Fig. 7: GRAMD1b acts at ER-TGN contact sites to remove excess cholesterol from the Golgi. | Nature Communications

Fig. 7: GRAMD1b acts at ER-TGN contact sites to remove excess cholesterol from the Golgi.

From: Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites

Fig. 7

a Confocal images of live QKO HeLa cells expressing either mRuby control, mRuby-tagged GRAMD1b (mRuby-GRAMD1b), mRuby-GRAMD1b carrying cholesterol binding-deficient StART-like domain [mRuby-GRAMD1b (5P)], or mRuby-GRAMD1b (5P) carrying cholesterol sensing-deficient GRAM domain [mRuby-GRAMD1b (R189W & 5P)] together with EGFP-GRAM-H (accessible cholesterol biosensor). Insets show at higher magnification the regions around the Golgi as indicated by white dashed boxes. Scale bars, 10 µm. b Quantifications of the ratio of PM signals (top) and Golgi signals (bottom) to the cytosolic signals of EGFP-GRAM-H, as shown in (a) (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment; Dunnett’s multiple comparisons test, **P < 0.0001). c Confocal images of live WT and QKO HeLa cells expressing EGFP-GRAM-H and mRuby-GRAMD1b (5P) that were treated with or without OSW-1 (20 nM for 1 h; OSBP inhibitor) as indicated. Yellow allows indicate the site of mRuby-GRAMD1b (5P) accumulation around the Golgi. Yellow arrowheads indicate the sites of mRuby-GRAMD1b (5P) accumulation at the PM. Note the dissociation of mRuby-GRAMD1b (5P) from the Golgi upon OSW-1 treatment. Scale bars, 10 µm. d Quantification of the ratio of Golgi signals to the endoplasmic reticulum (ER) signals of mRuby-GRAMD1b (5P),as shown in (c) (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment; two-tailed unpaired Student’s t-test **P = 0.0020). e Confocal images of the regions around the TGN of a QKO HeLa cell expressing miRFP-FKBP-GRAMD1b and tagBFP-TGN38-FRB together with EGFP-GRAM-H and mCherry-P4M (PI4P biosensor) that were treated with rapamycin (200 nM) for the indicated minutes. Scale bars, 2 µm. f Time course of normalized signals of EGFP-GRAM-H and iRFP-P4M in response to rapamycin, as assessed by confocal microscopy as shown in (e) (mean ± SEM, n = 18 cells; data are pooled from four independent experiments). Source data are provided as a Source Data file.

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