Fig. 6. Seipin depletion allows for late targeting proteins to target early from the ER to LDs in the absence of fusion machinery or ERES components.
a, GPAT4 targeting occurs at ER–LD connections independent of seipin. FRAP experiment of transiently expressed Halo–GPAT4 on LDs in endogenous GFP–seipin knock-in (KI) cells, after 6–10 h incubation in oleate-containing medium. Top: inlay images. Bottom: whole cell view. Yellow arrowheads indicate apparent ER–LD connections independent of seipin. Scale bars, 5 μm and 1 μm (inlay). Representative images from five independent experiments are shown. See also Supplementary Videos 4 and 5. b, Late targeting proteins target LDs early in the absence of seipin. Confocal imaging of live seipin knock-out (KO) cells transiently transfected with eGFP-tagged constructs at given timepoints after 1 mM oleic acid treatment. LDs were stained with MDH. Representative images are shown. Percentage of cells with LD targeting are indicated (mean, n = 3 independent experiments, 8–13 cells each). Scale bars, 5 μm and 1 μm (inlay). c, Absence of seipin provides an alternative pathway for late ER-to-LD targeting. Confocal imaging of live wild-type (WT) or seipin KO cells upon RNAi of ERES or fusion-machinery components, followed by transient transfection with eGFP-tagged constructs and a 20-h incubation in oleate-containing medium. Scale bars, 5 μm and 1 μm (inlay). d, Bar graph showing targeting ratios from the imaging experiment in c. Mean ± s.d., n = (left to right) 85, 33, 41, 42, 41, 42, 42 and 40; 84, 29, 35, 46, 48, 44, 49 and 44; 89, 42, 38, 41, 46, 47, 48 and 43 cells examined over three independent experiments. One-way ANOVA with Bonferroni correction, **P < 0.01 (from left to right: 0.0029 and 0.0067), ***P = 0.0005, compared with LacZ. e, Bar graph showing percentages of cells with LD targeting after a 0.5-h incubation in oleate-containing medium. Mean ± s.d., n = 6 experiments for LacZ and 3 for the rest. One-way ANOVA with Bonferroni correction, no significant differences. Representative images are shown in Extended Data Fig. 8a. Source numerical data are available in source data.