Extended Data Fig. 3: Formation of membrane sheets observed in vivo and in vitro.
From: Wetting regulates autophagy of phase-separated compartments and the cytosol
a, Live-cell images of droplet autophagy in RPE1 cells expressing GFP–LC3 and mCherry–p62. Autophagosome formation resulted in droplet division. b, Time series of the piecemeal process highlighted in the boxed regions (a) and shown in Fig. 2f. c, Several examples of droplet autophagy whereby two distinct droplets form. Small droplets (circled) with a diameter of 1.0 ± 0.15 μm (mean ± s.d., n = 4 sheets on distinct droplets) were sequestered within LC3-positive autophagosomes, with large droplets essentially free of remaining autophagosomal membranes. Duration of droplet splitting: 10.6 min ± 6.1 min (mean ± s.d.). d, Expansion of LC3 sheets. t = 0 min corresponds to the maximum sheet length. Rate = 0.2 ± 0.04 μm/min. Maximal length = 2.7 μm ± 0.7 μm. Duration 10.1 min ± 3.3 min. Droplets with two intensity peaks occurred within an additional 4.1 min ± 2.6 min (mean ± s.d.). n = 4 sheets on distinct droplets shown in b, c. e, Representative GUVs before phase separation. Interior tubules that form by PEG adsorption onto GUV membranes distribute randomly within the homogeneous interior solution. A fluorescent phospholipid (Atto633–DOPE) and polymer (FITC–dextran 500 kDa) were used to label the membrane (green) and internal solution (magenta). f, g, Representative GUV after phase separation. Vertical confocal (xz) section (f) and horizontal confocal (xy) sections (g) at the droplet surface of this GUV highlight the tubular membrane network that wets the droplet surface. Tubule formation and wetting tubules (e–g) confirm previous work17. h, Tubules visualized by a water-soluble dye (yellow; 5 μM sulforhodamine B) added externally to the GUV. i, The tubular network shown in g after an additional 4-min incubation. The de novo formation of two circular membrane structures representing membrane sheets is observed (arrows). j, Quantification of the relative abundance of membrane shapes at the interface after hyperosmotic deflation (1 h) and following an additional 16-h equilibration (three independent experiments with more than 50 GUVs each, mean ± s.d. with single data points). k, Representative examples of tubules and sheets at droplet surfaces (single xy sections at the droplet surface; the central image is a maximum intensity projection of two xy sections). Scale bars, 2 μm (b, c) and 5 μm (all other images). Representative images of n independent experiments (n = 5 (a–c); n = 10 (e–g, i, k) and n = 4 (h)).
