Lysobisphosphatidic Acid Controls Endosomal Cholesterol Levels
2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês
10.1074/jbc.m801463200
ISSN1083-351X
AutoresJulien Chevallier, Zeina Chamoun, Guowei Jiang, Glenn D. Prestwich, Naomi Sakai, Stefan Matile, Robert G. Parton, Jean Grüenberg,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoMost cell types acquire cholesterol by endocytosis of circulating low density lipoprotein, but little is known about the mechanisms of intra-endosomal cholesterol transport and about the primary cause of its aberrant accumulation in the cholesterol storage disorder Niemann-Pick type C (NPC). Here we report that lysobisphosphatidic acid (LBPA), an unconventional phospholipid that is only detected in late endosomes, regulates endosomal cholesterol levels under the control of Alix/AlP1, which is an LBPA-interacting protein involved in sorting into multivesicular endosomes. We find that Alix down-expression decreases both LBPA levels and the lumenal vesicle content of late endosomes. Cellular cholesterol levels are also decreased, presumably because the storage capacity of endosomes is affected and thus cholesterol clearance accelerated. Both lumenal membranes and cholesterol can be restored in Alix knockdown cells by exogenously added LBPA. Conversely, we also find that LBPA becomes limiting upon pathological cholesterol accumulation in NPC cells, because the addition of exogenous LBPA, but not of LBPA isoforms or analogues, partially reverts the NPC phenotype. We conclude that LBPA controls the cholesterol capacity of endosomes. Most cell types acquire cholesterol by endocytosis of circulating low density lipoprotein, but little is known about the mechanisms of intra-endosomal cholesterol transport and about the primary cause of its aberrant accumulation in the cholesterol storage disorder Niemann-Pick type C (NPC). Here we report that lysobisphosphatidic acid (LBPA), an unconventional phospholipid that is only detected in late endosomes, regulates endosomal cholesterol levels under the control of Alix/AlP1, which is an LBPA-interacting protein involved in sorting into multivesicular endosomes. We find that Alix down-expression decreases both LBPA levels and the lumenal vesicle content of late endosomes. Cellular cholesterol levels are also decreased, presumably because the storage capacity of endosomes is affected and thus cholesterol clearance accelerated. Both lumenal membranes and cholesterol can be restored in Alix knockdown cells by exogenously added LBPA. Conversely, we also find that LBPA becomes limiting upon pathological cholesterol accumulation in NPC cells, because the addition of exogenous LBPA, but not of LBPA isoforms or analogues, partially reverts the NPC phenotype. We conclude that LBPA controls the cholesterol capacity of endosomes. Endocytosed proteins and lipids destined for degradation, such as down-regulated signaling receptors and LDL, 4The abbreviations used are: LDL, low density lipoprotein; NPC, Niemann-Pick type C; LBPA, lysobisphosphatidic acid; BHK, baby hamster kidney; siRNA, short interfering RNA; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolylphosphatidylethanolamine; VSV, vesicular stomatitis virus. are sequentially transported to early and late endosomes, and eventually to lysosomes. A hallmark of endosomes along this degradation pathway is their multivesicular appearance due to the accumulation of intralumenal vesicles. These incorporate down-regulated receptors (1Hurley J.H. Emr S.D. Annu. Rev. Biophys. Biomol. Struct. 2006; 35: 277-298Crossref PubMed Scopus (454) Google Scholar, 2Gruenberg J. Stenmark H. Nat. Rev. Mol. Cell Biol. 2004; 5: 317-323Crossref PubMed Scopus (588) Google Scholar) and cholesterol (3Mobius W. van Donselaar E. Ohno-Iwashita Y. Shimada Y. Heijnen H.F. Slot J.W. Geuze H.J. Traffic. 2003; 4: 222-231Crossref PubMed Scopus (351) Google Scholar, 4Kolter T. Sandhoff K. Annu. Rev. Cell Dev. Biol. 2005; 21: 81-103Crossref PubMed Scopus (353) Google Scholar) and also contain large amounts of LBPA (or bis(monoacylglycero)phosphate), which is only detected in late endosomes (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar). In addition to their presence in intralumenal vesicles, LBPA and cholesterol have been linked functionally. Both are affected by sterol carrier protein-2 expression (6Gallegos A.M. Atshaves B.P. Storey S. Schoer J. Kier A.B. Schroeder F. Chem. Phys. Lipids. 2002; 116: 19-38Crossref PubMed Scopus (11) Google Scholar). Moreover, LBPA plays a role in cholesterol transport (7Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (251) Google Scholar). Some relationship between cholesterol and LBPA has also been revealed in the cholesterol storage disorder Niemann-Pick type C caused by mutations in NPC1 or NPC2, whose precise functions are not known (8Ikonen E. Holtta-Vuori M. Semin. Cell Dev. Biol. 2004; 15: 445-454Crossref PubMed Scopus (85) Google Scholar, 9Sturley S.L. Patterson M.C. Balch W. Liscum L. Biochim. Biophys. Acta. 2004; 1685: 83-87Crossref PubMed Scopus (131) Google Scholar, 10Futerman A.H. van Meer G. Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (637) Google Scholar). In tissues from NPC patients, cholesterol accumulation is paralleled by the accumulation of LBPA and other lipids, in particular sphingolipids (8Ikonen E. Holtta-Vuori M. Semin. Cell Dev. Biol. 2004; 15: 445-454Crossref PubMed Scopus (85) Google Scholar, 10Futerman A.H. van Meer G. Nat. Rev. Mol. Cell Biol. 2004; 5: 554-565Crossref PubMed Scopus (637) Google Scholar, 11Vanier M.T. Biochim. Biophys. Acta. 1983; 750: 178-184Crossref PubMed Scopus (153) Google Scholar, 12Vanier M.T. Millat G. Clin. Genet. 2003; 64: 269-281Crossref PubMed Scopus (497) Google Scholar). We previously found that LBPA functions may be controlled by Alix, which binds LBPA-containing bilayers (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar). Interestingly, Alix is involved in sorting into multivesicular endosomes (14Odorizzi G. J. Cell Sci. 2006; 119: 3025-3032Crossref PubMed Scopus (146) Google Scholar, 15Dikic I. BioEssays. 2004; 26: 604-607Crossref PubMed Scopus (16) Google Scholar). Moreover, both Alix and LBPA also play a role in intralumenal vesicle fission from and fusion with the endosome limiting membrane (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar, 16Abrami L. Lindsay M. Parton R.G. Leppla S.H. van der Goot F.G. J. Cell Biol. 2004; 166: 645-651Crossref PubMed Scopus (174) Google Scholar, 17Le Blanc I. Luyet P.-P. Pons V. Ferguson C. Emans N. Petiot A. Mayran N. Demaurex N. Fauré J. Sadoul R. Parton R.G. Gruenberg J. Nat. Cell Biol. 2005; 7: 653-664Crossref PubMed Scopus (262) Google Scholar). Here we report that Alix down-expression decreases both the lumenal membrane content of late endosomes and their LBPA level, whereas the cellular cholesterol level is reduced, presumably because the endosomal storage capacity is affected. Indeed, the addition of exogenous LBPA to Alix knockdown cells restores both the intralumenal membrane content of late endosomes and the cholesterol level. Conversely, we find that LBPA becomes limiting upon pathological cholesterol accumulation in NPC cells, because the addition of exogenous LBPA, but not of LBPA isoforms or analogues, reverts the NPC phenotype. We conclude that LBPA controls the cholesterol storage capacity of endosomes. Cells and Reagents—Baby hamster kidney cells (BHK-21), HeLa cells, and NPC skin fibroblasts were maintained as described previously (7Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (251) Google Scholar), as was the production and purification of vesicular stomatitis virus (VSV Indiana serotype) (18Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (455) Google Scholar). We previously described the synthesis of enantiopure 2,2′- and 3,3′-dioleoyl-LBPA (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar, 19Chevallier J. Sakai N. Robert F. Kobayashi T. Gruenberg J. Matile S. Org. Lett. 2000; 2: 1859-1861Crossref PubMed Scopus (46) Google Scholar), which can now be obtained from Echelon Biosciences Inc. (Salt Lake City, UT), and of (R,R)- and (S,S)-bisether analogues of LBPA (20Jiang G. Xu Y. Falguieres T. Gruenberg J. Prestwich G.D. Org. Lett. 2005; 7: 3837-3840Crossref PubMed Scopus (16) Google Scholar). Other lipids were purchased from Avanti Polar Lipids, Inc. We also previously described the 6C4 monoclonal antibody against LBPA (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar). The monoclonal antibodies against a cytoplasmic (P5D4) (21Kreis T.E. EMBO J. 1986; 5: 931-941Crossref PubMed Scopus (283) Google Scholar) or exoplasmic (17.2.21.4) (22Gruenberg J. Howell K.E. Eur. J. Cell Biol. 1985; 38: 312-321PubMed Google Scholar) VSV G-protein epitope were described, as well as the polyclonal antibody against G-protein (22Gruenberg J. Howell K.E. Eur. J. Cell Biol. 1985; 38: 312-321PubMed Google Scholar). Anti-apolipoprotein A2 was purchased from Biodesign International (Saco, ME). Reagents were obtained from the following sources: anti-mouse Dynabeads from Dynal (Oslo, Norway); n-octylpolyoxyethylene from Bachem (Bubendorf, Switzerland); silencer siRNA labeling kit Cy3 from Ambion, Inc. (Huntingdon, UK); fluorescently labeled secondary antibodies from Jackson ImmunoResearch; R18 from Molecular Probes (Eugene, OR); TRIzol from Invitrogen; and U18666A (3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one) from Biomol (Plymouth Meeting, PA). Analysis of the Genomic Sequence of ALIX siRNA Target in Hamster Cells—RNA from BHK or HeLa cells was extracted with TRIzol according to the manufacturer's instructions. Then 0.5 μg of total RNA was used for reverse transcription with Superscript™RT (Invitrogen) using random hexamer. The transcribed DNA was subjected to TaqMan reverse transcription-PCR using two primers (forward primer, 5′ GGT GCA GCT GAA GAA GAC CT 3′; reverse primer, 5′ CAG GTT CTG CTC TGC AAT 3′). For TaqMan real time PCR, we used the ICycler.IQ™ (Bio-Rad). Specific PCR bands were excised and purified. The DNA was cloned into a TOPO vector, according to the manufacturer's instructions (Invitrogen). TOP10 bacteria were then transformed and grown; DNA was isolated and then sequenced for analysis by Fasteris SA (Geneva, Switzerland). Microscopy—The analysis of cholesterol distribution after filipin staining by fluorescence microscopy was described, as was the analysis of LBPA (7Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (251) Google Scholar) or other antigens (17Le Blanc I. Luyet P.-P. Pons V. Ferguson C. Emans N. Petiot A. Mayran N. Demaurex N. Fauré J. Sadoul R. Parton R.G. Gruenberg J. Nat. Cell Biol. 2005; 7: 653-664Crossref PubMed Scopus (262) Google Scholar) by immunofluorescence microscopy. To visualize the content of late endosomes by electron microscopy, HRP was endocytosed for 15 min at 37 °C and then chased for 30 min in marker-free medium (18Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (455) Google Scholar). Cells were then fixed, and HRP was revealed cytochemically with 3,3′-diaminobenzidine as substrate and processed for plastic embedding (23Parton R.G. Schrotz P. Bucci C. Gruenberg J. J. Cell Sci. 1992; 103: 335-348Crossref PubMed Google Scholar). Micrographs were taken at random across the monolayer, and then sets of micrographs were analyzed in a blind fashion by an independent observer. Proteoliposomes Containing VSV-G Protein and LBPA—The major LBPA (>90%) isoform in BHK is 2,2′-dioleoyl-LBPA (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar, 19Chevallier J. Sakai N. Robert F. Kobayashi T. Gruenberg J. Matile S. Org. Lett. 2000; 2: 1859-1861Crossref PubMed Scopus (46) Google Scholar, 24Kobayashi T. Beuchat M.H. Chevallier J. Makino A. Mayran N. Escola J.M. Lebrand C. Cosson P. Gruenberg J. J. Biol. Chem. 2002; 277: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). This isoform was synthesized (19Chevallier J. Sakai N. Robert F. Kobayashi T. Gruenberg J. Matile S. Org. Lett. 2000; 2: 1859-1861Crossref PubMed Scopus (46) Google Scholar) and used to prepare liposomes by reverse phase evaporation with a phospholipid composition (DOPC/DOPE/LBPA, 60:20:20 mol) similar to that of late endosomes, where LBPA normally resides (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar). Because living cells do not take up liposomes efficiently, the G-glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the liposomes, to increase cell surface binding and targeting to the endocytic pathway. Thus, liposome delivery was independent of the lipid composition. To prepare the G-protein, purified VSV (800 μg) was sedimented for 2 h at 4 °C in a TLA100.3 centrifuge at 90,000 rpm. The supernatant was carefully discarded. The white pellet was resuspended in 0.6 ml of 0.1% n-octylpolyoxyethylene in HBS (20 mm Hepes, pH 7.4, 150 mm NaCl), by 6-8 passages through a yellow tip until the solution became clear. Then the tubes were gently mixed at room temperature on a rotary wheel (4 rpm) (in 0.6 ml) for 1 h to facilitate detergent extraction of the G-protein. The mixture was then centrifuged at 4 °C for 1 h at 50,000 rpm (TLA 100.3) to separate soluble and nonsoluble materials. The supernatants were collected, dialyzed six times against 250 ml of HBS at 4 °C to remove the detergent, and used to prepare proteoliposomes or aliquoted, flash-frozen in liquid N2, and stored at -90 °C. Lipsomes were prepared by reverse phase evaporation (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar), after dissolving lipids in CHCl3, and mixed at the following molar ratios: DOPC/DOPE (60:40 mol); DOPC/DOPE/cholesterol (60:20:20 mol); DOPC/DOPE/LBPA (60:20:20 mol); and DOPC/GM3 (70:30 mol). Lipids (total ≈10 mg) were then dried under reduced pressure and dissolved in 1.5 ml of water-saturated diethyl ether and 0.5 ml of HBS (aqueous phase) containing ≈50 μg of G-glycoprotein (equivalent to ≈200 μg of VSV with G = 26% of VSV mass). In some experiments, 10 μm R18, a lipophylic fluorescent dye, was added. The mixture was vortexed and then sonicated continuously for 3 min on ice with a probe-type sonicator (Sonifier 250, Branson) set on power 3. Diethyl ether was then evaporated slowly at 600 mbar for 30-60 min under nitrogen flux on a rotatory evaporator, until lipids become oily. Then 1 ml of HBS (containing the fluorescent dye, if necessary) was added, and the solution was evaporated twice for 30 min on a rotatory evaporator at a 500-mbar pressure and then at 300 mbar until no smell of ether was detected, indicating that full reversion had occurred. When needed, we quantified R18 (excitation 560 nm; emission 585 nm) incorporation into liposomes with a SPECTRAmax GEMINI XS spectrophotometer and SOFTmax Pro (Molecular Devices), as described previously (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar). When living cells (grown in 6-cm diameter Petri dishes) were treated with liposomes, the growth medium was replaced by 3 ml of Glascow minimum essential medium containing 10 mm Hepes, pH 7.4, and 0.2% bovine serum albumin, which was then complemented with 100 μl of the desired liposome preparation (0.66 mg of total lipid). The same operation was then repeated every 2 h, until the end of the incubation (after 8 h). To quantify the incorporation of VSV-G and to determine its trans-bilayer orientation, proteoliposomes (prepared by reverse phase evaporation as above) were immunopurified using Dynabeads coated with antibodies against an exoplasmic (17.2.21.4) or cytoplasmic (P5D4) epitope of the G-protein (18Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (455) Google Scholar). Similarly, we analyzed the antigenicity of LBPA incorporated into liposomes by incubating 50 μl of the liposome preparation with 15 μl of anti-LBPA antibody in 135 μl of HBS for 3 h at 4 °C. Then the antibody bound to liposomes was separated from free antibody by floatation in a step sucrose gradient (17Le Blanc I. Luyet P.-P. Pons V. Ferguson C. Emans N. Petiot A. Mayran N. Demaurex N. Fauré J. Sadoul R. Parton R.G. Gruenberg J. Nat. Cell Biol. 2005; 7: 653-664Crossref PubMed Scopus (262) Google Scholar). In each case, samples were analyzed by SDS-gel electrophoresis under nonreducing conditions, followed by Western blotting using a polyclonal antibody against VSV-G to detect the G-protein or anti-mouse antibodies to detect the anti-LBPA antibody. To determine the pH-dependent fusion properties of liposomes, we used the following: 1) as acceptor, DOPC/DOPE/LBPA (60:20:20) liposomes containing the G-protein; 2) as donor containing R18, liposomes were passed through a PD-10 Sephadex G-25 column (Amersham Biosciences) to remove the nonencapsulated dye R18. Then 100 μl of donor R18-liposomes were mixed at 4 °C with 50 μl of acceptor liposomes and 50 μl of HBS (neutral conditions) or 50 μl of Tris maleic acid, pH 5.6, in 150 mm NaCl (acidic conditions). The temperature was raised to 22 °C, and the emitted fluorescence (excitation, 560; emission 585) was measured over 90 min with a Photon Technology International fluorimeter (710 Photomultiplier system detection and LPS 220 lamp). Other Methods—SDS-gel electrophoresis (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and the quantification of LBPA by ELISA (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar) and cholesterol (26Gamble W. Vaughan M. Kruth H.S. Avigan J. J. Lipid Res. 1978; 19: 1068-1070Abstract Full Text PDF PubMed Google Scholar) were described. Down-regulation of Alix expression in BHK cells with siRNAs was as described in HeLa cells (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar) except that cells were split (1/20) the day before the experiment and then again (biochemistry, 1/4; immunofluorescence, 1/20) 48 h after siRNA addition. Gene expression was quantified from HeLa cells submitted or not to Alix siRNAs. For each condition, RNA was purified from two independent 10-cm dishes using the RNeasy minikit (Qiagen). Reverse transcription-PCR was performed using the Superscript II kit (Invitrogen) and oligo(dT) primer (Promega). The following amplification was made on an i-cycler (Bio-Rad) with the Quantitect SYBR Green PCR kit (Qiagen). The Quantitect Primers (Qiagen) for the following genes were used: SREBPF2, DHC7R, ACTB, HMGCR, SDHA, LDLR, and PDCD6IP (Alix). Formation of Intralumenal Membranes—To investigate LBPA functions in cholesterol storage and transport, we silenced Alix expression in BHK cells (supplemental Fig. S1A), because we had characterized LBPA in these cells. We had determined the atomic composition of the BHK major (>90%) LBPA isoform as 2,2′-dioleoyl-LBPA (supplemental Fig. S1B). We had also characterized the subcellular distribution and late endosomal content of LBPA (15 mol % of total phospholipids) in BHK cells (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar, 19Chevallier J. Sakai N. Robert F. Kobayashi T. Gruenberg J. Matile S. Org. Lett. 2000; 2: 1859-1861Crossref PubMed Scopus (46) Google Scholar, 24Kobayashi T. Beuchat M.H. Chevallier J. Makino A. Mayran N. Escola J.M. Lebrand C. Cosson P. Gruenberg J. J. Biol. Chem. 2002; 277: 32157-32164Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar) (see Fig. 1C). Upon Alix knockdown (Fig. 1C, inset), the LBPA staining intensity was decreased (Fig. 1A), as observed by others (27Cabezas A. Bache K.G. Brech A. Stenmark H. J. Cell Sci. 2005; 118: 2625-2635Crossref PubMed Scopus (93) Google Scholar) and us (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar) in human cells, as was the LBPA content of late endosomal fractions (Fig. 1C). Because LBPA is a major phospholipid of late endosome lumenal membranes (5Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (658) Google Scholar) and because it is decreased by Alix knockdown, we wondered whether the ultrastructure of late endosomes was affected in Alix knockdown cells. To this end, the lumen of late endosomes was labeled with endocytosed HRP pulsed for 15 min and then chased for 30 min at 37 °C. Cells were then processed for HRP cytochemistry and electron microscopy, and micrographs taken at random were analyzed in a blind fashion. In control, mock-treated cells, HRP-labeled late endosomes contained numerous lumenal inclusions and vesicles, exhibiting the characteristic multivesicular ultrastructure of this compartment in BHK cells (18Gruenberg J. Griffiths G. Howell K.E. J. Cell Biol. 1989; 108: 1301-1316Crossref PubMed Scopus (455) Google Scholar) (Fig. 1D). By contrast, in Alix knockdown cells, HRP-positive endosomes appeared like empty vacuoles with a few internal vesicles (Fig. 1E), consistent with observations that LBPA levels are then reduced (Fig. 1, A-C). These data agree well with our previous findings that Alix knockdown in HeLa cells reduces LBPA levels and the number of multilamellar late endosomes that are typical for these cells (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar). Alix and Cholesterol—We then investigated whether cholesterol was affected in Alix knockdown cells, because LBPA and cholesterol are linked functionally (6Gallegos A.M. Atshaves B.P. Storey S. Schoer J. Kier A.B. Schroeder F. Chem. Phys. Lipids. 2002; 116: 19-38Crossref PubMed Scopus (11) Google Scholar, 8Ikonen E. Holtta-Vuori M. Semin. Cell Dev. Biol. 2004; 15: 445-454Crossref PubMed Scopus (85) Google Scholar, 12Vanier M.T. Millat G. Clin. Genet. 2003; 64: 269-281Crossref PubMed Scopus (497) Google Scholar) and because LBPA and cholesterol are both present in intralumenal endosomal vesicles, although perhaps in different subpopulations (3Mobius W. van Donselaar E. Ohno-Iwashita Y. Shimada Y. Heijnen H.F. Slot J.W. Geuze H.J. Traffic. 2003; 4: 222-231Crossref PubMed Scopus (351) Google Scholar, 28Sobo K. Chevallier J. Parton R.G. Gruenberg J. van der Goot F.G. PLoS ONE. 2007; 2: e391Crossref PubMed Scopus (69) Google Scholar). Upon filipin staining of mock-treated control cells, cholesterol was mostly present at the plasma membrane by light microscopy but was also faintly detected in intracellular structures, presumably trans-Golgi network and endosomes (Fig. 1A), as expected (8Ikonen E. Holtta-Vuori M. Semin. Cell Dev. Biol. 2004; 15: 445-454Crossref PubMed Scopus (85) Google Scholar, 29Maxfield F.R. Tabas I. Nature. 2005; 438: 612-621Crossref PubMed Scopus (990) Google Scholar). Strikingly, the overall cholesterol staining intensity decreased in Alix knockdown cells concomitantly with decreased LBPA staining (Fig. 1A), and biochemical quantification showed that, much like LBPA, the total cellular cholesterol was then reduced by ≈30% (Fig. 1B). We could rule out the possibility that decreased cholesterol levels were because of an inhibition of LDL endocytosis, because Alix knockdown does not affect endocytosis nor transport along the endocytic pathway toward late endosomes and lysosomes or lysosomal degradation (13Matsuo H. Chevallier J. Mayran N. Le Blanc I. Ferguson C. Faure J. Blanc N.S. Matile S. Dubochet J. Sadoul R. Parton R.G. Vilbois F. Gruenberg J. Science. 2004; 303: 531-534Crossref PubMed Scopus (536) Google Scholar, 16Abrami L. Lindsay M. Parton R.G. Leppla S.H. van der Goot F.G. J. Cell Biol. 2004; 166: 645-651Crossref PubMed Scopus (174) Google Scholar, 17Le Blanc I. Luyet P.-P. Pons V. Ferguson C. Emans N. Petiot A. Mayran N. Demaurex N. Fauré J. Sadoul R. Parton R.G. Gruenberg J. Nat. Cell Biol. 2005; 7: 653-664Crossref PubMed Scopus (262) Google Scholar, 27Cabezas A. Bache K.G. Brech A. Stenmark H. J. Cell Sci. 2005; 118: 2625-2635Crossref PubMed Scopus (93) Google Scholar, 30Schmidt M.H. Hoeller D. Yu J. Furnari F.B. Cavenee W.K. Dikic I. Bogler O. Mol. Cell. Biol. 2004; 24: 8981-8993Crossref PubMed Scopus (104) Google Scholar). Similarly, Alix knockdown did not seem to affect de novo cholesterol synthesis, because the expression of genes involved in cholesterol synthesis and accumulation was unchanged (supplemental Fig. S2). Alternatively, Alix down-expression, with a concomitant depletion of LBPA (Fig. 1, A-C) and loss of lumenal membranes (Fig. 1E), may affect the capacity of multivesicular endosomes to store or retain cholesterol. This may affect NPC1 functions in cholesterol transport, because NPC1 is normally present both in intralumenal vesicles and on the limiting membrane of multivesicular endosomes (31Frolov A. Srivastava K. Daphna-Iken D. Traub L.M. Schaffer J.E. Ory D.S. J. Biol. Chem. 2001; 276: 46414-46421Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In turn, this may alter cellular cholesterol levels, because endosomal and cellular cholesterol levels are interdependent (29Maxfield F.R. Tabas I. Nature. 2005; 438: 612-621Crossref PubMed Scopus (990) Google Scholar). Upon release from endosomes, perhaps via NPC1 and NPC2, as well as ABCA1 (32Neufeld E.B. Stonik J.A. Demosky Jr., S.J. Knapper C.L. Combs C.A. Cooney A. Comly M. Dwyer N. Blanchette-Mackie J. Remaley A.T. Santamarina-Fojo S. Brewer Jr., H.B. J. Biol. Chem. 2004; 279: 15571-15578Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) or other members of this protein superfamily (33Rajagopal A. Simon S.M. 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FASEB J. 2005; 11: 1558-1560Crossref Scopus (73) Google Scholar) can then occur. We could not measure cholesterol in the medium, because of the necessary presence of fetal calf serum in these RNA interference experiments. However, we hypothesized that in the absence of Alix and LBPA, cholesterol may be inefficiently retained in endosomes, and thus cellular clearance may be accelerated. Cholesterol Accumulation—To test whether Alix and LBPA may play a role in cholesterol clearance from endosomes, we made use of U18666A, a drug that mimics the NPC phenotype by causing acute accumulation of LDL-derived cholesterol in the late endosomes of cells with a nonpathological genetic background (7Kobayashi T. Beuchat M.H. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (251) Google Scholar, 38Liscum L. Faust J.R. J. Biol. Chem. 1989; 264: 11796-11806Abstract Full Text PDF PubMed Google Scholar) (Fig. 2, A and B). Interestingly, LBPA and cholesterol levels changed concomitantly in U18666A-treated cells. Like cholesterol, LBPA increased in U18666A-treated cells incubated in the presence of serum (Fig. 2B). Conversely, cholesterol did not accumulate in U18666A-treated cells incubated in serum-free medium (Fig. 2B), as expected (38Liscum L. Faust J.R. J. Biol. Chem. 1989; 264: 11796-11806Abstract Full Text PDF PubMed Google Scholar). LBPA similarly showed no increase in U18666A-treated cells incubated in serum-free medium (Fig. 2B), demonstrating that LBPA accumulation in U18666A-treated cells did not result from some effects of the drug on lipid neo-synthesis, and further confirm the notion that LB
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