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Originally published In Press as doi:10.1194/jlr.M800145-JLR200 on June 3, 2008

Papers In Press, published online ahead of print September 1, 2008
J. Lipid Res., doi:10.1194/jlr.M800145-JLR200
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Journal of Lipid Research, Vol. 49, 2023-2037, September 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cellsboxs

Nicole Hartwig Petersen*, Nils J. Færgeman*, Liqing Yu{dagger} and Daniel Wüstner1,*

* Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, DK-5230 Denmark
{dagger} Department of Pathology, Section on Lipid Sciences, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1040

boxs The online version of this article (available at http://www.jlr.org) contains supplementary data.

Published, JLR Papers in Press, June 3, 2008.

This work was supported by grants from the Danish Heart Association Hjerteforeningen, the Diabetes Foundation Diabetesforeningen, as well as the Danish Research Agency Forskningsstyrelsen, Forskningsrådet for Natur og Univers (D.W.). L.Y. is supported by an American Heart Association Scientist Development grant.

1 To whom correspondence should be addressed. e-mail: wuestner{at}bmb.sdu.dk

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time ~2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.

Supplementary key words cholesterol • fluorescence microscopy • dehydroergosterol • cholestatrienol • kinetics • intestine • liver • absorption • bile • Niemann Pick C1-like 1

Abbreviations: Alexa546-Tf, Alexa546-tagged transferrin; BC, bile canaliculi; CTL, cholestatrienol; DHE, dehydroergosterol; DiIC12, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; EGFP, enhanced green fluorescent protein; ERC, endocytic recycling compartment; FCS, fetal calf serum; FRAP, fluorescence recovery after photobleaching; MCD, methyl-β-cyclodextrin; NPC1, Niemann Pick C1; NPC1L1, Niemann Pick C1-like 1; Rh-dextran, rhodamine-labeled dextran; ROI, region of interest; SSD, sterol-sensing domain; Tf, transferrin


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