Fig. 6

Plin4 interacts directly with neutral lipids in vitro forming oil droplets. a Images of tubes in which a drop of triolein (10 µl) was vigorously mixed with a solution (190 µl) of increasing concentration of Plin4-12mer. b Representative image of the Plin4-oil emulsion by negative staining electron microscopy. Scale bar: 0.5 µm. c, d Dynamic light scattering measurement of the size distribution of an aliquot withdrawn from the middle of the oil emulsion obtained with 0.5 mg ml⁻¹ Plin4-12mer, and comparison between three independent experiments, with dots representing peak maxima and vertical bars representing polydispersity. e Plin4-oil emulsion was visualized by confocal fluorescence microscopy. Unlabeled Plin4-12mer (0.3 mg ml⁻¹) was mixed Plin4-12mer-Alexa568 at a ratio 20:1 (magenta), and oil was stained with Bodipy (green). Left panel shows Plin4 and right panels show zoom-ins of merged images. Scale bars: 5 µm. f Plin4 in the oil emulsion is protected from degradation by trypsin. Plin4-12mer (1 mg ml⁻¹) was incubated in buffer only or vortexed with triolein as in a, then digested with 13 µg ml⁻¹ (×1) or 130 µg ml⁻¹ (×10) trypsin for the amount of time indicated. Samples were analyzed by SDS-PAGE with Sypro Orange staining. Five times less sample was loaded in the 0 min controls than in the other lanes. White arrowheads indicate the migration of molecular weight standards. Asterisks indicate the trypsin band. g, h Plin4-12mer (1 mg ml⁻¹) before (solution) or after (emulsion) the reaction depicted in (a) was mixed with sucrose and loaded on the bottom of a sucrose gradient. After centrifugation, four fractions were collected from the bottom and equal volumes were analyzed by SDS-PAGE with Sypro Orange staining. See Supplementary Fig. 7 for uncropped gels in f–h. i Model of a Plin4-12mer-covered oil droplet, drawn to scale. Calculation (see main text) suggests complete coverage of the oil surface by Plin4 AH