Analysis of the maturation kinetics of autophagosomes using dual-color imaging. (A) Example images showing formation, growth, and disappearance of colocalized Halo-ATG13 and GFP-p62 foci using dual-color live-cell imaging under EBSS starvation (1 h). Scale bar = 2 μm. (B) Histograms of the lifetimes of Halo-ATG2A and Halo-ATG13 foci that colocalized (green) or did not colocalize (dark gray) with virally transduced GFP-P62. (C) Percentage of HaloTagged autophagy protein foci that colocalized with P62 foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis. (D) Histograms of the lifetimes of Halo-ATG2A and Halo-ATG13 foci that colocalized (light blue) or did not colocalize (dark gray) with virally transduced GFP-LC3. (E) Percentage of HaloTagged autophagy protein foci that colocalized with LC3 foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis. (F–H) Histograms of the lifetimes of Halo-ATG13 foci that colocalized or did not colocalize (dark gray) with (F) GFP-GABARAPL1 (yellow), (G) GFP-LC3 (light blue), (H) GFP-p62 (green) stably expressed from an AAVS1 locus insertion. (I) Percentage of HaloTagged autophagy protein foci that colocalized with the adaptors GFP-GABARAPL1, GFP-LC3, and GFP-P62 stably expressed from an AAVS1 locus insertion. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis. (J) Quantification of the average HaloTagged autophagy protein foci lifetimes for GFP-P62 positive (green) and negative (dark gray) foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). (K) Quantification of the average HaloTagged autophagy protein foci lifetimes for GFP-LC3 positive (light blue) and negative (dark gray) foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). (L) Quantification of the average Halo-ATG13 foci lifetimes that are negative (dark gray) or positive for adaptor signals (GABARAPL1, yellow; LC3, light blue; P62, green). Data represent mean ± SD of three biological replicates (20–30 cells per replicate). (M) Quantification of the timing of the three distinct phases of autophagosome formation for the HaloTag proteins under EBSS starvation (1 h). Data represent mean ± SD of three biological replicates (20–30 cells per replicate).
Figure 6.

Analysis of the maturation kinetics of autophagosomes using dual-color imaging. (A) Example images showing formation, growth, and disappearance of colocalized Halo-ATG13 and GFP-p62 foci using dual-color live-cell imaging under EBSS starvation (1 h). Scale bar = 2 μm. (B) Histograms of the lifetimes of Halo-ATG2A and Halo-ATG13 foci that colocalized (green) or did not colocalize (dark gray) with virally transduced GFP-P62. (C) Percentage of HaloTagged autophagy protein foci that colocalized with P62 foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis. (D) Histograms of the lifetimes of Halo-ATG2A and Halo-ATG13 foci that colocalized (light blue) or did not colocalize (dark gray) with virally transduced GFP-LC3. (E) Percentage of HaloTagged autophagy protein foci that colocalized with LC3 foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis. (F–H) Histograms of the lifetimes of Halo-ATG13 foci that colocalized or did not colocalize (dark gray) with (F) GFP-GABARAPL1 (yellow), (G) GFP-LC3 (light blue), (H) GFP-p62 (green) stably expressed from an AAVS1 locus insertion. (I) Percentage of HaloTagged autophagy protein foci that colocalized with the adaptors GFP-GABARAPL1, GFP-LC3, and GFP-P62 stably expressed from an AAVS1 locus insertion. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). A two-tailed t test was used for statistical analysis. (J) Quantification of the average HaloTagged autophagy protein foci lifetimes for GFP-P62 positive (green) and negative (dark gray) foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). (K) Quantification of the average HaloTagged autophagy protein foci lifetimes for GFP-LC3 positive (light blue) and negative (dark gray) foci. Data represent mean ± SD of three biological replicates (20–30 cells per replicate). (L) Quantification of the average Halo-ATG13 foci lifetimes that are negative (dark gray) or positive for adaptor signals (GABARAPL1, yellow; LC3, light blue; P62, green). Data represent mean ± SD of three biological replicates (20–30 cells per replicate). (M) Quantification of the timing of the three distinct phases of autophagosome formation for the HaloTag proteins under EBSS starvation (1 h). Data represent mean ± SD of three biological replicates (20–30 cells per replicate).

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