Fig. 4: GERVs reproduce lipid droplet assembly.

a Confocal microscopy image of harvested Giant ER Vesicles (GERVs) containing seipin/KDEL. Scale bar: 5 µm. b Confocal microscopy image of an isolated Giant ER Vesicles (GERVs) containing seipin/sec61ß. Scale bar: 5 µm. c Top left: Representation of a membrane nanotube extracted from a seipin/sec61ß-based Giant ER Vesicle. The rectangular shape indicates the region of interest. Top right: Confocal microscopy snapshot of the nanotube extracted from the seipin/sec61ß-based Giant ER Vesicle. Middle: Fluorescence profiles drawn perpendicular to the membrane at both the flat region and the nanotube. Intensities are in arbitrary units. d Confocal microscopy image of a nanotube extracted from a seipin-based Giant ER Vesicle (GERV), which was fed with OCoA and DAG five minutes before tube pulling. LipidTox fluorescence is used to report for the hydrophobicity of the membrane. e The membrane tube shown in (d) is pulled to increase its curvature, resulting in the appearance of seipin puncta in the tube. Some seipin puncta are LipidTox-condensed (indicated by yellow arrows), while others lack LipidTox puncta (red arrows). All LipidTox puncta colocalize with seipin spots. Fluorescence profiles indicate the enrichment of LipidTox at specific spots in the tube. f Left: fraction of nucleation events observed following tube pulling and curvature increase. A nucleation event is considered when at least one LipidTox-positive puncta forms in the tubule. Right: Frequency of nascent LipidTox-positive nascent LD colocalization with a seipin cluster. g Confocal microscopy snapshots of a nanotube extracted from a Giant ER Vesicle (GERV) with seipin overexpressed, and supplied with the substrates for triacylglycerol synthesis. LipidTox fluorescence is monitored to visualize the formation of a nascent LD in the tube. Arrows indicate the appearance of an oil lens (t = 10 s) after the formation of a seipin spot (t = 5 s).