Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles.

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2's known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.

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

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 µm2/sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 µm2/s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of 13C-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.

SpatTrack: an imaging toolbox for analysis of vesicle motility and distribution in living cells.

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs 2D particle tracking, trajectory analysis and fitting of diffusion models to the calculated mean square displacement. It allows for spatial analysis of detected vesicle patterns including calculation of the radial distribution function and particle-based colocalization. Importantly, all analysis tools are supported by Monte Carlo simulations of synthetic images. This allows the user to assess the reliability of the analysis and to study alternative scenarios. We demonstrate the functionality of SpatTrack by performing a detailed imaging study of internalized fluorescence-tagged Niemann Pick C2 (NPC2) protein in human disease fibroblasts. Using SpatTrack, we show that NPC2 rescued the cholesterol-storage phenotype from a subpopulation of late endosomes/lysosomes (LE/LYSs). This was paralleled by repositioning and active transport of NPC2-containing vesicles to the cell surface. The potential of SpatTrack for other applications in intracellular transport studies will be discussed.