Fig. 1: Spartin targets to mature LDs via AH repeats in the senescence domain.
From: The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets
a, Cells expressing endogenously tagged spartin (with mScarlet-I) reveal preferential targeting to mature LDs. SUM159 cells treated with 0.5 mM OA for 30 min (top) or 24 h (bottom) and stained with BODIPY493/503. Scale: full-size, 20 μm; insets, 2 μm. b, Quantification of a. Mean ± s.d., n = 6 fields of view (24 h), 10 fields of view (30 min), three independent experiments, ****P = 0.0003, two-tailed unpaired t-test. c, Spartin and PLIN3 localize to different LD subpopulations. Cells expressing endogenously tagged spartin (with HaloTag) and PLIN3 (with mScarlet-I) treated with 0.5 mM OA (24 h) and stained with BODIPY493/503. HaloTag pre-labelled with 100 nM JF646. Scale bars as in a. d, Overlap of PLIN3 and spartin on LDs after 0.5 mM OA treatment, Pearson’s coefficient analysis. Mean ± s.d., n = fields of view, three independent experiments, ***P < 0.001, two-tailed unpaired t-test. e, Schematic representation of spartin (top) with long AH regions (purple) in the senescence domain (bottom). f, Localization of expressed spartin truncation mutants (with mScarlet-I tag) reveals spartin senescence domain is required for LD localization. Cells were treated with 0.5 mM OA (24 h) and stained with BODIPY493/503. Scale bars: full-size, 20 μm; insets, 2 μm. g, Quantification of f. Mean ± s.d., n = 4 cells (108–666), 5 cells (ΔUBR), 5 cells (380–666) and 5 cells (1–380). h, Helical wheel plot of spartin 33-mer repeats of AH1 (amino acids 431–463) and AH2 (amino acids 471–503), plotted as a 3–11 helix (36). AH(6HR→A), six mutations introduced into AH1 + AH2 (amino acids 431–503) and full length (FL). i, Cells expressing mScarlet-I-tagged spartin as in h show that AH1 and AH2 are sufficient for LD binding. Cells were treated with 0.5 mM OA (24 h) and stained with BODIPY493/503. Insets, overlay of spartin (magenta) and LDs (green). Scale bars as in a. j, Quantification of h. Mean ± s.d., n = 12 fields of view (AH1), 12 fields of view (AH1 + AH2), 14 fields of view (6HR-A[AHs]) and 13 fields of view (6HR-A[FL]), three independent experiments, ****P < 0.0001, one-way analysis of variance, Tukey’s multiple comparisons test. Source numerical data are available in source data.
