The ABCA1 Transporter Modulates Late Endocytic Trafficking
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m314160200
ISSN1083-351X
AutoresEdward B. Neufeld, John A. Stonik, Stephen J. Demosky, Catherine L. Knapper, Christian A. Combs, Adele Cooney, Marcella Comly, Nancy K. Dwyer, Joan Blanchette‐Mackie, Alan T. Remaley, Silvia Santamarina-Fojo, H Bryan Brewer,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoWe have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein. We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein. Cholesterol plays a critical role in the sorting and trafficking of other membrane lipids and proteins and serves to stabilize the structure of membrane domains required for signal transduction (1Simons K. Ikonen E. Science. 2000; 290: 1721-1726Crossref PubMed Scopus (1078) Google Scholar). The maintenance of optimal sterol levels for normal cellular function requires complex homeostatic cellular mechanisms that regulate cholesterol synthesis, intracellular trafficking, uptake, and efflux. The ABCA1 1The abbreviations used are: ABCA1, ATP-binding cassette protein A1; BSA, bovine serum albumin; DiI-LDL, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-low density lipoprotein; FBS, fetal bovine serum; GFP, green fluorescent protein; HDL, high density lipoprotein; LAMP2, lysosomal-associated membrane protein 2; MRP1, multidrug resistance protein 1; NP-C, Niemann-Pick type C disease; NPC1, Niemann-Pick C1 protein; AMEM, α-Minimal essential medium; LPDS, lipoprotein-deficient bovine serum; BODIPY, 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene. transporter plays a pivotal role in the energy-dependent efflux of cellular cholesterol and choline-containing phospholipids to apoA-I, the initial step in the formation of the nascent HDL particle (2Oram J.F. Yokoyama S. J. Lipid Res. 1996; 37: 2473-2491Abstract Full Text PDF PubMed Google Scholar, 3Mendez A.J J. Lipid Res. 1997; 38: 1807-1821Abstract Full Text PDF PubMed Google Scholar, 4Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (658) Google Scholar, 5Francis G.A. Knopp R.H. Oram J.F. J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (373) Google Scholar, 6Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer Jr., H.B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar). The role of ABCA1 in the maintenance of normal cellular sterol levels and HDL formation is strikingly illustrated when the transporter is mutated in Tangier disease (7Orso E. Broccardo C. Kaminski W.E. Bottcher A. Liebisch G. Drobnik W. Gotz A. Chambenoit O. Diederich W. Langmann T. Spruss T. Luciani M.-F. Rothe G. Lackner K.J. Chimini G. Schmitz G. Nat. Med. 2000; 24: 192-196Crossref Scopus (431) Google Scholar, 8Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer Jr., H.B. Duverger N. Denefle P. Assmann G. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1269) Google Scholar, 9Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1509) Google Scholar). This rare human genetic disorder is characterized by excess cholesterol ester accumulation in macrophages, low serum HDL levels, and increased risk of coronary heart disease (10Assmann G. von Eckardstein A. Brewer Jr., H.B. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill Inc., New York2001: 2937-2960Google Scholar). Studies of rare genetic diseases of cholesterol metabolism have revealed several proteins residing in late endosomes, and lysosomes play key roles in cellular cholesterol trafficking and metabolism. Niemann-Pick C proteins 1 and 2 (11Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 12Blom T.S. Linder M.D. Snow K. Pihko H. Hess M.W. Jokitalo E. Veckman V. Syvanen A.-C. Ikonen E. Hum. Mol. Gen. 2003; 12: 257-272Crossref PubMed Scopus (82) Google Scholar), which are defective in NP-C disease (13Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E.D. Neufeld E.B. Blanchette-Mackie E.J. Pentchev P.G. Scriver C.R Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001Google Scholar), redistribute LDL-derived lysosomal cholesterol to other cellular sites including the plasma membrane and the endoplasmic reticulum. MLN64, structurally related to steroidogenic acute regulatory protein defective in congenital adrenal hyperplasia, traffics lysosomal cholesterol to mitochondria (14Zhang M. Liu P. Dwyer N.K. Christenson L.K. Fujimoto T. Martinez F. Comly M. Hanover J.A. Blanchette-Mackie E.J. Strauss 3rd, J.F. J. Biol. Chem. 2002; 277: 33300-33310Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). ATP-binding cassette protein G1, although not yet linked to a genetic defect, also appears to reside in late endocytic vesicles and play a role in cholesterol trafficking (15Neufeld E.B. Sabol S. Remaley A.T. Ito T. Demosky S.J. Stonik J. Santamarina-Fojo S. Brewer H.B. Circulation. 2001; 104: 78Google Scholar). Oxysterol-binding protein-related protein ORP1L, also involved in sterol metabolism, resides in late endosomes and plays a role in macrophage late endosome membrane dynamics (16Johansson M. Bocher V. Lehto M. Chinetti G. Kuismanen E. Ehnholm C. Staels B. Olkkonen V.M. Mol. Biol. Cell. 2003; 14: 903-915Crossref PubMed Scopus (90) Google Scholar). To date, the cellular site(s) of function of the human ABCA1 transporter remain(s) to be determined. Our recent studies have established that ABCA1 resides on the plasma membrane as well as in endocytic vesicles that can shuttle between late endocytic compartments and the cell surface (17Neufeld E.B. Remaley A.T. Demosky Jr., S.J. Stonik J.A. Cooney A.M. Comly M. Dwyer N.K. Zhang M. Blanchette-Mackie J. Santamarina-Fojo S. Brewer Jr., H.B. J. Biol. Chem. 2001; 276: 27584-27590Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 18Neufeld E.B. Demosky Jr., S.J. Stonik J.A. Combs C. Remaley A.T. Duverger N. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2002; 297: 974-979Crossref PubMed Scopus (69) Google Scholar). Based on these findings, we proposed that endosomal ABCA1 might play a role in the apoA-I-mediated efflux of cellular lipids (17Neufeld E.B. Remaley A.T. Demosky Jr., S.J. Stonik J.A. Cooney A.M. Comly M. Dwyer N.K. Zhang M. Blanchette-Mackie J. Santamarina-Fojo S. Brewer Jr., H.B. J. Biol. Chem. 2001; 276: 27584-27590Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Several lines of research have provided evidence to support an endocytic pathway for the ABCA1-mediated cellular lipidation of apoA-I. Takahashi and Smith (19Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar) first provided evidence that cellular cholesterol efflux involves endocytosis and resecretion of apoA-I. More recently, Smith et al. (20Smith J.D. Waelde C. Horwitz A. Zheng P. J. Biol. Chem. 2002; 277: 17797-17803Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) have shown that apoA-I colocalizes with ABCA1-containing endosomes. Additional support for our conceptualization has been provided by recent studies that apoA-I-mediated lipid efflux is defective in the lysosomal storage diseases NP-C (21Chen W. Sun Y. Welch C. Gorelik A. Leventhal A.R. Tabas I. Tall A.R. J. Biol. Chem. 2001; 276: 43564-43569Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and Niemann-Pick type B disease (22Leventhal A.R. Chen W. Tall A.R. Tabas I. J. Biol. Chem. 2001; 276: 44976-44983Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In the present study, we examined the functionality of ABCA1 residing in endocytic vesicles in human Tangier disease fibroblasts that lack a functional ABCA1 transporter. These studies revealed defective lipid and protein trafficking in late endocytic vesicles in Tangier disease fibroblasts that can be corrected by adenovirally mediated expression of GFP-tagged ABCA1. Our present findings suggest that ABCA1 in late endocytic vesicles (late endosomes and lysosomes) can mobilize late endocytic lipids for apoA-I-mediated cellular efflux. The ABCA1 transporter residing in late endocytic vesicles appears to convert pools of late endocytic lipids, which would otherwise associate with NPC1, to pools that can associate with apoA-I to form the nascent HDL particle. Materials—α-Minimal essential medium (AMEM) was purchased from BIOSOURCE (Rockville, MD). Fetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. (Logan, UT). Lipoprotein-deficient bovine serum (LPDS) was prepared by Intracel Corp. (Rockville, MD). Glass microscope culture wells (Lab-Tek) were purchased from Thomas Scientific. Filipin was purchased from Polysciences (Warrington, PA). U18666A (3-β-(2-(diethylamino))ethoxy)androst-5-en-17-one), generously supplied by Dr. W. Andrus, The Upjohn Co., was stored as a 10 mg/ml stock solution in ethanol at -20 °C. Mouse anti-human LAMP2 antibodies, developed by Dr. J. T. August, were obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa (Iowa City, IA). Alexa-labeled secondary anti-human IgG antibodies, Alexa-568, and BODIPY-sphingomyelin were obtained from Molecular Probes (Eugene, OR). Tissue Culture—Wild type and Tangier disease fibroblasts were derived from volunteers and confirmed patients of the Molecular Disease Branch under the guidelines approved by the NHLBI Intramural Review Board, National Institutes of Health. The diagnosis of Tangier disease was based on clinical presentation (<5 mg/dl HDL cholesterol) and confirmed by biochemical assay (cholesterol efflux from skin fibroblasts <5% of wild type controls) (23Hovingh G.K. van Wijland M.J.A. Brownlie A. Bisoendial R.J. Hayden M.R. Kastelein J.J.P. Groen A.K. J. Lipid Res. 2003; 44: 1251-1255Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Four different wild type cell lines and five Tangier disease cell lines, including the proband (24Remaley A.T. Rust S. Rosier M. Knapper C. Naudin L. Broccardo C. Peterson K.M. Koch C. Arnould I. Prades C. Duverger N. Funke H. Assman G. Dinger M. Dean M. Chimini G. Santamarina-Fojo S. Fredrickson D.S. Denefle P. Brewer Jr., H.B. PNAS. 1999; 96: 12685-12690Crossref PubMed Scopus (230) Google Scholar), were used. All five Tangier disease fibroblasts presented similar cellular phenotypes. Fibroblasts were cultured in AMEM supplemented with 10% FBS, 2 mm glutamine, and 100 units of penicillin/streptomycin/ml in humidified 95% air and 5% CO2 at 37 °C. For biochemical analyses, fibroblasts seeded at a density of 5 × 104 cells/well in plastic 24-well dishes (Costar, Cambridge, MA) were incubated for 5-7 days in AMEM medium as above. For immunocytochemical analyses, fibroblasts were seeded at a density of 20,000 cells/well in AMEM, 5% LPDS medium in 9.5-cm glass microscope wells (Nunc, Inc., Naperville, IL). Some wild type and Tangier disease fibroblasts were transiently infected with adenovirus (Adv-ABCA1-GFP) containing the expression plasmid pTRE2 (Clontech, Palo Alto, CA), encoding a chimeric ABCA1-GFP protein (pTRE2-ABCA1-GFP) (18Neufeld E.B. Demosky Jr., S.J. Stonik J.A. Combs C. Remaley A.T. Duverger N. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2002; 297: 974-979Crossref PubMed Scopus (69) Google Scholar). Isolation and Fluorescent Labeling of Apolipoprotein—Lipoproteins were isolated by sequential ultracentrifugation as described previously (25Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar). ApoA-I purified from human plasma (26Brewer H.B. Ronan R. Meng M. Bishop C. Methods Enzymol. 1986; 128: 223-246Crossref PubMed Scopus (130) Google Scholar) was over 99% pure, as determined by SDS-PAGE and amino-terminal sequence analysis. The succinimidyl ester of Alexa 568 (Molecular Probes, Eugene, OR) was conjugated to apoA-I, according to the manufacturer's instructions. Alexa 568-labeled human apoA-I was purified by gel filtration and then spin-concentrated (Vivascience, Hannover, Germany) to 1 mg/ml in phosphate-buffered saline. Lipid Efflux Assay—Cholesterol efflux was performed as described previously (17Neufeld E.B. Remaley A.T. Demosky Jr., S.J. Stonik J.A. Cooney A.M. Comly M. Dwyer N.K. Zhang M. Blanchette-Mackie J. Santamarina-Fojo S. Brewer Jr., H.B. J. Biol. Chem. 2001; 276: 27584-27590Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Nearly confluent cells were labeled with [3H]cholesterol for 32 h, washed, infected with AdvABCA1-GFP for 1 h, and then incubated for 16 h in AMEM containing 1 mg/ml BSA (AMEM/BSA) in the presence or absence of 10 μg/ml apoA-I or 10 μg/ml Alexa 568-apoA-I. Percentage efflux was calculated by subtracting the radioactive counts of blank media (AMEM/BSA) from the radioactive counts in the presence of apoA-I, and then dividing the result by the sum of the radioactive counts in the medium plus the cell fraction. Transport of Endocytosed Sphingomyelin—The transport of sphingomyelin along the endocytic pathway was assessed as described by Pagano and Chen (27Pagano R.E. Chen C.S. Ann. N. Y. Acad. Sci. 1998; 845: 152-160Crossref PubMed Scopus (70) Google Scholar). Briefly, wild type and Tangier disease fibroblasts maintained in lipoprotein-depleted serum, as described above, were incubated with 5 μm BODIPY-sphingomyelin in Ham's F-12 medium without serum for 30 min at 37 °C, washed, chased in Ham's F-12 medium without serum for 60 min at 37 °C, washed, and then incubated in 5% defatted BSA for 30 min at 37 °C. After washing, cells were fixed in 3% paraformaldehyde for 10 min, washed, and then imaged. Immunocytochemical Analyses—Cells in glass chamber slides were washed in phosphate-buffered saline and fixed in 3% paraformaldehyde for 30 min. Cells were immunolabeled, using an indirect procedure, in which all incubations were performed either in blocker solution containing filipin (0.05%) and goat IgG (2.5 mg/ml) or 10% FBS in phosphate-buffered saline containing saponin (0.2%). Primary antibodies used were raised against NPC1 (11Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar) and human LAMP2. Secondary Alexa 568-labeled antibodies were used at 1:100 dilution. Fluorescence was viewed with a ×40 (NA 1.3) oil immersion objective on a Zeiss 410 or 510 laser scanning confocal microscope, using a UV laser (Enterprise model Coherent, Inc.), an argon laser, and a HeNe laser, with excitation wavelengths of 364, 488, and 543 or 568 nm, for filipin, enhanced green fluorescent protein, and BODIPY, and Alexa 568 fluorescence, respectively. Time-lapse Confocal Fluorescence Microscopy—For live cell imaging, cells prepared in glass chamber slides were maintained at 37 °C in an enclosed chamber using a feedback heater-blower (World Precision Instruments, Sarasota, FL). Time-lapse images were obtained using a 60× (NA 1.4) oil objective on an Olympus IX-70 microscope equipped with a cooled charge-coupled device camera (Orca ER) and a spinning disc confocal head. The optical slice thickness was ∼1 μm. Monochromatic 488 and 568 nm excitation light for GFP and Alexa 568 was provided by argon laser excitation controlled with an acousto-optical tunable filter, and detected using 505-540- and 580-620-nm BP emission filters, respectively. A total of 60 GFP, or DiI, or Alexa 568 images were acquired at a rate of 1/s. For dual color imaging, GFP and Alexa 568 images were obtained sequentially at an acquisition rate determined by their respective intensities. Capture, animation, and export to QuickTime movie were performed using the Metamorph software (Universal Imaging, Downington, PA). Zoom, pseudocoloring, drawing, and text in QuickTime movies were added using Adobe AfterEffects software. Cholesterol Is Retained in Late Endocytic Vesicles in Tangier Disease Fibroblasts—We first examined the distribution of cellular cholesterol, revealed by cholesterol-specific cytochemical staining with filipin (11Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), in wild type and Tangier disease human skin fibroblasts (Fig. 1A). Under conditions of cholesterol loading, Tangier disease fibroblasts accumulated cholesterol in perinuclear vesicles (Fig. 1A, c), established previously to represent late endocytic compartments (11Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). To determine whether cholesterol in late endocytic vesicles can traffic to the surface, intracellular pools of cholesterol that traffic to the plasma membrane were depleted in living wild type and Tangier disease fibroblasts, using cyclodextrin as an extracellular acceptor (28Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). As shown in Fig. 1A, the excess cholesterol retained in perinuclear vesicles in Tangier disease fibroblasts could not be depleted by cyclodextrin (Fig. 1A, d). This finding suggests that trafficking of cholesterol from late endocytic vesicles to the cell surface is perturbed in Tangier disease cells. The Motility of Late Endocytic Vesicles in Tangier Disease Fibroblasts Is Impaired—We have shown above that Tangier disease fibroblasts accumulate cholesterol in late endocytic compartments and have impaired trafficking of cholesterol to the cell surface. Excess cholesterol in late endocytic vesicles has been reported to impair their movement from the perinuclear region toward the cell periphery (29Lebrand C. Corti M. Goodson H. Cosson P. Cavalli V. Mayran N. Faure J. Gruenberg J. EMBO J. 2002; 21: 1289-1300Crossref PubMed Scopus (278) Google Scholar). To determine whether late endosomes in Tangier disease fibroblasts exhibit impaired motility, living wild type and Tangier disease fibroblasts were incubated with DiI-LDL, in order to label late endocytic vesicles, and then monitored by time-lapse confocal fluorescence microscopy. DiI-LDL-labeled late endocytic vesicles in living Tangier disease fibroblasts remained clustered in the perinuclear region (Fig. 1B) and exhibited impaired movement toward the cell surface (Movie 1, see Supplemental Material). These findings suggest that the cellular cholesterol sequestered in Tangier disease fibroblast late endocytic vesicles impairs their movement toward the cell surface. Cholesterol Retained in Tangier Disease Late Endocytic Vesicles Is Detergent-resistant—We next assessed the ability of detergent to extract cholesterol from late endocytic compartments in lipoprotein-depleted wild type and Tangier disease fibroblasts (Fig. 1C). Cholesterol retained in cells after cold Triton X-100 extraction has been shown to be associated with membrane microdomains enriched with sphingomyelin and other sphingolipids (30Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar). As shown in Fig. 1C, the majority of the cholesterol that accumulates in late endocytic vesicles in Tangier disease fibroblasts is detergent-resistant and thus appears to be associated with sphingolipids. Sphingomyelin Trafficking Is Perturbed in Tangier Disease Late Endocytic Vesicles—We next examined whether sphingomyelin was also retained in late endocytic compartments in Tangier disease fibroblasts (Fig. 1D). BODIPY-sphingomyelin was incorporated into the plasma membrane of living, lipoprotein-depleted, wild type and Tangier disease fibroblasts, and then allowed to traffic to intracellular compartments. Fluorescent sphingolipids incorporated into the plasma membrane of wild type fibroblasts trafficked to the Golgi, as reported previously (27Pagano R.E. Chen C.S. Ann. N. Y. Acad. Sci. 1998; 845: 152-160Crossref PubMed Scopus (70) Google Scholar) (Fig. 1D). Tangier disease fibroblasts presented a distinctive phenotype. Plasma membrane-derived BODIPY-sphingomyelin was retained in endocytic vesicles as well as in a markedly hypertrophied Golgi (Fig. 1D). Schmitz and co-workers (31Robenek H. Schmitz G. Arterioscler. Thromb. 1991; 11: 1007-1020Crossref PubMed Scopus (46) Google Scholar) have reported previously that the Golgi is hypertrophied in Tangier disease fibroblasts. Cholesterol Retained in Tangier Disease Late Endocytic Vesicles Recruits NPC1—The sterol-sensing NPC1 protein resides in a distinctive subset of late endosomes (11Neufeld E.B. Wastney M. Patel S. Suresh S. Cooney A. Dwyer N.K. Roff C.F. Ohno K. Morris J.A. Carstea E.D. Incardona J.P. Strauss III, J.F. Vanier M.T. Patterson M.C. Brady R.O. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1999; 274: 9627-9635Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 32Zhang M. Dwyer N.K. Neufeld E.B. Love D.C. Cooney A. Comly M. Patel S. Watari H. Strauss III, J.F. Pentchev P.G. Hanover J.A. Blanchette-Mackie E.J. J. Biol. Chem. 2000; 276: 3417-3425Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) that plays a critical role in the relocation of endocytosed LDL-derived lysosomal cholesterol to the plasma membrane and the endoplasmic reticulum (13Patterson M.C. Vanier M.T. Suzuki K. Morris J.A. Carstea E.D. Neufeld E.B. Blanchette-Mackie E.J. Pentchev P.G. Scriver C.R Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001Google Scholar). To probe further the functionality of ABCA1 in late endocytic compartments, we examined the effect of lipoprotein depletion on the distribution of cholesterol and NPC1 protein in Tangier disease fibroblasts. Maintenance of wild type fibroblasts in lipoprotein-free medium depletes cholesterol and NPC1 from late endocytic vesicles (32Zhang M. Dwyer N.K. Neufeld E.B. Love D.C. Cooney A. Comly M. Patel S. Watari H. Strauss III, J.F. Pentchev P.G. Hanover J.A. Blanchette-Mackie E.J. J. Biol. Chem. 2000; 276: 3417-3425Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), as shown in Fig. 2. LDL uptake in lipoprotein-depleted wild type fibroblasts enriches the membranes of late endocytic vesicles with cholesterol, which then retains NPC1 (32Zhang M. Dwyer N.K. Neufeld E.B. Love D.C. Cooney A. Comly M. Patel S. Watari H. Strauss III, J.F. Pentchev P.G. Hanover J.A. Blanchette-Mackie E.J. J. Biol. Chem. 2000; 276: 3417-3425Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), as shown in Fig. 2. The excess cholesterol that accumulates in Tangier disease late endocytic vesicles (Fig. 1) might be expected to retain excess NPC1. As shown in Fig. 2, cholesterol retained in late endocytic vesicles of lipoprotein-depleted Tangier disease fibroblasts did indeed retain NPC1. Most interesting, the structure of late endocytic vesicles in ABCA1-mutant Tangier disease fibroblasts is abnormal (Fig. 3). Late endocytic vesicles in Tangier disease fibroblasts often appear to be twisted, interconnected, and often dilated tubular structures.Fig. 3ApoA-I reduces NPC1 in wild type but not in Tangier disease late endocytic compartments. Wild type (A-F) and Tangier disease (G-L) fibroblasts were maintained in LPDS medium for 4-5 days and then incubated for an additional 24 h in LPDS medium containing 50 μg/ml LDL for 24 h. Cells were then incubated in medium containing 0.1% BSA in the absence (A-C and G-I) or presence (D-F and J-L) of 10 μg/ml apoA-I for 24 h, and then fixed and stained with filipin to reveal the cellular distribution of cholesterol (C, F, I, and L) and immunostained for NPC1 (red, A, D, G, and J) and LAMP2 (green, B, E, H, and K) as described under "Experimental Procedures." Note the reduction in late endosomal NPC1 in the presence of apoA-I in wild type (D versus A) but not in Tangier disease (J versus G) fibroblasts, as well as the massive accumulation of NPC1 (G and J) and cholesterol (I and L) in Tangier disease late endocytic vesicles (H and K). The tubulated late endocytic vesicles seen in Tangier disease fibroblasts (J-L, arrowheads) are shown enlarged in (J′-L′). Note that the large, late endocytic vesicle, whose lumen is indicated by the asterisk, is marked by LAMP2 (K′) and NPC1 (J′) and is cholesterol-enriched on its surface membrane (L′). The twisted late endocytic tubule (K′) indicated by the small arrowhead also contains NPC1 (J′) and cholesterol (L′).View Large Image Figure ViewerDownload Hi-res image Download (PPT) ApoA-I-mediated Mobilization of Late Endocytic Cholesterol Is Defective in Tangier Disease Fibroblasts—To explore further the potential role of the ABCA1 transporter in the efflux of cholesterol from late endocytic vesicles, w
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