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• W1984590580 abstract "We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. Biol. Chem. 275, 4013–4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5–10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum. We previously studied the early trafficking of low density lipoprotein (LDL)-derived cholesterol in mutant Chinese hamster ovary cells defective in Niemann-Pick type C1 (NPC1) using cyclodextrin (CD) to monitor the arrival of cholesterol from the cell interior to the plasma membrane (PM) (Cruz, J. C., Sugii, S., Yu, C., and Chang, T.-Y. (2000) J. Biol. Chem. 275, 4013–4021). We found that newly hydrolyzed cholesterol derived from LDL first appears in certain CD-accessible pool(s), which we assumed to be the PM, before accumulating in the late endosome/lysosome, where NPC1 resides. To determine the identity of the early CD-accessible pool(s), in this study, we performed additional experiments, including the use of revised CD incubation protocols. We found that prolonged incubation with CD (>30 min) caused cholesterol in internal membrane compartment(s) to redistribute to the PM, where it became accessible to CD. In contrast, a short incubation with CD (5–10 min) did not cause such an effect. We also show that one of the early compartments contains acid lipase (AL), the enzyme required for liberating cholesterol from cholesteryl ester in LDL. Biochemical and microscopic evidence indicates that most of the AL is present in endocytic compartment(s) distinct from the late endosome/lysosome. Our results suggest that cholesterol is liberated from LDL cholesteryl ester in the hydrolytic compartment containing AL and then moves to the NPC1-containing late endosome/lysosome before reaching the PM or the endoplasmic reticulum. In mammalian cells, low density lipoprotein (LDL) 1The abbreviations used are: LDL, low density lipoprotein; AL, acid lipase; PM, plasma membrane; NPC1, Niemann-Pick type C1; CL, cholesteryl linoleate; [3H]CL-LDL, [3H]cholesteryl linoleate-labeled low density lipoprotein; CHO, Chinese hamster ovary; CD, cyclodextrin; FBS, fetal bovine serum; DNP, dinitrophenyl; DAMP, 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine; V-ATPase, vacuolar ATPase; MPR, mannose 6-phosphate receptor; CI-MPR, cation-independent mannose 6-phosphate receptor; HDL, high density lipoprotein; Hf, human fibroblast; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GFP, green fluorescent protein; TGN, trans-Golgi network.1The abbreviations used are: LDL, low density lipoprotein; AL, acid lipase; PM, plasma membrane; NPC1, Niemann-Pick type C1; CL, cholesteryl linoleate; [3H]CL-LDL, [3H]cholesteryl linoleate-labeled low density lipoprotein; CHO, Chinese hamster ovary; CD, cyclodextrin; FBS, fetal bovine serum; DNP, dinitrophenyl; DAMP, 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine; V-ATPase, vacuolar ATPase; MPR, mannose 6-phosphate receptor; CI-MPR, cation-independent mannose 6-phosphate receptor; HDL, high density lipoprotein; Hf, human fibroblast; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GFP, green fluorescent protein; TGN, trans-Golgi network. binds to its receptor at the cell surface and is recruited into clathrin-coated endocytotic vesicles. After endocytosis, LDL enters the endosomal/lysosomal system, where cholesteryl ester, a major lipid found in LDL, is hydrolyzed by the enzyme acid lipase (AL) (1Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4350) Google Scholar). Mutations in AL cause cholesteryl ester to eventually accumulate in the lysosome (2Brown M.S. Sobhani M.K. Brunschede G.Y. Goldstein J.L. J. Biol. Chem. 1976; 251: 3277-3286Abstract Full Text PDF PubMed Google Scholar, 3Anderson R.A. Byrum R.S. Coates P.M. Sando G.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2718-2722Crossref PubMed Scopus (106) Google Scholar). After the hydrolytic action by AL, the transport of LDL-derived cholesterol from the endosome/lysosome to the plasma membrane (PM) or to the endoplasmic reticulum for re-esterification requires the protein named Niemann-Pick type C1 (NPC1). Mutations in NPC1 cause unesterified cholesterol and other lipids to accumulate in the late endosome and lysosome. Despite significant advances, the events that led to eventual accumulation of cholesterol in the late endosome/lysosome remain unclear. To delineate the early trafficking events of LDL-derived cholesterol, we previously performed pulse-chase experiments using [3H]cholesteryl linoleate-labeled LDL ([3H]CL-LDL) in Chinese hamster ovary (CHO) mutant cells defective in the npc1 locus, CT43, along with their parental cells, 25RA (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). To monitor the arrival of [3H]cholesterol at the PM, we utilized a cyclodextrin (CD)-based intact cell assay. CD is a water-soluble molecule that has a high affinity for cholesterol and has been widely used to monitor the arrival of cholesterol at the PM from the cell interior (5Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar, 6Neufeld 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, 7Lange Y. Ye J. Rigney M. Steck T. J. Biol. Chem. 2000; 275: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8Frolov 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 (41) Google Scholar). Our results show that [3H]cholesterol, newly released from the hydrolysis of [3H]CL-LDL, emerges in the early pool(s) in a manner unaffected by the npc1 mutation. Subsequently (within 2 h), in the parental cells, [3H]cholesterol is distributed to the PM and the endoplasmic reticulum. In CT43 cells, [3H]cholesterol accumulates in the characteristic aberrant endosome/lysosome. [3H]Cholesterol that is present in the early pool(s) is extractable by CD, whereas [3H]cholesterol that accumulates in the aberrant endosome/lysosome is resistant to extraction by CD. Based on this CD sensitivity test, the early pool(s) was assumed to be the PM (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). These results led us to hypothesize that, in NPC1 cells, cholesterol liberated from cholesteryl ester in LDL first moves to the PM independent of NPC1 and then moves back to the cell interior and accumulates in the aberrant late endosome/lysosome. Using a similar CD-based assay, other investigators independently reached the same conclusion (7Lange Y. Ye J. Rigney M. Steck T. J. Biol. Chem. 2000; 275: 17468-17475Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The original CD-based assay used by us and by others involved continuous incubation of cells with CD for 30 min or longer. Thus, it is possible that prolonged incubation of cells with CD may cause redistribution of cellular cholesterol, so cholesterol originally residing in internal membranes moves to the PM and becomes extractable by CD. Recently, Haynes et al. (9Haynes M.P. Phillips M.C. Rothblat G.H. Biochemistry. 2000; 39: 4508-4517Crossref PubMed Scopus (113) Google Scholar) showed that, in CHO cells, depending on the incubation time used (ranging from 30 s to 20 min), CD is capable of extracting cellular cholesterol from two or three kinetically distinct pools; rearrangement of cholesterol between these pools could occur under various treatments. In this work, we further investigated the early trafficking events of LDL-derived cholesterol. To follow the fate of newly hydrolyzed cholesterol more precisely, we redesigned the procedures for the pulse-chase experiment and the CD treatment. We also performed biochemical and immunofluorescence experiments to define the hydrolytic compartment(s) involved in producing LDL-derived cholesterol. A model, revised from the one previously proposed by this laboratory (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), describing the early itinerary of LDL-derived cholesterol in the context of the endocytic pathway is presented. Materials—Fetal bovine serum (FBS), protease inhibitor mixture, Nonidet P-40, 2-hydroxypropyl-β-cyclodextrin, monoclonal antibody against dinitrophenyl (DNP), paraformaldehyde, and human apoA-I were purchased from Sigma. The acyl-CoA:cholesterol O-acyltransferase inhibitor F12511 was a gift of Pierre Fabre Research (Castres, Cedex, France). Percoll and [1,2,6,7-3H]CL were from Amersham Biosciences. Optiprep (Nycomed) was from Axis-Shield. FuGENE 6 transfection reagent was from Roche Applied Science. The ProLong antifade kit, Alexa 488- or Alexa 568-conjugated goat anti-rabbit or anti-mouse IgG, LysoTracker Red (DND-99), 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), and Zenon rabbit IgG labeling kits were from Molecular Probes, Inc.. Monoclonal antibodies against EEA1, caveolin-1, and syntaxin-6 were from BD Biosciences. Monoclonal antibody against Na+/K+-ATPase was from Upstate Biotechnology, Inc. Monoclonal antibody against hamster LAMP2 (lysosomal-associated membrane protein-2) was from the Developmental Studies Hybridoma Bank maintained by the University of Iowa. Rabbit polyclonal antibodies against AL were produced as described (10Du H. Witte D.P. Grabowski G.A. J. Lipid Res. 1996; 37: 937-949Abstract Full Text PDF PubMed Google Scholar). Monoclonal antibody against vacuolar ATPase (V-ATPase) was a generous gift from Professor Satoshi Sato (Kyoto University, Kyoto, Japan); this antibody (OSW2) is directed against the 100–116-kDa subunit of the V0 domain of V-ATPase and has been shown to specifically recognize the vacuolar type proton pump (11Sato S.B. Toyama S. J. Cell Biol. 1994; 127: 39-53Crossref PubMed Scopus (24) Google Scholar). Rabbit polyclonal antibodies against the cation-independent mannose 6-phosphate receptor (CI-MPR) and against Rab9 were generous gifts from Professor Suzanne Pfeffer (Stanford University) (12Dintzis S.M. Velculescu V.E. Pfeffer S.R. J. Biol. Chem. 1994; 269: 12159-12166Abstract Full Text PDF PubMed Google Scholar). Delipidated FBS was prepared as described (13Chin J. Chang T.-Y. J. Biol. Chem. 1981; 256: 6304-6310Abstract Full Text PDF PubMed Google Scholar). LDL (density of 1.019–1.063 g/ml) was prepared from fresh human plasma by sequential flotation as previously described (14Cadigan K.M. Heider J.G. Chang T.-Y. J. Biol. Chem. 1988; 263: 274-282Abstract Full Text PDF PubMed Google Scholar). High density lipoprotein (HDL; density of 1.063–1.21 g/ml) was prepared by the same flotation method and purified by heparin affinity chromatography. Cell Lines and Cell Culture—25RA is a CHO cell line that is resistant to the cytotoxicity of 25-hydroxycholesterol (15Chang T.-Y. Limanek J.S. J. Biol. Chem. 1980; 255: 7787-7795Abstract Full Text PDF PubMed Google Scholar) and that contains a gain-of-function mutation in SCAP (SREBP cleavage-activating protein) (16Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). The CT43 mutant cell line was isolated as one of the cholesterol trafficking mutants from mutagenized 25RA cells (17Cadigan K.M. Spillane D.M. Chang T.-Y. J. Cell Biol. 1990; 110: 295-308Crossref PubMed Scopus (91) Google Scholar). It contains the same gain-of-function mutation in SCAP. In addition, it contains a premature translational termination mutation near the 3′-end of the npc1 coding sequence, producing a nonfunctional truncated NPC1 protein (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). CHO cells were maintained in medium A (Ham's F-12 medium plus 10% FBS and 10 μg/ml gentamycin) as monolayers at 37 °C with 5% CO2. When medium B (Ham's F-12 medium supplemented with 5% delipidated FBS plus 35 μm oleic acid and 10 μg/ml gentamycin) was used at lower temperatures (18 °C or lower), Ham's F-12 medium (titrated to pH 7.4 without sodium bicarbonate) was used, and cells were placed in a water bath without CO2. A human fibroblast (Hf) cell line derived from an NPC patient (No. 93.22) was the generous gift of Dr. Peter Pentchev (National Institutes of Health). Hf cell lines isolated from patients with Wolman's disease (GM00863A and GM01606A) and with mucolipidosis II (I-cell disease) (GM02013D) were from the NIGMS Human Genetic Cell Repository (Camden, NJ). Hepatocyte-like HepG2 and monocytic THP-1 cells were obtained from American Type Culture Collection (Manassas, VA). Hf and HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS and 100 units/ml penicillin/streptomycin at 37 °C with 10% CO2. THP-1 cells were maintained under the same conditions, except that RPMI 1640 medium was used instead. Prior to the experiment, THP-1 was treated with 100 nm phorbol 12-myristate 13-acetate and 100 nm 1α,25-dihydroxyvitamin D3 (both from Sigma) for at least 72 h to induce differentiation (18Maung K. Miyazaki A. Nomiyama H. Chang C.C. Chang T.-Y. Horiuchi S. J. Lipid Res. 2001; 42: 181-187Abstract Full Text Full Text PDF PubMed Google Scholar). LDL-derived Cholesterol Trafficking Assays—[3H]CL-LDL with specific radioactivity of ∼5 × 104 cpm/μg of protein was prepared as previously described (19Faust J.R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1977; 252: 4861-4871Abstract Full Text PDF PubMed Google Scholar). Cells were plated in 6- or 12-well dishes as previously described (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Cruz J.C. Chang T.-Y. J. Biol. Chem. 2000; 275: 41309-41316Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Prior to each experiment, the cells were cultured for 2 days in medium B (to deplete stored cholesterol within the cell). Cells were chilled on ice, labeled with 30 μg/ml [3H]CL-LDL in medium B for 5 h at 18 °C, and washed once with 1% bovine serum albumin-containing cold phosphate-buffered saline (PBS) at 4 °C and three more times with cold PBS. Cells were then fed cold medium B and placed in a water bath for the indicated chase times at 37 °C. During the chase period, rapid metabolism of [3H]CL-LDL occurs in a time-dependent manner. To obtain reproducible results, we found that it was essential to use healthy cells grown at late log phase for plating and to control the temperature and pH of the growth media in a precise manner. To control the temperature, we incubated tissue culture plates or dishes on platforms covered with water in a constant temperature water bath. To control the pH in a precise manner, we used media devoid of sodium bicarbonate and titrated to pH 7.4 within 1 week before usage. An acyl-CoA:cholesterol O-acyltransferase inhibitor (2 μm F12511) was included whenever cells were incubated at 37 °C. F12511 was previously shown to inhibit acyl-CoA:cholesterol O-acyltransferase activity at the submicromolar level (21Chang C.C. Sakashita N. Ornvold K. Lee O. Chang E.T. Dong R. Lin S. Lee C.Y. Strom S.C. Kashyap R. Fung J.J. Farese Jr., R.V. Patoiseau J.F. Delhon A. Chang T.-Y. J. Biol. Chem. 2000; 275: 28083-28092Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Labeled cellular lipids were extracted and analyzed by TLC as described (17Cadigan K.M. Spillane D.M. Chang T.-Y. J. Cell Biol. 1990; 110: 295-308Crossref PubMed Scopus (91) Google Scholar); the percent hydrolysis was calculated as [3H]cholesterol counts divided by the sum of [3H]CL and [3H]cholesterol counts. For cholesterol efflux experiments, cells were incubated with 4% 2-hydroxypropyl-β-cyclodextrin (CD) in medium B in the presence of the acyl-CoA:cholesterol O-acyltransferase inhibitor at 37 °C for the indicated times. The labeled lipids were extracted and analyzed as described (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 17Cadigan K.M. Spillane D.M. Chang T.-Y. J. Cell Biol. 1990; 110: 295-308Crossref PubMed Scopus (91) Google Scholar). The percent cholesterol efflux was calculated as [3H]cholesterol counts in the medium divided by the sum of [3H]CL counts in the cell and [3H]cholesterol counts in the cell and in the medium. Isolation of the PM—To isolate the PM from the cells, we employed the 30% Percoll gradient procedure essentially as described (20Cruz J.C. Chang T.-Y. J. Biol. Chem. 2000; 275: 41309-41316Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). All procedures were performed at 4 °C. Briefly, after the pulse-chase experiment, cells in two 150-mm dishes were collected. The cells were scraped in buffer containing 0.25 m sucrose, 1 mm EDTA, and 20 mm Tricine (pH 7.8) and broken with 15 strokes using a stainless steel tissue grinder (Dura-Grind, Wheaton). The post-nuclear supernatant was loaded onto a 30% Percoll gradient. After centrifugation at 84,000 × g for 30 min, 25 fractions were collected from the top. The PM fractions usually corresponded to fractions 9 and 10, as evidenced by a visible white membrane band; this band showed high enrichment in Na+/K+-ATPase and caveolin-1 protein (20Cruz J.C. Chang T.-Y. J. Biol. Chem. 2000; 275: 41309-41316Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In addition, we performed biotinylation of PM proteins in intact cells at 4 °C for 10 min using sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce), which showed that only fractions 9 and 10 were highly enriched in the biotinylated proteins (data not shown). The 3H-labeled lipids were extracted using chloroform/methanol and analyzed by TLC as previously described (20Cruz J.C. Chang T.-Y. J. Biol. Chem. 2000; 275: 41309-41316Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). 11% Percoll Gradient Analyses—All procedures were performed at 4 °C. The fractionation method was performed as described previously (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 22Spillane D.M. Reagan Jr., J.W. Kennedy N.J. Schneider D.L. Chang T.-Y. Biochim. Biophys. Acta. 1995; 1254: 283-294Crossref PubMed Scopus (20) Google Scholar). Briefly, after the pulse-chase experiment, cells from one 150-mm dish were scraped into homogenization buffer (0.25 m sucrose, 1 mm EDTA, and 20 mm Tris (pH 7.4)) and homogenized with 15 strokes using the same stainless steel tissue grinder described above. To minimize breakage of membrane vesicles, 250 mm sucrose was included in the buffer. To increase recovery, the pellet was resuspended in buffer and homogenized a second time. The combined post-nuclear supernatant from cells was loaded onto 11% Percoll and centrifuged at 20,000 × g for 40 min using a Beckman Model Ti-70.1 rotor. 10 fractions were collected from the top. >80% of the PM marker (Na+/K+-ATPase) was concentrated in fractions 1 and 2, whereas >80% of the late endosomal/lysosomal markers (LAMP1/LAMP2) were concentrated in fractions 9 and 10 as previously described (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The 3H-labeled lipids were extracted using chloroform/methanol and analyzed by TLC as previously described (20Cruz J.C. Chang T.-Y. J. Biol. Chem. 2000; 275: 41309-41316Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Optiprep Gradient Analyses—The procedure was based on a previously described method (23Sheff D.R. Daro E.A. Hull M. Mellman I. J. Cell Biol. 1999; 145: 123-139Crossref PubMed Scopus (382) Google Scholar) with modifications. Cells grown in one 150-mm dish were homogenized at 4 °C as described above. The post-nuclear supernatant (1 ml) was placed onto 9 ml of a linear 5–20% Optiprep gradient prepared in homogenization buffer at 4 °C. Gradients were centrifuged at 27,000 rpm for 20 h at 4 °C using a Beckman SW 41 rotor. 20 fractions (0.5 ml each) were carefully collected from the top. Immunoblot analyses were performed using antibodies against individual organelle markers as indicated. The 3H-labeled lipids were extracted using chloroform/methanol and analyzed by TLC as previously described (20Cruz J.C. Chang T.-Y. J. Biol. Chem. 2000; 275: 41309-41316Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Immunoblot and Spectrofluorometric Analyses of Percoll Fractions— For immunoblot analysis, each Percoll fraction was ultracentrifuged either at 100,000 × g for 90 min or at 150,000 × g for 30 min to remove the Percoll particles. Afterward, the samples (located on top of the Percoll particles) were carefully collected using Pasteur pipettes. Proteins present in these fractions were concentrated by chloroform/methanol precipitation (24Chang C.C. Lee C.Y. Chang E.T. Cruz J.C. Levesque M.C. Chang T.-Y. J. Biol. Chem. 1998; 273: 35132-35141Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The precipitated proteins were dissolved in lysis buffer (100 mm Tris (pH 8.0), 0.2 m NaCl, 1% Nonidet P-40, 1 mm EDTA, and 1× protease inhibitor mixture), separated on SDS-polyacrylamide gel, and immunoblotted with polyclonal anti-AL antibodies (1:1000). To quantitate the LysoTracker signal (a late endosomal/lysosomal marker), we used a highly sensitive fluorometer to measure the fluorescence intensities present in various Percoll fractions. The method is briefly described as follows. Cells were incubated with 100 nm LysoTracker Red for 2 h at 37 °C and then fractionated on a Percoll gradient at 4 °C. The Percoll fractions were ultracentrifuged at 150,000 × g for 30 min to remove the Percoll particles. Each fraction was then quantitated for its fluorescence at Ex577 nm/Em590 nm using a PC1 photon counting spectrofluorometer from ISS Inc. (Champaign, IL). For detection of the green fluorescent protein (GFP) signal in GFP-transfected or NPC1-GFP-transfected cells, a modified method was needed (because Percoll particles exhibited autofluorescent signals that strongly interfered with the GFP signal). Each Percoll fraction was solubilized with the non-fluorescent detergent Thesit (Roche Applied Science) at 0.2%, and the solubilized material was ultracentrifuged at 150,000 × g for 6 h. The fluorescent signal present in the supernatant was quantitated in the fluorometer at Ex488 nm/Em507 nm. Construction and Transfection of GFP-tagged NPC1—The construct encoding mouse NPC1 protein fused with GFP was created and sub-cloned into the pREX-IRES vector by a procedure described elsewhere (25Sugii, S., Reid, P. C., Ohgami, N., Shimada, Y., Maue, R. A., Ninomiya, H., Ohno-Iwashita, Y., and Chang, T.-Y. (2003) J. Lipid Res., in pressGoogle Scholar). CT43 cells were transfected with the npc1-gfp cDNA using FuGENE 6 according to the manufacturer's instructions. Control experiments showed that expression of NPC1-GFP, but not GFP alone, completely rescued the cholesterol accumulation defect in CT43 cells, indicating that the NPC1-GFP fusion protein is functional (25Sugii, S., Reid, P. C., Ohgami, N., Shimada, Y., Maue, R. A., Ninomiya, H., Ohno-Iwashita, Y., and Chang, T.-Y. (2003) J. Lipid Res., in pressGoogle Scholar). Transfected cells were used within 2–3 days of transfection for imaging analysis and within 4 days for Percoll gradient analysis. Fluorescence Microscopy—Cells were grown on glass coverslips in 6-well plates or in 60-mm dishes and processed for fluorescence studies. For LysoTracker labeling, cells were preincubated with 200 nm LysoTracker Red in the medium at 37 °C for 2 h prior to the experiment. For DAMP staining, intact cells were incubated with 50 μm DAMP for 30 min at 37 °C (26Anderson R.G. Falck J.R. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4838-4842Crossref PubMed Scopus (182) Google Scholar, 27Anderson R.G. Pathak R.K. Cell. 1985; 40: 635-643Abstract Full Text PDF PubMed Scopus (219) Google Scholar); its signal was detected with monoclonal antibody against DNP, followed by Alexa 568-conjugated secondary antibody. For immunostaining, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, washed three times again, and permeabilized either with methanol (chilled at -20 °C) for 1 min or with 1% Triton X-100 in PBS at room temperature for 10 min. After three more washes, the cells were blocked with 10% goat serum in PBS for 30 min at room temperature and incubated with predetermined concentrations of various primary antibodies in the blocking medium for 1 h. When anti-V-ATPase antibody was used as the primary antibody, the incubation time was for 20 min only. Antibody dilutions used in immunofluorescence were as follows: LAMP2 (1:200), AL (1:500 to 1:1000), EEA1 (1:50), syntaxin-6 (1:50), caveolin-1 (1:100), V-ATPase (1:1000), CI-MPR (1:500), and DNP (1:100). For double labeling studies using rabbit anti-AL and rabbit anti-CI-MPR antibodies, Zenon rabbit IgG labeling kits (Alexa 488 and Alexa 568, respectively) were employed according to the manufacturer's protocol. For other labeling studies, cells were washed with PBS three times, treated with various Alexa-conjugated secondary IgGs, and then washed three times. The coverslips were mounted with a drop of ProLong antifade medium onto the glass slides before image processing. Samples were viewed and photographed using a Zeiss Axiophot microscope with a ×63 objective equipped with a CCD camera (DEI-750, Optronics Engineering, Goleta, CA). Fluorescein isothiocyanate and rhodamine filters were used to visualize GFP/Alexa 488 and LysoTracker Red/Alexa 568, respectively. The images were processed using MetaView Version 4.5 software (Universal Imaging Corp., Downing-town, PA). In selective experiments as indicated, the samples were also viewed under a Bio-Rad MRC-1024 krypton/argon laser scanning confocal microscope. The images were constructed using LaserSharp software and further processed using Adobe Photoshop Version 5.02. Early Trafficking of LDL-derived Cholesterol Probed with a Long Versus Short Incubation with CD—We grew CT43 and 25RA cells in cholesterol-free medium for 2 days and pulse-labeled them with [3H]CL-LDL for 5 h at 18 °C. At this temperature, LDL was internalized, but accumulated in pre-lysosomal compartments without significant hydrolysis of CL. When the temperature was increased to 37 °C, CL in LDL was rapidly hydrolyzed to free cholesterol and transported to designated locations (4Cruz J.C. Sugii S. Yu C. Chang T.-Y. J. Biol. Chem. 2000; 275: 4013-4021Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In numerous experiments, we found that the half-time of hydrolysis averaged 25 ± 5 min in both cell types; a typical result is shown in Fig. 1F. During the warm-up period (i.e. immediately after the labeling), if cells were continuously incubated with CD for various time periods as indicated (0–120 min), 25RA and CT43 cells showed the same degree of cholesterol efflux toward CD; the efflux significantly increased from 30 min on (Fig. 1A). When cells were chased at 37 °C for 30 min before adding CD for up to 120 min, a slight defect in cholesterol efflux (starting at 15 min after adding CD) occurred in CT43 cells (Fig. 1B). In a separate experiment, a slight efflux defect in CT43 cells was also found in cells chased for 15 min before CD treatment (data not shown). In contrast, a severe efflux defect in CT43 cells occurred when cells were chased at 37 °C for 60 min (Fig. 1C) or for 120 min (Fig. 1D) before adding CD. In a separate experiment, a severe efflux defect in CT43 cells was also shown in cells chased for 45 min before CD treatment (data not shown). We next compared cholesterol effluxes in 25RA and CT43 cells using a procedure that involves a short incubation with CD (28Yancey P.G. Rodrigueza W.V. Kilsdonk E.P. Stoudt G.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1996; 271: 16026-16034Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). In a control experiment" @default.
• W1984590580 created "2016-06-24" @default.
• W1984590580 creator A5013353605 @default.
• W1984590580 creator A5027881339 @default.
• W1984590580 creator A5047179498 @default.
• W1984590580 creator A5048991469 @default.
• W1984590580 creator A5072490126 @default.
• W1984590580 date "2003-07-01" @default.
• W1984590580 modified "2023-10-16" @default.
• W1984590580 title "Distinct Endosomal Compartments in Early Trafficking of Low Density Lipoprotein-derived Cholesterol" @default.
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