Fig. 3: SBP-LiveDrop accesses LDs via seipin-mediated ER–LD contact sites.
From: Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets
a, Representative MINFLUX single-molecule trajectory of SBP-LiveDrop entering the LD surface. Time progression is colour-coded (start to end, black to yellow). LDs (green) are stained with BODIPY 493/503. Scale bar, 250 nm. b, A schematic of synchronized SBP-LiveDrop trafficking from the ER to LDs using the RUSH system. SBP-LiveDrop is retained on the ER membrane through interactions with an ER-localized hook. Biotin addition competes for streptavidin binding, releasing SBP-LiveDrop and enabling real-time monitoring of its trafficking to the LD surface. c, Live-cell confocal images of SBP-LiveDrop before and 10 min after biotin-induced release. Cells were pretreated with 250 µM oleic acid overnight to induce LD formation. SBP-LiveDrop was labelled with the JFX554 Halo ligand and released by adding 80 µM biotin. LDs were stained with 1 µM BODIPY 493/503. Scale bars, 10 µm (left) and 5 µm (middle). d, The motion types of SBP-LiveDrop classified by DeepSPT. Single-molecule trajectories were categorized into four groups: free-only (blue), confined-only (orange), single transition (free-to-confined or vice versa) and multiple state-switching trajectories. e, SBP-LiveDrop single-molecule trajectories plotted over a seipin density map. Cells expressing sparsely labelled SBP-LiveDrop and endogenously tagged seipin were imaged simultaneously using HILO microscopy. An LD prediction algorithm identified trajectories moving from the ER to LDs (Methods). Motion states are colour-coded; other tracks are shown in light grey. Scale bar, 10 µm. f, Examples of SBP-LiveDrop trajectories entering LDs through seipin-rich regions. Trajectories are colour-coded by motion state: free (blue) and confined (orange). Stars denote starting positions; orange circles indicate free-to-confined transitions used as proxies for LD entry. Scale bar, 500 nm. g, Confocal images of SBP-LiveDrop in WT and seipin KO cells. Cells were incubated overnight with 250 µM oleic acid and imaged by spinning-disc confocal microscopy 30 min after biotin release. Arrowheads indicate LDs in seipin KO cells that failed to recruit SBP-LiveDrop, consistent with a loss of ER–LD membrane contact. Scale bars, 10 µm (left) and 5 µm (right).
