Extended Data Fig. 4: Membrane sheets close to form double-membrane vesicles in vitro.
From: Wetting regulates autophagy of phase-separated compartments and the cytosol
a, Fluorescence intensity line profiles of Sh1 and Sh2 (as indicated in Fig. 3b). The profiles intersect the two sides of the sheet membrane and the single bilayer of the GUV, as labelled. b, Luminal staining of sheets by addition of the water-soluble, membrane-impermeable dye sulforhodamine B outside GUVs (yellow). The orange line indicates the position of the intensity profile. c, Remodelling of a droplet-pinned, predominantly tubular structure stained by sulforhodamine B into a circular membrane sheet, as observed by confocal time-lapse imaging (inverted intensity). Top panels, droplet surface imaged before (left) and after (right) remodelling. Bottom panels, time series of remodelling. d, The size of sheets can be stable for extended periods. Line profiles of Sh1 and Sh2 obtained from xy sections shown in left panels of Fig. 3b, c. e, Membranes wet droplet surfaces in a GUV-free assay. Droplets sedimenting on a pre-hydrated membrane were observed by confocal microscopy. Maximum intensity projections of distinct upper droplet hemispheres showing surface pinning of membranes in the form of (from left to right) small vesicles, narrow tubules, wide tubules while remodelling into a sheet, or two sheets with small vesicles. f, A representative vertical confocal section showing the profile of a sedimented droplet with a surface-attached, droplet-sequestering membrane sheet (left). The corresponding maximum intensity projection is shown at right. g, In some cases, both sides of wetting double-membrane sheets are resolvable. Left, a single confocal section with an orange line indicating the fluorescence intensity profile. Centre, line profile exhibiting a membrane double peak. Right, maximum intensity projection of the same droplet. h, Left, confocal section of the centre plane of a droplet undergoing sequestration. Also shown are maximum intensity projections of the upper (centre) and lower (right) droplet hemispheres. i, Fluorescence intensity line profile of a double membrane vesicle and the preceding sheet, Sh3 (shown in Fig. 3b, c). The size of the structure decreased by approximately half during remodelling. A fluorescent phospholipid (Atto633–DOPE) and polymer (FITC-dextran 500 kDa) were used to label the membrane (green) and droplet (magenta). j, Luminal staining of a double membrane vesicle by addition of the water-soluble, membrane-impermeable dye sulforhodamine B outside GUVs (yellow). The orange line indicates the intensity profile segment. k, Multiple GUVs with numerous wetting sheets are shown under isotonic conditions (left) and 7 min after exposure to hypoosmotic conditions (right). The sheets are partially out-of-focus because the z position of the slightly non-horizontal droplet surface differs between GUVs. A few minutes after the decrease in Σcd associated with hypoosmotic conditions, all sheets closed into autophagosome-like double-membrane vesicles. Confocal fluorescence microscopy of Atto633–DOPE-stained membranes (intensities inverted). l, Intensity plots for segments intersecting double-membrane vesicles and corresponding GUVs as indicated in k (right). m, Left, membrane fluorescence intensity of GUVs delimiting membranes and their internal double-membrane vesicles. Gradient determined by linear regression (n = 9 GUVs examined, several double-membrane vesicles within one GUV are shown as mean ± s.d.). Right, size measurements of double-membrane vesicles relative to preceding sheets as illustrated in i. Gradient determined by linear regression (n = 13 sheet-vesicle pairs). Scale bars, 20 μm (k) and 5 μm (all other images). Representative results from n independent experiments (n = 5 (k–m), n = 3 (c) and n = 2 (b, e–h, j).
