, HyD‐LIR(Fy)‐GFP, or HyD‐LIR(TP)‐GFP. Twenty‐four hours after transfection, the cells were deprived of serum in the presence or absence of chloroquine (CQ) for 8 h. The cell lysates were then subjected to Western blot analyses (C) with an anti‐GFP, anti‐LC3, anti‐p62, or anti‐GAPDH antibody. Autophagic flux indicates differences in the levels of p62 (D) or LC3‐II (E) in the presence and absence of CQ. The levels of LC3‐II and p62 in the GFP‐, GFP‐LC3, or HyD‐LIR(Fy/TP)‐GFP‐expressing cells were normalized to that of GAPDH in cells expressing GFP or one of the GFP‐tagged sensors. The data are presented as the mean ± SEM of three independent experiments. Starvation, serum starvation.

Source data are available online for this figure.
Figure 4
Figure 4. Binding of HyD‐LIR(Fy)‐GFP or HyD‐LIR(TP)‐GFP with each LC3/GABARAP protein and their cellular localization to mRFPLC3/GABARAP‐positive autophagosomes in MEFs upon serum starvation in a LIR‐dependent manner

Pull‐down assay with anti‐GFP antibody using HEK293T cell lysates expressing either HyD‐LIR(Fy)‐GFP (A1) or mutant LIR HyD‐LIR(Fy)m‐GFP (A2) together with either mRFP‐LC3A, mRFP‐LC3B, mRFP‐LC3C, mRFP‐GABARAP, mRFP‐GABARAPL1, or mRFP‐GABARAPL2. IgG (mouse IgG) was used as a negative control. Western blotting was performed using anti‐LC3(A, B, C), GABARAP, GABARAPL1, or GABARAPL2 antibody or anti‐GFP antibody.

Confocal images showing cellular localization of either HyD‐LIR(Fy)‐GFP (or HyD‐LIR(Fy)m‐GFP) into mRFP‐LC3/GABARAP‐positive autophagosomes in MEFs upon autophagy induction (serum starvation for 4 h). Scale bar: 10 μm.

The bar graphs illustrate A/C ratios of the fluorescent intensity (B) of HyD‐LIR(Fy)(or LIR(Fy)m)‐GFP. ***< 0.001 according to Kruskal–Wallis test followed by Dunn's multiple comparison test. For the quantitative analysis of the A/C ratio, 20 randomly selected cells in each group were used. The values are presented as the mean ± SEM.

Pull‐down assay with anti‐GFP antibody using HEK293T cell lysates expressing either HyD‐LIR(TP)‐GFP (D1) or HyD‐LIR(TP)m‐GFP (D2) together with either mRFP‐LC3A, mRFP‐LC3B, mRFP‐LC3C, mRFP‐GABARAP, mRFP‐GABARAPL1, or mRFP‐GABARAPL2. IgG (mouse IgG) was used as a negative control. Western blotting was performed using anti‐LC3(A, B, C), GABARAP, GABARAPL1, or GABARAPL2 antibody or anti‐GFP antibody.

Confocal images showing cellular localization of either HyD‐LIR(TP)‐GFP (or HyD‐LIR(TP)m‐GFP) into mRFP‐LC3/GABARAP‐positive autophagosomes in MEFs upon autophagy induction (serum starvation for 4 h). Scale bar: 10 μm.

The bar graph illustrates A/C ratios of the fluorescent intensity (E) of HyD‐LIR(TP)(or LIR(TP)m)‐GFP. ***< 0.001 according to Kruskal–Wallis test followed by Dunn's multiple comparison test. For the quantitative analysis of the A/C ratio, 20 randomly selected cells in each group were used. The values are presented as the mean ± SEM.

Source data are available online for this figure.
Figure 5
Figure 5. Effective localization of HyD‐LIR(Fy)‐GFP and HyD‐LIR(TP)‐GFP to endogenous LC3/GABARAP‐positive autophagosomes during autophagy induction with serum starvation in WT MEFs, but not in autophagy‐deficient MEFs

Confocal images show cellular localization of HyD‐LIR(Fy)‐GFP (A) or HyD‐LIR(TP)‐GFP (B) to endogenous LC3A/B‐positive autophagosomes in wild‐type (WT), Atg5 −/−, or Atg7 −/− MEFs upon serum starvation (8 h). Confocal images show cellular localization of HyD‐LIR(TP)‐GFP (B) to endogenous GABARAPL1‐positive autophagosomes in wild‐type (WT) MEFs upon serum starvation (8 h). The merged image was enlarged. Scale bar: 10 μm.

The bar graph illustrates the ratios of sensor‐positive LC3A/B or GABARAPL1 spots. The values are presented as the mean ± SEM. The numbers on the bars indicate the number of cells used for the experiment.

Electron microscopy images show immunogold labeling of HyD‐LIR(Fy)‐GFP (D) or HyD‐LIR(TP)‐GFP (E) in autophagic vacuoles (AU) in WT (left) and Atg5 −/− MEFs (right) during serum starvation (8 h). Arrows: immunogold labeling of LC3/GABARAP‐bound GFP inside the autophagosomes. Arrowheads: immunogold labeling of LC3/GABARAP‐bound GFP in the autophagosomal membrane. R: region, M: mitochondrion, RER: rough endoplasmic reticulum.

Autophagic flux assay in MEFs expressing GFP, HyD‐LIR(Fy)‐GFP, or HyD‐LIR(TP)‐GFP upon starvation with EBSS (in the presence or absence of bafilomycin A1 (BafA1) for 4 h). The cell lysates were then subjected to Western blot analyses with an anti‐GFP, anti‐LC3, anti‐p62, or anti‐actin antibody.

Autophagic flux indicates differences in the levels of p62 (G) or LC3‐II (H) in the presence and absence of CQ. The bar graphs illustrate the percentage of level of p62 or LC3‐II. The levels of LC3‐II or p62 in the GFP‐ or HyD‐LIR(Fy/TP)‐GFP‐expressing cells were normalized to that of actin in cells expressing GFP or one of the GFP‐tagged sensors. The data are presented as the mean ± SEM of three independent experiments.

Source data are available online for this figure.
Figure EV3
Figure EV3. Efficient localization of HyD‐LIR(Fy)‐GFP or HyD‐LIR(TP)‐GFP to autophagic vacuoles in WT and Atg5 −/− MEFs upon various conditions of autophagy induction

Confocal images showing cellular localization of HyD‐LIR(Fy)‐GFP (A) and HyD‐LIR(TP) (B) in WT and Atg5 −/− MEFs upon autophagy induction with amino acid deprivation (8 h), PP242 (20 μM, 4 h), EBSS starvation (2 or 4 h), or rapamycin (100 nM, 4 h) in the presence or absence of CQ (50 μM), BafA1 (100 nM) or NH4Cl (10 mM). Scale bar: 10 μm.

Figure 6
Figure 6. Efficient localization of HyD‐LIR(TP)‐mRFP to GFPDFCP1‐positive, GFPSTX17‐positive, or LysoTracker‐positive autophagic vacuoles in MEFs upon autophagy activation by serum starvation

Confocal images showing cellular localization of either mRFP‐LC3B (or GFP‐LC3B) or HyD‐LIR(TP)‐mRFP together with GFP‐DFCP1 (A), GFP‐STX17 (C), or Lysotracker (E) in MEFs upon serum starvation (in the absence or presence of CQ). The bar graphs illustrate the ratios of GFP‐DFCP1‐positive (B), GFP‐STX17‐positive mRFP dots per cell (D), and ratios of LysoTracker‐positive GFP spots (F). The data are presented as the mean ± SEM of three independent experiments. ***< 0.001, according to one‐way ANOVA followed by Tukey's post hoc test. **P = 0.013, according two‐tailed Mann–Whitney U‐test. The numbers on the bars indicate the number of cells used for the experiment.

Proteinase K protection assay in HEK293T cells expressing HyD‐LIR(TP)‐EGFP upon serum starvation (in the absence or presence of CQ, 8 h) with (or without) proteinase K (25 μg/ml) or Triton X‐100 (0.5%). Western blot analysis was performed using anti‐GFP or anti‐p62 antibodies.

Source data are available online for this figure.
Figure 7
Figure 7. Detection of dynamic autophagosomes in live MEFs upon serum starvation without LC3/GABARAPs overexpression by using HyD‐LIR(TP)‐GFP

Confocal images showing cellular localization of GFP‐LC3B or HyD‐LIR(TP)‐GFP in MEFs upon serum starvation (8 h). Scale bar: 10 μm.

The bar graphs illustrate (B) the number of GFP‐LC3B‐ or HyD‐LIR(TP)‐GFP‐positive spots and (C) A/C ratio of fluorescence intensity of GFP‐fused sensors. The data are presented as the mean ± SEM of three independent experiments. *= 0.02, according to Kruskal–Wallis test followed by Dunn's multiple comparison test; ***< 0.001, according to one‐way ANOVA followed by Tukey's post hoc test. The numbers on the bars indicate the number of cells used for the experiment.

Confocal images in live cells showing HyD‐LIR(TP)‐GFP‐ or GFP‐LC3B‐positive autophagosomes (dots) at 0, 1, 2, 5, 10, or 20 min after autophagy induction with rapamycin (100 nM) for 20 min. Cell images between 0 and 20 min (pseudo‐red image) after autophagy induction were merged. Scale bar: 10 μm.

Quantification of the ratio of the number of co‐localized dots in images at the indicated time to that in the image at time 0 min in cells expressing HyD‐LIR(TP)‐GFP or GFP‐LC3B (E). Bar graph illustrates the ratio of the number of co‐localized dots in images at 1, 2, and 5 min compared to that of co‐localized dots at 0 min (F). The data are presented as the mean ± SEM of three independent experiments. **= 0.007, ***< 0.001, according to two‐tailed unpaired t‐test. The numbers on the bars indicate the number of cells used for the experiment.

Figure 8
Figure 8. Co‐localization of HyD‐LIR(TP)‐GFP or HyD‐LIR(Fy)‐GFP with MitoTracker‐positive spots in mitophagy induced by CCCP or phenanthroline treatment

Confocal images showing cellular localization of GFP, GFP‐LC3B, HyD‐LIR(TP)‐GFP or HyD‐LIR(Fy)‐GFP together with MitoTracker in MEFs (A) upon mitophagy induction (CCCP (20 μM) or CCCP+CQ (50 μM)) and in HeLa cells (C) upon mitophagy induction (50 μM phenanthroline). Scale bar, 10 μm. The bar graphs (B, D) illustrate the number of co‐localized spots (GFP and MitoTracker‐positive‐spots). ***< 0.001, according to one‐way ANOVA followed by Tukey's post hoc test. The data are presented as the mean ± SEM. The numbers on the bars indicate the number of cells used for the experiment.

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