Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells.
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 approximately 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.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, Denmark.
Nicole Hartwig
NH
Petersen
Nils J
NJ
Faergeman
Nils J
NJ
Faegeman
Liqing
L
Yu
Daniel
D
Wüstner
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D000090204:Mesothelin; D008856:Microscopy, Fluorescence; D051381:Rats
D026901:Membrane Transport Proteins; C000715529:Msln protein, rat; C482444:NPC1L1 protein, rat; C010905:dehydroergosterol; D000090204:Mesothelin; D004875:Ergosterol