Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts. (July 2018)

• Record Type: Journal Article
• Authors: Berzina, Zane; Solanko, Lukasz M.; Mehadi, Ahmed S.; Jensen, Maria Louise V.; Lund, Frederik W.; Modzel, Maciej; Szomek, Maria; Solanko, Katarzyna A.; Dupont, Alice; Nielsen, Gitte Krogh; Heegaard, Christian W.; Ejsing, Christer S.; Wüstner, Daniel
• Is Part Of: Chemistry and physics of lipids. Volume 213(2018)
• Page Start: 48
• Page End: 61
• Publication Date: 2018-07
• Subjects: ACAT acyl Coenzyme A acyl transferase -- Alexa647-Tf Alexa647-tagged transferrin -- 13C3-cholesterol cholesterol with heavy carbon (C13) at position 3 -- CHO Chinese Hamster Ovarian -- ER endoplasmic reticulum -- D diffusion coefficient -- DHE dehydroergosterol -- DHE/CD dehydroergosterol-cyclodextrin complex -- DHE/BSA complex of dehydroergosterol with bovine serum albumin -- DIT dynamic intensity threshold -- FRAP fluorescence recovery after photobleaching -- LE/LYSs late endosomes/lysosomes -- LPDS lipoprotein depleted serum -- MSD mean square displacement -- NPC1 Niemann Pick type C1 protein -- NPC2 Niemann Pick type C2 protein -- PC phosphatidylcholine -- NPC2+/+ cells human fibroblasts from healthy subjects -- NPC2-/- cells human fibroblasts from patients lacking functional NPC2 -- OG-dextran Oregon-green-tagged dextran -- PC phosphatidylcholine -- PDF probability density function -- PM plasma membrane -- REs recycling endosomes -- Rh-dextran tetramethyl-rhodamine-tagged dextran -- ROI region of interest -- SM sphingomyelin -- TGN trans-Golgi network; Cholesterol -- Vesicle tracking -- Fluorescence recovery after photobleaching -- Diffusion -- Niemann-Pick disease type C2 -- Lysosome -- Non-vesicular -- Lipid mass spectrometry -- Kinetics; Lipids -- Periodicals; Lipids -- Periodicals; Lipides -- Périodiques; Lipids; Periodicals; Electronic journals; 547.77
• DOI: 10.1016/j.chemphyslip.2018.03.006
• Languages: English
• ISSNs: 0009-3084

Abstract

Highlights: Plasma-membrane derived sterol is transported to endo-lysosomes but becomes trapped in these organelles in NPC2 deficient cells. A small sterol pool remains mobile as shown by fluorescence recovery after photobleaching (FRAP). Quantitative lipid mass spectrometry reveals NPC2-dependent but little esterification of plasma-membrane derived sterol. Internalized NPC2 rescues sterol storage and increases the mobile fraction of DHE in FRAP experiments. Our data supports that NPC2 mobilizes sterol from vesicles inside late endosomes for non-vesicular export to other compartments. Abstract: Niemann-Pick disease type C2 is a lipid storage disorder in which mutations in the NPC2 protein cause accumulation of lipoprotein-derived cholesterol in late endosomes and lysosomes (LE/LYSs). Whether cholesterol delivered by other means to NPC2 deficient cells also accumulates in LE/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous diffusion in disease cells (D ∼ 4.6∙10 −4 μm 2 /sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 μm 2 /s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of 13 C-labeled cholesterol but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM and LE/LYSs depends on a functional NPC2 protein. NPC2 likely acts inside LE/LYSs from where it increases non-vesicular sterol exchange with other organelles.