Fig. 3: ESYT1/2-VAPB form a complex at the LD-Mitochondria-ER interface. | Nature Communications

Fig. 3: ESYT1/2-VAPB form a complex at the LD-Mitochondria-ER interface.

From: Proximity proteomics reveals a mechanism of fatty acid transfer at lipid droplet-mitochondria- endoplasmic reticulum contact sites

Fig. 3

a Left panel: Single plane view of Airyscan imaging of HepG2 cells stably expressing eGFP-ESYT, eGFP-ESYT2 or eGFP-VAPB (blue). HepG2 cells were fixed and stained for LDs with HCS LipidTox red Neutral Lipid Stain (green), mitochondria with TOMM20 antibody (yellow), and ER with Calnexin antibody (magenta). White arrows highlight tripartite interfaces of LD, mitochondria, and ER. A total of 16 cells from four independent experiments were imaged and a representative of image is indicated. Large scale bars, 2 μm and inset scale bars 0.5 μm. Right panel: Line scan analysis of images presented to the left. A and B on x-axis corresponds to the line scan in panel a inset. b Left panel: Donor lifetime maps of the FLIM data acquisitions between donor eGFP-ESYT1, eGFP-ESYT2, and eGFP-VAPB with acceptor VAPB-mCH FRET pair pseudo-colored according to the palette defined in the phasor plot in (Fig. 2h) spatially map the hetero protein-protein interaction (red). Right panel: Identification of the LDs and mitochondrial interface based on PLIN2-AF405 and TOMM20-AF647 immunofluorescence intensity mask analysis. c Quantification of the fraction of ESYT1/VAPB (upper panel), ESYT2/VAPB (middle panel), and VAPB/VAPB interaction inside the LDs and mitochondrial interface defined by PLIN2-AF405 and TOMM20-AF647 intensity masks (panel B, right) versus whole cell protein interaction fraction with and without Forksolin stimulation across multiple HepG2 cells. ESYT1/VAPB -FSK n = 10; ESYT1/VAPB + FSK n = 7; ESYT2/VAPB -FSK n = 10; ESYT2/VFSK+fork n = 9; VAPB/VAPB -FSK n = 9; VAPB/VAPB + FSK n = 7 cells. *P < 0.05 paired t-test, one-tailed. See also Supplementary Fig. 3.

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