Figure 5. Two new microscopy assays show... | Rockefeller University Press

Figure 5.

$Figure 5. Two new microscopy assays show that autophagy cargos accumulating in single and double ESCRT mutant cells are accessible to protease and anti-cargo antibodies. (A) Modified protease-protection assay. Following the protease (PK) assay, fractions were washed to remove degraded peptides, and the presence of GFP-Atg8 in membrane fractions was determined by fluorescence microscopy (right, number of Atg8 dots used for quantification shown in B). Protease protection of GFP-Atg8 can be seen when PK is added to membranes without detergent. Scale bar, 2 µm. Arrows point to GFP-Atg8 positive dots/particles. (B) Bar graph shows percentage of GFP-Atg8 dots protected from the protease for the strains from A; >2000 DIC particles per condition were quantified (9 fields, 3 fields × 3 repeats). (C) Accessibility to antibodies against autophagic cargo assay. Membrane fractions from cells expressing GFP-Atg8 (for AP marking) were subjected to immunofluorescence microscopy using anti-Ape1 antibodies (right, number of Atg8 dots used for quantification in D). Particles (DIC) in which Ape1 and Atg8 colocalized show that the cargo was accessible to the antibodies (white arrows). Scale bar, 2 µm. (D) Bar graph showing GFP-Atg8 colocalization with anti-Ape1 (% of Atg8 dots) for the strains from C; >800 GFP-Atg8 dots per strain were quantified. In B and D, columns represent mean and error bars represent SD. Results in this figure represent three independent experiments. ***P < 0.001.$

Two new microscopy assays show that autophagy cargos accumulating in single and double ESCRT mutant cells are accessible to protease and anti-cargo antibodies. (A) Modified protease-protection assay. Following the protease (PK) assay, fractions were washed to remove degraded peptides, and the presence of GFP-Atg8 in membrane fractions was determined by fluorescence microscopy (right, number of Atg8 dots used for quantification shown in B). Protease protection of GFP-Atg8 can be seen when PK is added to membranes without detergent. Scale bar, 2 µm. Arrows point to GFP-Atg8 positive dots/particles. (B) Bar graph shows percentage of GFP-Atg8 dots protected from the protease for the strains from A; >2000 DIC particles per condition were quantified (9 fields, 3 fields × 3 repeats). (C) Accessibility to antibodies against autophagic cargo assay. Membrane fractions from cells expressing GFP-Atg8 (for AP marking) were subjected to immunofluorescence microscopy using anti-Ape1 antibodies (right, number of Atg8 dots used for quantification in D). Particles (DIC) in which Ape1 and Atg8 colocalized show that the cargo was accessible to the antibodies (white arrows). Scale bar, 2 µm. (D) Bar graph showing GFP-Atg8 colocalization with anti-Ape1 (% of Atg8 dots) for the strains from C; >800 GFP-Atg8 dots per strain were quantified. In B and D, columns represent mean and error bars represent SD. Results in this figure represent three independent experiments. ***P < 0.001.