Nov 6, 2018

Analysis of cholesterol export from endo-lysosomes by Niemann Pick C2 protein using combined fluorescence and X-ray microscopy

BioRxiv : the Preprint Server for Biology
Alice DupontDaniel Wüstner

Abstract

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in late endosomes and lysosomes (LE/LYSs). How its capacity to transport cholesterol between membranes is linked to endo-lysosomal membrane trafficking is not known. Using quantitative fluorescence imaging combined with soft X-ray tomography (SXT); we show that NPC2 mediated sterol efflux is accompanied by large changes in distribution, size and ultrastructure of endocytic organelles. We observed clearance of intra-luminal lipid deposits, a decrease in number of autophagosomes, formation of membrane contact sites (MCSs) to the endoplasmic reticulum and extensive tubulation of LE/LYSs in three-dimensional SXT reconstructions of NPC2 treated human fibroblasts. The cells could recycle the cholesterol analog dehydroergosterol (DHE) from LE/LYSs slowly also in the absence of NPC2 protein but internalized NPC2 synchronized and accelerated this process significantly. Most fluorescent NPC2 was retained in LE/LYSs while DHE was selectively removed from these organelles, at least partially by non-vesicular exchange with other membranes. During sterol efflux LE/LYSs were reallocated to the cell periphery, where they could fuse with newly formed endosomes. Surface shedding of ...Continue Reading

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Mentioned in this Paper

Tomography, X-Ray
Fluorescent Dyes
Biochemical Pathway
Size
Tissue Membrane
Specimen Type - Fibroblasts
Repair of Peripheral Nerve by Tubulation
Membrane
Three-dimensional
Reconstructive Surgical Procedures

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