Abstract
BackgroundPathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics.Methodology/Principal FindingsCholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus.Conclusions/SignificanceThese results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.
Highlights
Mammalian cells harbor a complex endocytic pathway that fulfills a variety of functions such as uptake of nutrients, recycling of proteins and lipids back to the plasma membrane, downregulation of signaling receptors, detection and destruction of pathogens [1,2,3]
Treatment with 3 mg/ml U18666A for 18 hours led to the accumulation of cholesterol, detected using the fluorescent probe filipin, in late endosomes colocalizing with the unconventional lipid lysobisphosphatidic acid (LBPA) [25] (Fig 1A)
The increased resistance to detergent solubilization of late endosomal GPI-anchored proteins upon U18666A treatment raised the possibility that the dynamics of GPI-domains in intralumenal vesicles were altered—we have previously shown that GPI-anchored proteins reside mostly in intralumenal membranes [11]
Summary
Mammalian cells harbor a complex endocytic pathway that fulfills a variety of functions such as uptake of nutrients, recycling of proteins and lipids back to the plasma membrane, downregulation of signaling receptors, detection and destruction of pathogens [1,2,3]. In order to ensure proper targeting of proteins and lipids to the correct destination in the cell, sorting occurs at three stages of the pathway: the plasma membrane, the early endosomes and the late endosomes. Of these compartments, the late endosome appears to be the most complex in terms of morphology [4]. Platforms organized by the small GTPases Rab and Rab have been observed [9] In addition to this compartmentalization of the limiting membrane of the organelle–which is in contact with the cytoplasm–compartmentalization between the limiting membrane and the intralumenal membranes occurs [10]. These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation
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