Extended Data Fig. 6: Autophagosomal membranes wet and sequester p62 droplets in a physiological context.
From: Wetting regulates autophagy of phase-separated compartments and the cytosol
a, Confocal images of fixed RPE1 cells stably expressing GFP–LC3 and mCherry–p62 or mCherry–p62(ΔLIR). Cytosolic p62 droplet formation (droplets were not found within LAPM1-positive lysosomes or engulfed by LC3-positive vesicles) was induced by addition of the reversible VPS34 inhibitor SAR405 (10 μM) for 4 h. Arrows in the left panels indicate magnified panels in the right panels. b, Droplet formation of p62 mutants unable to bind ubiquitin (M404V) or to self-oligomerize via the PB1 domain (R21A) is inhibited. Indicated cells were treated with SAR405 for 4 h. Corresponding wild-type and ΔLIR data are shown in Extended Data Fig. 7b. c, Western blots of total cell lysates of each stable RPE1 cell line expressing GFP–LC3 and an siRNA-resistant mCherry–p62 variant (wild type (WT), ΔLIR, M404V or R21A) with or without endogenous p62 knockdown using siRNA. d, Left, knockdown efficiency of siRNA treatment. Right, quantification of mCherry–p62 expression levels (WT, ΔLIR, M404V or R21A) relative to endogenous p62. Data are mean ± s.d. from three independent experiments. e, f, RPE1 cells treated with full medium (DMEM/F12) after 4-h VPS34 inhibition by SAR405 and subsequent formation of cytosolic p62 droplets. GFP–LC3 localized to the surface of p62 (e) and p62(ΔLIR) (f) droplets within 15 min of SAR405 washout. Arrows in the top panels indicate magnified panels in the bottom panels. g, Piecemeal sequestration of a p62 droplet by GFP–LC3 after SAR405 washout. Live-cell imaging of droplets was initiated 15 min after SAR405 washout and z-stacks of a whole cell were acquired every 15 s. 0 min was defined as the time point at which the GFP–LC3 signal exhibited maximum length. Representative images from two (a, b, e, f, g) or three (c, d) independent experiments. Scale bars, 10 μm (a, b), 5 μm (e, f) or 1 μm (g, magnified images in a, e, f).
