Journal: The EMBO Journal
Article Title: ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P 2 exchange
doi: 10.15252/embj.2020106871
Figure Lengend Snippet: A Schematic of live‐cell dextran and transferrin pulse‐chase in degron‐ORP2 cells. B Exemplary confocal images from movies of +/− IAA‐treated degron‐ORP2 cells labeled with AF647‐transferrin (magenta) and fluorescein dextran (green) as in Fig . Trajectories of manually tracked double‐positive organelles at 30 min chase time point of the corresponding time series shown in Movie (‐IAA; ORP2 present) and Movie (+IAA; ORP2 depleted). C Duration of contacts between transferrin and dextran organelles. Mean ± SEM, n = 28 contacts from 6 cells (+IAA); 36 contacts from 5 cells (‐IAA). Student’s t ‐test. Number of contacts between dextran and transferrin organelles. Mean ± SEM. n = 42 (‐IAA); 39 (+IAA) dextran organelles. Student’s t ‐test. D Control and degron‐ORP2 cells were co‐plated. After 1 day in 5% LPDS, cells were loaded with 50 μg/ml LDL and IAA for the indicated times, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. Asterisk indicates ORP2‐depleted cell. E Quantification of EV5D. Mean ± SD, n = 13–23 cells from 2 independent experiments. Student’s t ‐test. F, G TIRF imaging of NPC1‐GFP distribution in live cells with or without 10 μM PF‐228 for 4 h, and quantification of NPC1 vesicles within 10 μm distance from the cell edge (dotted lines). Transiently transfected mCherry‐FAK was used to mark the cell edge. Mean ± SD, n = 6 cells. Student’s t ‐test. H Representative epifluorescent images of NPC1‐mCherry organelles in GFP‐ORP2 or GFP‐ORP2‐mHHK overexpressing cells quantified in Fig . Arrowheads indicate tubular NPC1 organelles. I After 1 day in 5% LPDS, control and degron‐ORP2 cells were loaded with 50 μg/ml LDL and IAA for the indicated times, lysed and subjected to immunoblotting with pFAK antibody. Mean ± SD, n = 3 independent experiments. Paired Student’s t ‐test. J Control and degron‐ORP2 cells were co‐plated and incubated without (−LDL) or with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for 1 h. Representative confocal images showing increased pFAK intensity at perinuclear endomembranes upon LDL loading in control cells but not in the ORP2‐depleted degron cell (asterisks). Red inset shows endomembrane endo‐GFP‐ORP2 and pFAK signals in the control cell, and blue inset shows corresponding signals in the ORP2‐depleted cell. Orange arrowheads indicate mature FAs. K Representative confocal images of GFP‐ORP2‐mHHK or GFP‐ORP2‐∆ELSK‐transfected cells quantified in Fig . Dashed lines indicate cell outlines. Asterisks indicate cells depleted of endogenous ORP2. L AF488‐FERM binding to liposomes by liposome‐co‐sedimentation. Lipid composition and concentration as well as protein concentration were the same as in Fig , in 0, 5, and 10% PI(4,5)P 2 ‐containing liposomes. S; supernatant, P; pellet. Numbers under the blots indicate the fraction of FAK FERM bound to liposomes (P) of total FAK FERM (S + P). Source data are available online for this figure.
Article Snippet: Antibodies: pFAK (BD Transduction Laboratory #611807, clone 18 for Western blotting and Thermo Fisher #44‐624G for immunofluorescence), FAK (Sigma 05‐537, Clone 4.47), integrin β1 (Santa Cruz sc‐18887, K20 for immunofluorescence and Sigma MAB2252, clone N29 for Western blotting), PI(4,5)P 2 (Echelon, Z‐G045, clone 2C11), ORP2 (Novus Biologicals, NBP1‐92236), NPC1 (Abcam, ab134113), Lamp1 (DSHB, H4A3), GFP (Abcam, ab290 and ab1218); see Appendix␣Fig for specificity controls of pFAK, FAK, and integrin β1antibodies.␣Lipids: 1‐palmitoyl‐2oleoyl‐sn‐ glycero‐3‐phosphocholine (POPC), 1‐palmitoyl‐2oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE), 1‐palmitoyl‐2oleoyl‐sn‐glycero‐3‐phospho‐L‐serine (sodium salt) (POPS), L‐a‐phosphatidylinositol‐4,5‐bisphosphate (brain, porcine, ammonium salt; brain PI(4,5)P 2 ), cholesterol (ovine) were from Avanti Polar Lipids.
Techniques: Pulse Chase, Labeling, Staining, Confocal Microscopy, Imaging, Transfection, Western Blot, Incubation, Binding Assay, Sedimentation, Concentration Assay, Protein Concentration