Fig. 4: Cargo movement across seipin-mediated ER–LD contact sites is bidirectional.

a, A schematic of the reverse RUSH assay. Cells were cultured in biotin-containing medium to allow SBP-LiveDrop accumulation on LDs. Subsequent addition of avidin displaced biotin from the ER hook, enabling re-capture of any SBP-LiveDrop molecules that returned to the ER membrane. b, Confocal images of SBP-LiveDrop in cells incubated overnight with biotin (top) or after avidin treatment (bottom). Cells were treated with biotin and 250 µM oleic acid overnight to induce LD formation. LD-localized SBP-LiveDrop was labelled with 100 nM JFX554 before avidin addition. After an overnight avidin incubation, cells were imaged using confocal microscopy. LDs were stained with BODIPY 493/503 (green). Scale bars, 10 µm (left) and 2 µm (right). c, Representative HILO single-molecule tracks of SBP-LiveDrop exhibiting bidirectional trafficking between the ER and LDs. Left: multiple trajectories showing both entry into and exit from the same LD. Right: spatial coordinates of motion switches are plotted for ER-to-LD (blue) and LD-to-ER (orange) events, overlaid on the LD channel. Stars indicate trajectory start positions. Scale bar, 500 nm. d, An example of SBP-LiveDrop bidirectional movement across a seipin-containing ER–LD contact site. Motion switch coordinates in both directions colocalize with a seipin density hotspots near the LD. Scale bars, 1 µm (left) and 250 nm (right). e, A model of nanodomain-mediated protein confinement on ER and LD membranes. Seipin maintains ER–LD membrane continuity and regulates lipid and protein flux between the two organelles, thereby shaping the membrane environments that govern protein partitioning and motion. On the LD monolayer, increased local presentation of triglycerides enhances interactions with conserved tryptophan residues, leading to tighter clustering and stronger confinement.