HDL-mediated cholesterol uptake and targeting to lipid droplets in adipocytes
2003; Elsevier BV; Volume: 44; Issue: 10 Linguagem: Inglês
10.1194/jlr.m300267-jlr200
ISSN1539-7262
AutoresGeorges Dagher, Nathalie Donne, Christophe Klein, Pascal Ferré, Isabelle Dugail,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoAdipocytes express high levels of the HDL scavenger receptor class B type I in a differentiation-dependent manner. We thus have analyzed the routes of HDL cholesterol trafficking at different phases of adipocyte differentiation in the 3T3-L1 cell line. One novel and salient feature of this paper is the observation of a widespread distribution in the cell cytoplasm of Golgi markers, caveolin-2, and a fluorescent cholesterol analog NBD-cholesterol (NBD-chol), observed in the early phases of adipocyte formation, clearly distinct from that observed in mature fat cells (i.e., with fully formed lipid vesicles). Thus, in cells without visible lipid droplets, Golgi markers (Golgi 58K, Golgin 97, trans-Golgi network 38, Rab 6, and BODIPY-ceramide), caveolin-2, and NBD-chol all colocalize in a widespread distribution in the cell. In contrast, when lipid droplets are fully formed at latter stages, these markers clearly are distributed to distinct cell compartments: a compact juxtanuclear structure for the Golgi markers and caveolin-2, while NDB-chol concentrates in lipid droplets.In addition, disorganization of the Golgi using three different agents (Brefeldin, monensin, and N-ethyl-maleimide) drastically reduces NBD-chol uptake at different phases of adipocyte formation, strongly suggesting that the Golgi apparatus plays a critical role in HDL-mediated NBD uptake and routing to lipid droplets. Adipocytes express high levels of the HDL scavenger receptor class B type I in a differentiation-dependent manner. We thus have analyzed the routes of HDL cholesterol trafficking at different phases of adipocyte differentiation in the 3T3-L1 cell line. One novel and salient feature of this paper is the observation of a widespread distribution in the cell cytoplasm of Golgi markers, caveolin-2, and a fluorescent cholesterol analog NBD-cholesterol (NBD-chol), observed in the early phases of adipocyte formation, clearly distinct from that observed in mature fat cells (i.e., with fully formed lipid vesicles). Thus, in cells without visible lipid droplets, Golgi markers (Golgi 58K, Golgin 97, trans-Golgi network 38, Rab 6, and BODIPY-ceramide), caveolin-2, and NBD-chol all colocalize in a widespread distribution in the cell. In contrast, when lipid droplets are fully formed at latter stages, these markers clearly are distributed to distinct cell compartments: a compact juxtanuclear structure for the Golgi markers and caveolin-2, while NDB-chol concentrates in lipid droplets. In addition, disorganization of the Golgi using three different agents (Brefeldin, monensin, and N-ethyl-maleimide) drastically reduces NBD-chol uptake at different phases of adipocyte formation, strongly suggesting that the Golgi apparatus plays a critical role in HDL-mediated NBD uptake and routing to lipid droplets. Cholesterol is a crucial component of cell membranes, and as such plays a key role in the regulation of signal transduction (1Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Google Scholar) and gene expression (2Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Google Scholar). The homeostasis of cell cholesterol is maintained through complex mechanisms that imply biosynthesis, uptake, and constant recycling between membranes. Many intracellular organelles take part in the regulation of cholesterol trafficking. Among these, the endosomal lysosomal pathway is involved in the uptake of exogenous cholesterol from LDL; caveolae are engaged in cholesterol trafficking to and from the plasma membrane; the endoplasmic reticulum (ER), where some of the enzymes of the cholesterol biosynthetic pathway reside, is of particular importance because it includes a cell cholesterol sensor system linked to the cleavage activating protein sterol-regulatory element binding proteins (SREBP) system (3Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89: 331-340Google Scholar); and the Golgi apparatus plays a crucial role in sorting processes (4Anderson R.G. Pathak R.K. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments.Cell. 1985; 40: 635-643Google Scholar, 5Fielding P.E. Fielding C.J. Intracellular transport of low density lipoprotein derived free cholesterol begins at clathrin-coated pits and terminates at cell surface caveolae.Biochemistry. 1996; 35: 14932-14938Google Scholar, 6Mendez A. Uint L. Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus.J. Lipid Res. 1996; 37: 2510-2524Google Scholar, 7Mendez A.J. Monensin and Brefeldin A inhibit high density lipoprotein-mediated cholesterol efflux from cholesterol-enriched cells.J. Biol. Chem. 1995; 270: 5891-5900Google Scholar). Recently, previously unrecognized organelles have been identified as new players in cholesterol traffic: the cytoplasmic lipid droplets (8Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms.Prog. Lipid Res. 2001; 40: 325-438Google Scholar). The adipocyte is the unique cell type in which triglyceride-rich lipid droplets are present continuously and physiologically in the cytoplasm. Free cholesterol is also found in adipocyte lipid droplets (9Le Lay S. Robichon C. Le Liepvre X. Dagher G. Ferre P. Dugail I. Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of 3T3–L1 cells.J. Lipid Res. 2003; 44: 1499-1507Google Scholar, 10Prattes S. Horl G. Hammer A. Blaschitz A. Graier W.F. Sattler W. Zechner R. Steyrer E. Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures.J. Cell Sci. 2000; 113: 2977-2989Google Scholar). On the other hand, the intracellular distribution of cholesterol between the plasma membrane and the lipid droplet is altered in pathological states such as obesity, as pointed out by previous studies from our laboratory (11Boizard M. Le Liepvre X. Lemarchand P. Foufelle F. Ferre P. Dugail I. Obesity-related overexpression of fatty-acid synthase gene in adipose tissue involves sterol regulatory element-binding protein transcription factors.J. Biol. Chem. 1998; 273: 29164-29171Google Scholar). As a signature of this defect, we have shown that the cholesterol-regulated transcription factor SREBP-2 was selectively activated in hypertrophied adipocytes from obese rodents. In addition, we have shown that some metabolic abnormalities that characterize adipocyte dysfunction in obesity can be mimicked in normal fat cells by altering their intracellular cholesterol balance (12Le Lay S. Krief S. Farnier C. Lefrere I. Le Liepvre X. Bazin R. Ferre P. Dugail I. Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes.J. Biol. Chem. 2001; 276: 16904-16910Google Scholar). For these reasons, we thought that the adipocyte cell type might provide useful information on the intracellular cholesterol trafficking. We took advantage of the existence of committed adipose cell lines that undergo adipose differentiation in culture to investigate the targeting of cholesterol to lipid droplets. Because adipocytes accumulate cholesterol from exogenous sources and express high levels of the HDL scavenger receptor class B type I (SR-BI) (13Babitt J. Trigatti B. Rigotti A. Smart E.J. Anderson R.G. Xu S. Krieger M. Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.J. Biol. Chem. 1997; 272: 13242-13249Google Scholar, 14Graf G.A. Connell P.M. van der Westhuyzen D.R. Smart E.J. The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae.J. Biol. Chem. 1999; 274: 12043-12048Google Scholar), we examined the targeting of an HDL-associated fluorescent cholesterol analog, NBD-cholesterol (NBD-chol), to the lipid droplets at different stages of the adipocyte differentiation program. The results of this study show that the distribution of the Golgi complex, caveolin-2, and cholesterol exhibit marked changes in distribution during the differentiation process of adipocytes. The Golgi complex and caveolin-2 exhibit a dispersed distribution and colocalization with cholesterol in the first phase of adipocyte formation. In contrast, in fully differentiated lipid laden cells, the Golgi complex, as well as caveolin-2, occupies a juxtanuclear location, while cholesterol distributes preferentially in lipid droplets. These data suggest that the intracellular routes of HDL cholesterol significantly differ during fat cell differentiation, and that different mechanisms targeting cholesterol to the lipid droplets might exist, depending on its maturation. Human plasma HDLs were obtained from Calbiochem, La Jolla, CA. NBD-chol, [22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3-ol)], LysoTracker Red DND-99, cholera toxin subunit B Alexa Fluor 594 conjugate, monoclonal antibody to Golgin 97, calnexin, BODIPY FLC5 ceramide, and Alexa Fluor 488 and 546 goat anti-mouse or goat anti-rabbit IgG conjugate were supplied by Molecular Probes (Eugene, OR). Golgi 58K antibody was from Sigma, and monoclonal anti-caveolin-2 IgG antibodies were from Transduction Labs (Lexington, KY). Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, heat-inactivated fetal bovine serum (FBS), and trypsin-EDTA were from Gibco (Invitrogen, France). All other chemicals were from Sigma. A rabbit monoclonal antibody to Rab 6 was supplied by L. Johannes (Institut Curie, France), and a goat monoclonal antibody to the trans-Golgi network (TGN) 38 was supplied by M. Borneins [Institut Curie, France (15Jasmin B.J. Cartaud J. Bornens M. Changeux J.P. Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation.Proc. Natl. Acad. Sci. USA. 1989; 86: 7218-7222Google Scholar)]. 3T3-L1 mouse preadipocytes (2 × 104 cells/well) were grown to confluence on either glass coverslips or 8-well Labtek plates (PolyLABO, France) in high-glucose DMEM supplemented with 10% FBS, 20 mM HEPES, and 100 U/ml penicillin/streptomycin. Differentiation of confluent preadipocytes (designated as day 0) was induced by adding methyl-isobutylxanthine (100 μM), dexamethasone (0.25 μM), and insulin (1 μg/ml) to the above solution. After 48 h, the culture medium was replaced with DMEM supplemented with 10% FBS and 1 μg/ml of insulin for 1–12 days. As previously described, cytoplasmic triglyceride droplets were visible by day 4, and 70% to 80% of cells were fully differentiated by day 8 (16Hwang C-S. Loftus T.M. Mandrup S. Lane M.D. Adipocyte differentiation and leptin expression.Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Google Scholar). Cells were studied at three different phases of adipocyte differentiation. Phase I comprises days 3–4 after induction of differentiation [i.e., days 1–2 after 1-methyl-3-isobuthylxanthine (MIX)-dexamethasone (DEX) retrieval]. At this stage, no lipid droplets could be observed in the cytoplasm. Phase II and phase III refer to days 7–10 and days 12–14, respectively, after retrieval of MIX-DEX. At these latter phases, cells were fully differentiated and displayed the characteristic lipid droplets of adipocytes. HDLs loaded with NBD-chol were reconstituted as described previously (17Pitas R.E. Innerarity T.L. Weinstein J.N. Mahley R.W. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy.Arteriosclerosis. 1981; 1: 177-185Google Scholar). In brief, 5 μM NBD-chol was incorporated into human HDL (Calbiochem), and the NBD-chol-loaded HDLs were obtained after ultracentrifugation and extensive dialysis against serum-free culture medium. The protein concentration of NBD-chol-loaded HDL was determined according to Bradford using BSA as a standard (18Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Google Scholar). To assess the uptake and HDL-derived accumulation of NBD-chol, 3T3 adipocytes were incubated in DMEM and 10% lipoprotein-deficient serum (LPDS) at 37°C in the presence of HDL loaded with 5 μM NBD-chol. LPDS was prepared by ultracentrifugation as described elsewhere (12Le Lay S. Krief S. Farnier C. Lefrere I. Le Liepvre X. Bazin R. Ferre P. Dugail I. Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes.J. Biol. Chem. 2001; 276: 16904-16910Google Scholar). NBD-chol uptake kinetics were determined using laser confocal fluorescence microscopy on single, living cells. A field on the coverslip chamber containing 10–15 cells was randomly selected, and the position of the microscope objective was focused to view the median section of the cells. Twelve sets were analyzed from a single differentiation. At least five sets of differentiated cultures were recorded for each condition. Cholesterol influx was initiated by addition of NBD-chol and was followed by acquisition of digital images at different time intervals using a time course module (Zeiss AIM software). The average pixel intensity [reactive oxygen intermediates (ROI)] of each single-cell as a function of time was determined using Zeiss software and expressed as mean ± SE. The kinetic of NBD-chol uptake was analyzed by the Marquardt algorithm using UltraFit software from Biosoft (Cambridge, UK). The calculated time constants were analyzed statistically using Student's t-test. Adipose tissue expresses very low levels of ACAT mRNA (19Uelmen P.J. Oka K. Sullivan M. Chang C.C.Y. Chang T.Y. Chan L. Tissue-specific expression and cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice.J. Biol. Chem. 1995; 270: 26192-26201Google Scholar) and virtually no ACAT activity (20Little M.T. Hahn P. Ontogeny of acyl-CoA: cholesterol acyltransferase in rat liver, intestine, and adipose tissue.Am. J. Physiol. 1992; 262: G599-G602Google Scholar). Given that adipocytes have a very low ability to esterify cholesterol, and that 3T3-L1 adipocytes contain essentially free cholesterol (9Le Lay S. Robichon C. Le Liepvre X. Dagher G. Ferre P. Dugail I. Regulation of ABCA1 expression and cholesterol efflux during adipose differentiation of 3T3–L1 cells.J. Lipid Res. 2003; 44: 1499-1507Google Scholar), intracellular fluorescence is considered to be caused by intact, internalized NBD-chol. In some experiments, cellular NBD-chol efflux was evaluated as follows: cells were incubated in the presence of HDL NBD-chol (5 μM) for 2 h. Before each efflux experiment, cells were washed three times with DMEM. NBD-chol and efflux kinetics were determined using laser confocal fluorescence microscopy on single, living cells. A field on the coverslip chamber containing 10–15 cells was randomly selected, and the position of the microscope objective was focused to view the median section of the cells. Twelve sets were analyzed from a single differentiation. At least three sets of differentiated cultures were recorded for each condition. Cholesterol efflux was initiated by the addition of cholesterol-free HDL and was followed by acquisition of digital images at different time intervals using a time course module (Zeiss AIM software). The average pixel intensity (ROI) of each single cell as a function of time was determined using Zeiss software and expressed as mean ± SE as described for the uptake experiments. The NBD-chol basal rate was assessed in the absence of HDL and subtracted from that observed in the presence of HDL. To study the involvement of cellular energy in the uptake of NBD-chol, 3T3 adipocytes were depleted of their intracellular ATP stores by incubation in glucose-free DMEM with 50 mM deoxyglucose (2 h), 50 μM orthovanadate (90 min), or 3 mM sodium azide (30 min). To determine whether cholesterol uptake is mediated by the Golgi complex, we assessed NBD-chol uptake in the presence of Brefeldin (2 μg/ml, 30 min) and of monensin (5 μM, 30 min). These compounds are known to disrupt the organization of the Golgi complex and were added for the indicated time before exposure to NBD-chol. The effects of cytochalasin D (20 μM, 60 min), a compound known to disrupt the actin network (21Yahara I. Harada F. Sekita S. Yoshihira K. Natori S. Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro.J. Cell Biol. 1982; 92: 69-78Google Scholar), and N-ethyl-maleimide (NEM) (1 mM, 20 min), a V-type ATPase inhibitor (22Stone D.K. Xie X.S. Proton translocating ATPases: issues in structure and function.Kidney Int. 1988; 33: 767-774Google Scholar), on NBD-chol influx were also assessed. Cells were grown to confluence on coverslips and differentiated as described above. To assess intracellular NBD-chol distribution, cells were exposed to DMEM containing HDL NBD-chol (5 μg/ml) for 1 h at 37°C. Cells were then washed three times with PBS and fixed with formaldehyde in PBS (3%, 45 min.), followed by exposure to PBS plus glycine (50 mM, 30 min) and then PBS plus BSA (2%, 30 min). Cells were then permeabilized with saponin (0.1%, 5 min) in PBS. For immunolabeling, the cells were incubated in PBS containing one of the following antibodies: Golgi 58K (1:50; v/v), Golgin 97 (1:125; v/v), anti-TGN 38 (1:500; v/v), Rab-6 (1:200; v/v), calnexin (1:200; v/v), and monoclonal antibody 65 anti-caveolin-2 (1:500; v/v). Bound primary antibodies were then visualized with the appropriate Alexa-Fluor (molecular probes) secondary antibodies at a dilution of 1:200 (v/v). Coverslips were mounted onto slides with either VECTASHIELD or SlowFade and observed with a Zeiss laser scanning confocal microscope (LSM 500). Colocalization was quantified as described previously (23Manders E.E.M. Verbeek F.J. Aten J.A. Measurement of co-localisation of objects in dual-colour confocal images.J. Microscopy. 1993; 169: 375-382Google Scholar). Differentiated cells were loaded with 5 μM BODIPY CFL5 ceramide at 4°C for 15 min, and the medium was then removed and cells washed three times with DMEM solution. The temperature was then increased to 25°C. Immediately thereafter, the live cells were analyzed by fluorescence microscopy. To measure the diffusional mobility of NBD-chol in membranes, we used the method of fluorescence recovery after photobleaching (FRAP). In brief, fluorescent molecules in a small area are irreversibly photobleached by an intense laser flash, and fluorescence recovery through the exchange of bleached for nonbleached molecules is measured using low-light illumination. Mobility parameters are then derived from the kinetics of fluorescence recovery, as described previously (24Ellenberg J. Lippincott-Schwartz J. Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras.Methods. 1999; 19: 362-372Google Scholar). The mean fluorescence intensities are plotted per area over time, and the experimental data are fitted to the formula for one-dimensional diffusion (equation 1). where I(t) is the fluorescence intensity as a function of time; zero of time t is taken as the midpoint of the bleach; I(final) is the final intensity reached after complete recovery; W is the strip width; and D is the effective one-dimensional diffusion constant. The diffusion constant D can be obtained directly by spreadsheet programs using least square minimization algorithms. The diffusion constant for NBD-chol was calculated for lipid droplets and cytoplasmic membranes in living cells and compared with that obtained for 2′,7′-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF), a fluorescent probe for intracellular pH. As a control, diffusion of NBD-chol to bleached region was assessed in paraformaldehyde fixed cells. Under these conditions, no recovery of fluorescence should be observed. Recovery was also assessed from the nucleus as a region devoid of a fluorescent probe. The time course of NBD-chol uptake from HDL best fits a single exponential curve indicative of a first order relationship (P < 0.001, a coefficient of determination equal to 0.959, and an F value of 189 (Fig. 1). The suitability of a second- and a third-order fit between the fluorescence intensity and time was also analyzed in detail, and in these instances, the P values were 0.51 and 0.80, respectively. First-order kinetic analysis of NBD-chol uptake in cells gives a time constant ranging from 0.01–0.09 min−1, with a mean ± SD of 0.036 ± 0.018 min–1 (n = 90). Similarly, NBD-chol efflux from cells preloaded with fluorescent lipid followed first order kinetics with a time constant of 0.020 ± 0.010 min–1 (mean ± SD). Furthermore, addition of nonlabeled HDL competitively inhibits HDL NBD-chol uptake (Fig. 1C). Similarly, addition of a neutralizing antibody to SR-BI significantly inhibits NBD-chol uptake (Fig.1C). This suggests the involvement of SR-BI in the uptake of HDL-derived NBD-chol. The distribution of NBD-chol in 3T3-L1 cells largely depends on the phase of differentiation. By 60 min of incubation with NBD-chol, many cells in phase I (with no clearly defined lipid droplets) showed prominent punctuate localizations throughout the cytoplasm (Fig. 2A, B). In terminally differentiated adipocytes (phases II and III), highly fluorescent lipid droplets could be observed (Fig. 2C–H). The pattern of NBD-chol distribution in these cells resembled that of lipid droplets visible by transmission microscopy (Fig. 2D, F) or stained with Nile red, a selective lipid droplet stain (Fig. 2H). In order to assess the involvement of cellular energy in the trafficking of cholesterol, cells in phase II were depleted of ATP by incubation with either deoxyglucose, Na-azide, or Na-vanadate. These agents drastically reduced the uptake (Fig. 3A)and efflux (data not shown) of NBD-chol. These data establish that HDL-derived cholesterol uptake in adipocytes is an energy-dependent process. On the other hand, cytochalasin D, known to interfere with actin-type filaments, (21Yahara I. Harada F. Sekita S. Yoshihira K. Natori S. Correlation between effects of 24 different cytochalasins on cellular structures and cellular events and those on actin in vitro.J. Cell Biol. 1982; 92: 69-78Google Scholar) did not significantly modify HDL cholesterol uptake (Fig. 3B). The routes of HDL-derived sterols to the adipocyte lipid droplet are not yet characterized. In this study, we wished to assess the involvement of several cell structures in this trafficking. First, we ensured that under our experimental conditions, the routing of NBD-chol did not follow the lysosomal pathway, which is known to be involved mainly in LDL uptake. To this end, cells were labeled with LysoTracker Red, and NBD-chol uptake from HDL was followed for a period of 60 min. No colocalization of cholesterol within lysosomes could be observed during this time interval. Figure 4depicts lysosomal distribution (Fig. 4A) and NBD-chol (Fig. 4B) at 60 min after initiation of NBD-chol uptake, with no colocalization between these two fluorescent dyes (Fig. 4D). These results suggest that uptake and routing of sterol from HDL follows a pathway different from that of LDL. We next wished to determine the role of the Golgi complex in the uptake, intracellular transport, and storage of HDL-derived NBD-chol in adipocytes. Cells were pretreated with several agents known to disrupt Golgi function and structure. These include Brefeldin A (BFA) (25Pelham H.R. Multiple targets for Brefeldin A.Cell. 1991; 67: 449-451Google Scholar) and monensin, which disorganize the Golgi complex. As shown in Fig. 3B, treatment with BFA or monensin drastically reduced cholesterol uptake by these cells, thus implicating the Golgi complex in this process and possibly in intracellular trafficking. A similar reduction in the rate of NBD-chol uptake was observed with NEM (Fig. 3B), reported to inhibit the NEM-sensitive factor involved in the transport of molecules from the TGN to other cell organelles (26Ikonen E. Tagaya M. Ullrich O. Montecucco C. Simons K. Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells.Cell. 1995; 81: 571-580Google Scholar). We next assessed the colocalization of NBD-chol with the Golgi complex using antibodies to Golgin 97. Interestingly, in phase I, when no visible lipid droplets have yet accumulated in cells, fluorescent labeling with antibody to Golgin 97 revealed a dispersed Golgi complex spread all over the cytoplasm, and NBD-chol was found to be colocalized within these Golgi structures (Fig. 5A–C). The percent of cholesterol colocalized with Golgi (chol/Golgi) in phase I was 86% while that of Golgi/chol was 57% (Fig. 5C). In phase II, antibody to Golgin 97 was dispersed in the cytoplasm, and NBD-chol was localized in nascent lipid droplets and cytoplasm. (Fig. 5D–E). Whereas the Golgi complex formed a compact juxtanuclear structure in phase III cells (Fig. 5G–I), NBD-chol is present primarily in lipid droplets (Fig. 5H) with no colocalization (Fig. 5I). In order to further establish the scattering of the Golgi complex in phase I, we used either a live marker of the Golgi complex (BODIPY FLC5 ceramide) or several antibodies to Golgi markers: Golgi 58K, Rab 6, or TGN 38. As shown in Fig. 6, a scattered pattern of all of these antibodies was observed in the cytoplasm, strongly suggesting a dispersed Golgi complex in cells in phase I of the differentiation process (Fig. 6A–C). Similar patterns were obtained using BODIPY ceramide in living cells (Fig. 6D), thus validating the distribution observed by immunofluorescence in fixed cells. This markedly differs from the juxtanuclear location of the Golgi complex in phase III cells with prominent lipid droplets (see Figs. 5G, 6E). Projections of confocal serial sections of cells in phase I showed that the different fluorescent Golgi markers were scattered over the entire volume of the cell, excluding the nucleus, and were colocalized with NBD-chol (data not shown). These data establish that the distribution of Golgi complex exhibits marked changes during the differentiation process. A dispersed distribution colocalized with cholesterol was observed in phase I, whereas in fully differentiated lipid laden cells, cholesterol segregates from the juxtanuclear Golgi complex and distributes preferentially in lipid droplets. Caveolins are cholesterol binding proteins and have been shown to be distributed mainly at the plasma membrane and also in the Golgi complex. They exist as three distinct isoforms, caveolin-2 being the most abundant subtype in adipocytes (27Razani B. Lisanti M.P. Caveolins and caveolae: molecular and functional relationships.Exp. Cell Res. 2001; 271: 36-44Google Scholar, 28Schlegel A. Lisanti M.P. The caveolin triad: caveolae biogenesis, cholesterol trafficking, and signal transduction.Cytokine Growth Factor Rev. 2001; 12: 41-51Google Scholar). In order to further document the differential localization of cholesterol and Golgi markers during the course of adipocyte differentiation, we stained cells for caveolin-2. In phase I cells, endogenous caveolin-2 was detected by immunostaining with a monoclonal antibody against human caveolin-2 (mAb 65), exhibiting a scattered distribution and colocalizing with NBD-chol (Fig. 7A–C). During later phases, NBD-chol was mainly present in lipid droplets while caveolin-2 exhibited a marked juxtanuclear distribution and was also observed at the cell membrane (Fig. 7D–F). Thus, the distribution of caveolin-2 at different phases resembles that of Golgi markers. Because NBD-chol was found to be distributed throughout the cytoplasm and colocalized with the Golgi complex in cells at phase I, we wished to assess whether the probe was integrated to membranous elements rather than clustered in micelles in the cytosol. Fluorescent recovery after photobleaching was used to measure the rate at which NBD-chol diffused into photobleached areas. Figure 8depicts FRAP of NBD-chol. Outlined areas were irreversibly photobleached by an intense laser flash, and fluorescence recovery through exchange of bleached for nonbleached molecules was assessed in the cytoplasm and in lipid droplets. As a control, an area of the nucleus devoid of NBD-chol was also photobleached. The value of diffusion constant (Fig. 8D) was calculated as described in the Materials and Methods. The mean value of D ± SD in the cytoplasm was 0.13 ± 0.12 × 10−8 cm2 sec−1. When calculated separately for lipid droplets, it was 0.08 ± 0.06 × 10−8 cm2 sec−1 and 0.46 ± 0.32 × 10−8 cm2 sec−1 for cytoplasm (P ≤ 0.05, n = 8). These values are of a magnitude similar to those observed for membrane-integrated elements, such as NBD-phosphatidylserine, NBD-phosphatidylcholine, neutral lipids, cholecytokinin, or laminin receptor (24Ellenberg J. Lippincott-Schwartz J. Dynamics and mobility of nuclear envelope proteins in interphase and mitotic cells revealed by green fluorescent protein chimeras.Methods. 1999; 19: 362-372Google Scholar, 29Cezanne L. Lopez A. Loste F. Parnaud G. Saurel O. Demange P. Tocanne J.F. Organization and dynamics of the proteolipid complexes formed by annexin V and lipids in planar supported lipid bilayers.Biochemistry. 1999; 38: 2779-2786Google Scholar, 30Fulbright R.M. Axelrod D. Dunham W.R. Marcelo C.L. Fatty acid alteration and the lateral diffusion of lipids in the plasma membrane of keratinocytes.Exp. Cell Res. 1997; 233: 128-134Google Scholar, 31Roettger B.F. Pinon D.I. Burghardt T.P. Miller L.J. Regulation of lateral mobility and cellular traffickin
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