Fig. 5. GPAT4 targeting to LDs occurs via ER–LD membrane connections at Sec23-defined spots upon the rescue of Tango1 depletion.
a, ERES components enrich in LD fractions upon Tango1 depletion. Heat map for abundance of ERES organizers in LD fractions upon LacZ versus Tango1 RNAi, as measured by mass spectrometry and normalized to LacZ control. b, Sec16 strongly localizes around LDs upon Tango1 depletion. Immunofluorescence of Sec16 in wild-type cells upon RNAi of Tango1 or Tango1 plus another ERES component, followed by a 20-h incubation in oleate-containing medium. Representative images from three independent experiments are shown. Scale bars, 5 μm and 1 μm (inlay). c, Schematic diagram of cell–cell fusion assay to rescue Tango1 depletion. d, Representative images for the cell–cell fusion assay, showing soluble marker (mCherry) as fusion control, Halo–GPAT4 and Sec23–eGFP, at a timepoint before fusion (t = 0 min) as well as pre-GPAT4 targeting, post-GPAT4 targeting and enrichment phases. Scale bar, 5 μm. See also Supplementary Videos 2 and 3. e, Quantification of experiments in d 10 min after cell–cell fusion. Left: bar graph showing percentages of LDs that undergo rescue of GPAT4 targeting that are marked (or not marked) by Sec23 puncta. Right: bar graph compares percentages of Sec23-negative and Sec23-positive LDs that undergo GPAT4 targeting rescue. Mean ± s.d., n = 9 cells examined over seven independent experiments. Two-tailed, paired Student’s t-test, ***P = 0.0004. f, Inlay of the imaging experiment in d showing Sec23 spot on LD and the apparent ER–LD membrane connection through which GPAT4 targeting rescue occurs. Scale bar, 1 μm. See also Supplementary Videos 2 and 3. Source numerical data are available in source data.