High-throughput quantification of autophagy factor foci lifetime and diffusion dynamics. (A) Upon labeling with fluorescent dye (JF646), cells expressing HaloTagged autophagy factors were starved (EBSS) and imaged at four frames per minute for 1 h. TrackIT was used to detect foci based on threshold intensity (left) and connected into tracks using the nearest neighbor algorithm (right). Scale bar = 5 μm. (B) Example images of foci for Halo-ATG2A (upper panel) and Halo-ATG13 (bottom panel). Scale bar = 1 μm. (C) Histograms of foci lifetime for the HaloTagged autophagy proteins in control conditions (Control) and after 1 h nutrient starvation (EBSS). Three biological replicates (20–30 cells per replicate) were performed for each HaloTag cell line. The number of data points (n) is indicated in each graph in the figure panels. (D) Quantification of the number of foci formed per cell by autophagy factors over the course of 1 h imaging in control (Control) and nutrient starvation (EBSS) conditions (N = 3 biological replicates, mean ± SD). A two-tailed t test was used for statistical analysis (*P < 0.05). (E) Histograms of diffusion coefficients of the foci formed by autophagy factors under nutrient starvation. Histograms were fitted with Gaussian curves. For the proteins other than Halo-ATG2A, we fixed the mean of one subpopulation (in red) to match Halo-ATG2A mean. An additional subpopulation (in purple) represents non-ATG2A-like foci. The black line represents the cumulative fitting. (F) Distribution of ATG2A-like and non-ATG2A-like foci diffusion coefficients (N = 3 biological replicates, mean ± SD). (G) Co-localization of ATG13 foci with the ER in control conditions (siCTR) or CHMP2A knock-down (siCHMP2A) cells. The ER was marked with mEmerald-SEC61, and high-resolution images were generated using the CARE algorithm. Halo-ATG13 foci were scaled at low (1×) and high (2.5×) brightness. Scale bar = 5 μm. (H) Histogram of step-size distribution of Halo-ATG2A-positive (H-ATG2A+, red) and Halo-ATG2A-negative (H-ATG2A−, gray) GFP-ATG13 foci. Three biological replicates (20–30 cells per replicate) were performed for each experiment. (I) Fraction of GFP-ATG13 foci showing accumulation of Halo-ATG2A (N = 3 biological replicates, mean ± SD). (J) GFP-ATG13 foci lifetime for Halo-ATG2A+ and Halo-ATG2A− populations (N = 3 biological replicates, mean ± SD).
Figure 4.

High-throughput quantification of autophagy factor foci lifetime and diffusion dynamics. (A) Upon labeling with fluorescent dye (JF646), cells expressing HaloTagged autophagy factors were starved (EBSS) and imaged at four frames per minute for 1 h. TrackIT was used to detect foci based on threshold intensity (left) and connected into tracks using the nearest neighbor algorithm (right). Scale bar = 5 μm. (B) Example images of foci for Halo-ATG2A (upper panel) and Halo-ATG13 (bottom panel). Scale bar = 1 μm. (C) Histograms of foci lifetime for the HaloTagged autophagy proteins in control conditions (Control) and after 1 h nutrient starvation (EBSS). Three biological replicates (20–30 cells per replicate) were performed for each HaloTag cell line. The number of data points (n) is indicated in each graph in the figure panels. (D) Quantification of the number of foci formed per cell by autophagy factors over the course of 1 h imaging in control (Control) and nutrient starvation (EBSS) conditions (N = 3 biological replicates, mean ± SD). A two-tailed t test was used for statistical analysis (*P < 0.05). (E) Histograms of diffusion coefficients of the foci formed by autophagy factors under nutrient starvation. Histograms were fitted with Gaussian curves. For the proteins other than Halo-ATG2A, we fixed the mean of one subpopulation (in red) to match Halo-ATG2A mean. An additional subpopulation (in purple) represents non-ATG2A-like foci. The black line represents the cumulative fitting. (F) Distribution of ATG2A-like and non-ATG2A-like foci diffusion coefficients (N = 3 biological replicates, mean ± SD). (G) Co-localization of ATG13 foci with the ER in control conditions (siCTR) or CHMP2A knock-down (siCHMP2A) cells. The ER was marked with mEmerald-SEC61, and high-resolution images were generated using the CARE algorithm. Halo-ATG13 foci were scaled at low (1×) and high (2.5×) brightness. Scale bar = 5 μm. (H) Histogram of step-size distribution of Halo-ATG2A-positive (H-ATG2A+, red) and Halo-ATG2A-negative (H-ATG2A−, gray) GFP-ATG13 foci. Three biological replicates (20–30 cells per replicate) were performed for each experiment. (I) Fraction of GFP-ATG13 foci showing accumulation of Halo-ATG2A (N = 3 biological replicates, mean ± SD). (J) GFP-ATG13 foci lifetime for Halo-ATG2A+ and Halo-ATG2A− populations (N = 3 biological replicates, mean ± SD).

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