Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells
2008; Elsevier BV; Volume: 49; Issue: 9 Linguagem: Inglês
10.1194/jlr.m800145-jlr200
ISSN1539-7262
AutoresNicole Hartwig Petersen, Nils J. Færgeman, Liqing Yu, Daniel Wüstner,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoNiemann-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. 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. ERRATAJournal of Lipid ResearchVol. 49Issue 11PreviewThe Journal acknowledges that one of the authors' names was misspelled in the article "Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells" (J. Lipid Res. 2008; 49:2023-2027). The list of authors should read: Full-Text PDF Open Access Niemann-Pick C1-like 1 (NPC1L1) was recently identified as a protein mediating intestinal cholesterol absorption (1Altmann S.W. Davis H.R.J. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1-Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1442) Google Scholar). NPC1L1 is a target of the potent cholesterol absorption inhibitor ezetimibe, which has been widely used to lower plasma total cholesterol and LDL-cholesterol (2Garcia-Calvo M. Lisnock J. Bull H.G. Hawes B.E. Burnett D.A. Braun M.P. Crona J.H. Davis H.R.J. Dean D.C. Detmers P.A. et al.The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).Proc. Natl. Acad. Sci. USA. 2005; 102: 8132-8137Crossref PubMed Scopus (657) Google Scholar, 3Davis H.R. Veltri E.P. Zetia: inhibition of Niemann-Pick C1 Like 1 (NPC1L1) to reduce intestinal cholesterol absorption and treat hyperlipidemia.J. Atheroscler. Thromb. 2007; 14: 99-108Crossref PubMed Scopus (146) Google Scholar). In the liver, NPC1L1 protein localizes at the canalicular membrane of hepatocytes (4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. 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Both intracellular and cell surface locations have been reported for NPC1L1 protein (1Altmann S.W. Davis H.R.J. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1-Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1442) Google Scholar, 2Garcia-Calvo M. Lisnock J. Bull H.G. Hawes B.E. Burnett D.A. Braun M.P. Crona J.H. Davis H.R.J. Dean D.C. Detmers P.A. et al.The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).Proc. Natl. Acad. Sci. USA. 2005; 102: 8132-8137Crossref PubMed Scopus (657) Google Scholar, 4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 5Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. 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Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane.Biochim. Biophys. Acta. 2007; 1771: 1140-1147Crossref PubMed Scopus (50) Google Scholar, 9Yamanashi Y. Takada T. Suzuki H. Niemann-Pick C1-like 1 overexpression facilitates ezetimibe-sensitive cholesterol and beta-sitosterol uptake in CaCo-2 cells.J. Pharmacol. Exp. Ther. 2007; 320: 559-564Crossref PubMed Scopus (103) Google Scholar). Some studies suggest that NPC1L1 acts at the cell surface, mediating sterol entry into cells (1Altmann S.W. Davis H.R.J. Zhu L.J. Yao X. Hoos L.M. Tetzloff G. Iyer S.P. Maguire M. Golovko A. Zeng M. et al.Niemann-Pick C1-Like 1 protein is critical for intestinal cholesterol absorption.Science. 2004; 303: 1201-1204Crossref PubMed Scopus (1442) Google Scholar, 2Garcia-Calvo M. Lisnock J. Bull H.G. Hawes B.E. Burnett D.A. Braun M.P. Crona J.H. Davis H.R.J. Dean D.C. Detmers P.A. et al.The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).Proc. Natl. Acad. Sci. USA. 2005; 102: 8132-8137Crossref PubMed Scopus (657) Google Scholar, 4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 5Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A. Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (306) Google Scholar, 9Yamanashi Y. Takada T. Suzuki H. Niemann-Pick C1-like 1 overexpression facilitates ezetimibe-sensitive cholesterol and beta-sitosterol uptake in CaCo-2 cells.J. Pharmacol. Exp. Ther. 2007; 320: 559-564Crossref PubMed Scopus (103) Google Scholar, 10Davis Jr., H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.J. Biol. Chem. 2004; 279: 33586-33592Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). Others report that NPC1L1 resides mainly in intracellular compartments in some cultured nonpolarized cells, where NPC1L1 protein, like Niemann Pick C1 protein (NPC1), may mediate intracellular sterol transport (8Knöpfel M. Davies J.P. Duong P.T. Kværnø L. Carreira E.M. Phillips M.C. Ioannou Y.A. Hauser H. Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane.Biochim. Biophys. Acta. 2007; 1771: 1140-1147Crossref PubMed Scopus (50) Google Scholar, 11Davies J.P. 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Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). In nonpolarized human hepatoma HepG2 cells, immunofluorescence revealed colocalization of NPC1L1 with rab5, a marker for sorting endosomes (10Davis Jr., H.R. Zhu L.J. Hoos L.M. Tetzloff G. Maguire M. Liu J. Yao X. Iyer S.P. Lam M.H. Lund E.G. et al.Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.J. Biol. Chem. 2004; 279: 33586-33592Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar). Sorting endosomes are a population of early endosomes that iteratively deliver recycling receptors, lipids, and other membrane proteins to the long-living ERC by pinching off small vesicles and tubules (13Dunn K.W. McGraw T.E. Maxfield F.R. Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome.J. Cell Biol. 1989; 109: 3303-3314Crossref PubMed Scopus (259) Google Scholar). The sorting endsomes are transient organelles that lose their fusion competence for incoming vesicles with a half-time of approximately 8 min, and eventually mature into late endosomes (14Salzmann N.H. Maxfield F.R. Fusion accessibility of endocytic compartments along the recycling and lysosomal endocytic pathways in intact cells.J. Cell Biol. 1989; 109: 2097-2104Crossref PubMed Scopus (75) Google Scholar, 15Mukherjee S. Ghosh R.N. Maxfield F.R. Endocytosis.Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1311) Google Scholar). After endocytosis, fluorescent Tf is first transported to sorting endosomes with a half-time of about 2 min (15Mukherjee S. Ghosh R.N. Maxfield F.R. Endocytosis.Physiol. Rev. 1997; 77: 759-803Crossref PubMed Scopus (1311) Google Scholar). 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Ioannou Y.A. The role of the Niemann-Pick C1-like 1 protein in the subcellular transport of multiple lipids and their homeostasis.Curr. Opin. Lipidol. 2006; 17: 221-226Crossref PubMed Scopus (25) Google Scholar, 25Brown J.M. Rudel L.L. Yu L. NPC1L1 (Niemann-Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells.Biochem. J. 2007; 406: 273-283Crossref PubMed Scopus (45) Google Scholar). The amount of free cholesterol was doubled in McA cells expressing NPC1L1-EGFP, compared with control cells when cultured in cholesterol-containing medium (4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). This suggests that the presence of NPC1L1 alone is sufficient to increase cellular cholesterol levels in hepatoma cells. Acute cholesterol depletion using the cholesterol chelator cyclodextrin was used previously to facilitate cellular uptake of unesterified cholesterol by NPC1L1 and to be able to monitor this process using radioactive cholesterol tracer (4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 25Brown J.M. Rudel L.L. Yu L. NPC1L1 (Niemann-Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells.Biochem. J. 2007; 406: 273-283Crossref PubMed Scopus (45) Google Scholar). Here, we have performed a kinetic imaging study on transport of NPC1L1-EGFP in hepatoma cells. Focus was set on a detailed comparison of trafficking of NPC1L1 with that of fluorescent Tf under control and cholesterol depletion conditions. We also investigated transport of NPC1L1 in polarized human hepatoma HepG2 cells. The HepG2 cell line is able to form apical bile canaliculi (BC)-like structures resembling the canaliculi biliferi of the liver (28Sormunen R. Eskelinen S. Lehto V.P. Bile canaliculus formation in cultured HEPG2 cells.Lab. Invest. 1993; 68: 652-662PubMed Google Scholar). This cell line is therefore often used to investigate polarized transport processes as they occur in hepatocytes (24Wüstner D. Quantification of polarized trafficking of transferrin and comparison with bulk membrane transport in hepatic cells.Biochem. J. 2006; 400: 267-280Crossref PubMed Scopus (7) Google Scholar, 29van IJzendoorn S.C. Zegers M.M. Kok J.W. Hoekstra D. 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Finally, we used the intrinsically fluorescent close cholesterol analogs dehydroergosterol (DHE) and cholestatrienol (CTL) to determine whether NPC1L1 plays a role in sterol transport from the plasma membrane. Fluorescent sterols like DHE or CTL can be transferred quickly and efficiently to the plasma membrane from cyclodextrin donor complexes, allowing for kinetic sterol trafficking studies using pulse-chase protocols (35Hao M. Lin S.X. Karylowski O.J. Wüstner D. McGraw T.E. Maxfield F.R. Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.J. Biol. Chem. 2002; 277: 609-617Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 36Wüstner D. Mondal M. Tabas I. Maxfield F.R. Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells.Traffic. 2005; 6: 396-412Crossref PubMed Scopus (83) Google Scholar). We show that NPC1L1 shuttles dynamically between the plasma membrane and the ERC, and we provide evidence for a function of NPC1L1 in vesicular sterol transport from the plasma membrane to the recycling endosomes in polarized and nonpolarized hepatoma cells. Rhodamine-labeled dextran (Rh-dextran; 70 kDa), 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC12), Alexa-546, and Alexa-488 were purchased from Molecular Probes, Inc. CTL was obtained from Avanti Polar Lipids. Medium 1 contained 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 20 mM HEPES (pH 7.4). Medium 2 was identical to medium 1 except that it contained no glucose but 5 mM sodium azide and 50 mM 2-deoxyglucose for energy depletion of cells (see below). Fetal calf serum (FCS) and DMEM were from GIBCO BRL (Life Technologies, Paisley, Scotland). All other chemicals were from Sigma Chemical (St. Louis, MO). Transferrin (Tf) was iron loaded as previously described (37Yamashiro D.J. Tycko B. Fluss S.R. Maxfield F.R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway.Cell. 1984; 37: 789-800Abstract Full Text PDF PubMed Scopus (420) Google Scholar). Succinimidyl ester of Alexa-546 was then conjugated to the iron-loaded Tf following the manufacturer's instructions. HepG2 and McArdle RH7777 (McA) cells were grown in DMEM with 4.5 g/l glucose, supplemented with 10% heat-inactivated FCS and antibiotics. Mammalian hepatoma cells expressing plasmid of human NPC1L1 protein, with EGFP appended at its C terminus (NPC1L1-EGFP) and McA cells stably expressing NPC1L1-EGFP, have been reported previously (4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Cells were routinely passaged in plastic tissue culture dishes. NPC1L1-EGFP fusion protein was stably expressed in HepG2 cells after transfection of NPC1L1-EGFP-expressing plasmid (4Yu L. Bharadwaj S. Brown J.M. Ma Y. Du W. Davis M.A. Michaely P. Liu P. Willingham M.C. Rudel L.L. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake.J. Biol. Chem. 2006; 281: 6616-6624Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) using the lipofectamine transfection system according to the manufacture's instructions (Invitrogen A/S, Taastrup, Denmark). For experiments, cells were plated onto glass coverslips coated with poly-d-lysine and used after 3 days, when the highest degree of polarization is reached in HepG2 cells (22Wüstner D. Mukherjee S. Maxfield F.R. Müller P. Hermann A. Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells.Traffic. 2001; 2: 277-296Crossref PubMed Scopus (36) Google Scholar). A stock solution of DHE or CTL (5 mM) was made in ethanol and stored under nitrogen. For labeling cells with DHE or CTL, analogs were loaded on methyl-β-cyclodextrin (MCD), giving a DHE- or CTL-MCD (DHE/MCD and CTL/MCD) solution, respectively, as described previously (35Hao M. Lin S.X. Karylowski O.J. Wüstner D. McGraw T.E. Maxfield F.R. Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.J. Biol. Chem. 2002; 277: 609-617Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). McA or HepG2 cells were routinely labeled with DHE/MCD or CTL/MCD for 1 min at 37°C. To visualize kinetically defined sorting endosomes, McA or HepG2 cells were labeled with 20 μg/ml Alexa546-tagged transferrin (Alexa546-Tf) for 1 min, washed, and chased for 5 min at 37°C. This short incubation ensured that internalized Tf was transported from the cell surface to sorting endosomes before being targeted to the perinuclear ERC. To stain the ERC and subapical recycling compartment, respectively, cells were labeled with 20 μg/ml Alexa546-Tf for 15 min, washed, and chased for 30 min at 37°C, or, alternatively, cells were labeled for 30 min, washed, and directly imaged after various chase times (chase-out experiment). For triple labeling experiments, cells were first labeled with fluorescent sterol, then washed and labeled for 5 min at 37°C with 20 μg/ml Alexa546-Tf, and then washed and chased for 30 min at 37°C. To determine colocalization of NPC1L1-EGFP with lysosomal markers, cells were prelabeled with 2.5 mg/ml Rh-dextran for 1 h at 37°C, washed, and imaged on a wide-field microscope as described below. In polarized hepatoma cells, this procedure is also suitable for visualizing the BC, because Rh-dextran is transported to the BC lumen after prolonged chase (30Wüstner D. Herrmann A. Hao M. Maxfield F.R. Rapid nonvesicular transport of sterol between the plasma membrane domains of polarized hepatic cells.J. Biol. Chem. 2002; 277: 30325-30336Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 32Wüstner D. Mondal M. Huang A. Maxfield F.R. Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells.J. Lipid Res. 2004; 45: 427-437Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 38Wüstner D. Mathematical analysis of hepatic high density lipoprotein transport based on quantitative imaging data.J. Biol. Chem. 2005; 280: 6766-6779Abstract Full Text
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