Extended Data Fig. 7: Disruption of the LIR region perturbs droplet autophagy.
From: Wetting regulates autophagy of phase-separated compartments and the cytosol
a, Localization of endogenous ubiquitin and siRNA-resistant mCherry–p62 (left) or siRNA-resistant mCherry–p62(ΔLIR) (right) in RPE1 cells. The mCherry–p62(ΔLIR) lacks amino acids 321–348 that make up the LIR. Stable p62 expression is quantified in Extended Data Fig. 6d. b, Left, autophagy was inhibited by SAR405 treatment for 4 h before release by DMEM washout (additional data in Extended Data Fig. 6b). Right, droplet numbers per cell were determined by ubiquitin immunofluorescence before and after 4h SAR405 treatment, and also following SAR405 washout. Exponential fits (lines) yield time constants ± s.d. of 37.03 ± 10.39 min (ΔLIR), 19.25 ± 1.17 min (WT), 17.24 ± 0.02 min (M404V) and 16.14 ± 6.94 min (R21A) that characterize droplet turnover and are plotted as inset. n values, given as fields of view (and cell numbers) at −240, 0, 30, 60 and 120 min after SAR405 treatment: WT, n = 6 (118), 5 (127), 5 (175), 6 (178), 7 (154); ΔLIR, n = 10 (120), 5 (109), 9 (206), 7 (169), 6 (172); M404V, n = 6 (127), 5 (129), 7 (171), 5 (158), 5 (123); R21A, n = 5 (125), 4 (135), 6 (163), 5 (118), 5 (135). Data are mean ± s.d. c, The region within the white broken boundary (left) was examined by electron tomography, showing that the droplet is not sequestered by LC3-positive membranes (arrows). These data correspond to the image presented in Fig. 4b. d, Time series (30-s interval) of a wild-type MEF exhibiting a WIPI2-positive structure sequestering a subset of a p62 droplet. Droplet deformation and division are highlighted by arrows (wild-type MEF expressing WIPI2–mRuby3 and GFP–p62). e, f, Correlated confocal (e) and scanning electron (f) microscopy of autophagosomes attached to the p62 droplet indicated in the leftmost panel. The regions indicated by numbers are shown at high magnification in the rightmost panels of f. The autophagosome interior is filled with low-density material consistent with sequestration of droplet material, not ribosomes present in the cytosol (MEF expressing WIPI2–mRuby3 and GFP–p62). g, Time series (at 30-s intervals) of an ATG3(KO) MEF in which a droplet-bound WIPI2-positive structure is observed to expand, forming a cup-shape intermediate before closing without droplet sequestration (ATG3(KO) MEF expressing WIPI2–mRuby3 and GFP–p62). h, Several additional examples of droplet-bound WIPI2-positive structures that expand towards the cytosol, subsequently closing to form autophagosomes without droplet sequestration (circles), as quantified in Fig. 4c (ATG3(KO) MEFs expressing WIPI2–mRuby3 and GFP–p62). Scale bars for fluorescence images, 2 μm; scale bar for electron microscopy, 0.5 μm (c, left panel in f), 0.2 μm (right panels in f). Representative images from three (d, g, h) or two (c, e, f) independent experiments. i, Size of p62 droplets increases in ATG3(KO) cells. Quantification of the diameter of p62 droplets in wild-type and ATG3(KO) MEFs. Data medians are shown within boxes representing the interquartile range (25th to 75th percentile), with whiskers denoting the 10th and 90th percentiles. Significance was determined by the unpaired two-tailed Student’s t-test (P = 6.2 × 10−10). Data were collected in three independent experiments and n ≥ 100 p62 droplets were observed for each cell type (control n = 105, ATG3(KO) n = 100).
