Fig. 4: The ORD of ORP9 must extract PI4P to maintain proper distribution of cellular cholesterol. | Nature Communications

Fig. 4: The ORD of ORP9 must extract PI4P to maintain proper distribution of cellular cholesterol.

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

Fig. 4

a Schematic representation of the rapamycin-induced recruitment strategy used for the recruitment of ER-anchored ORD of ORP9 (ER-mCherry-FKBP-ORP9) to the PM. ER-mCherry-FKBP-ORP9 was rapidly recruited to the PM by rapamycin-induced dimerization of FRB and FKBP. ER-mCherry-FKBP-ORP9 was expressed in QKO HeLa cells together with a tagBFP-tagged FRB module that is targeted to the PM (PM-FRB-tagBFP). b Time course of normalized signals of EGFP-GRAM-H (accessible cholesterol biosensor) and iRFP-P4M (PI4P biosensor) in response to rapamycin, as assessed by TIRF microscopy of QKO HeLa cells expressing ER-mCherry-FKBP-ORP9 and PM-FRB-tagBFP together with EGFP-GRAM-H and iRFP-P4M. Rapamycin addition (200 nM) is indicated (mean ± SEM, n = 29 cells for each condition; data are pooled from three independent experiments). c Time course of normalized iRFP-P4M signals in response to rapamycin, as assessed by TIRF microscopy of QKO HeLa cells expressing either ER-mCherry-FKBP-ORP9 or ER-mCherry-FKBP-ORP9 carrying PI4P binding-deficient ORD [ER-mCherry-FKBP-ORP9 (HH/AA)] together with PM-FRB-tagBFP and iRFP-P4M. Rapamycin addition (200 nM) is indicated [mean ± SEM, n = 24 cells (WT), n = 30 (HH/AA); data are pooled from two independent experiments]. d Confocal images of live QKO HeLa cells expressing EGFP-GRAM-H together with either mCherry control, mCherry-ORP9, mCherry-ORP9 carrying PI4P binding-deficient ORD [mCherry-ORP9 (HH/AA)], or mCherry-ORP9 carrying phosphatidylserine binding-deficient ORD [mCherry-ORP9 (AAA)]. Insets show at higher magnification the regions around the Golgi as indicated by white dashed boxes. Scale bars, 10 µm. e Quantification of the ratio of PM signals (left) and Golgi signals (right) to the cytosolic signals of EGFP-GRAM-H, as shown in (d) {mean ± SEM, n = 20 cells for mCherry, mCherry-ORP9, and mCherry-ORP9 (HH/AA); n = 10 cells for mCherry-ORP9 (AAA) (PM), n = 18 cells for mCherry, mCherry-ORP9 and mCherry-ORP9 (HH/AA); n = 10 cells for mCherry-ORP9 (AAA) (Golgi); data are pooled from two independent experiments for mCherry, mCherry-ORP9, and mCherry-ORP9 (HH/AA); one experiment for mCherry-ORP9 (AAA); Dunnett’s multiple comparisons test, **P < 0.0001, *P = 0.0251 [PM: mCh vs mCh-ORP9 (AAA)]. n.s. denotes not significant}. Source data are provided as a Source Data file.

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