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• W2117788306 abstract "Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by β-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs. Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by β-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs. The maintenance of lipid homeostasis within the cell is controlled through combined synthesis, influx, efflux, and storage. Cells store excess fatty acids and cholesterol in lipid droplets (LDs), 2The abbreviations used are: LDlipid dropletERendoplasmic reticulumTVEtubulovesicular elementsPFAparaformaldehydeBHKbaby hamster kidney cellsBSAbovine serum albuminGFPgreen fluorescent proteinYFPyellow fluorescent proteinMRFPmonomeric red fluorescent proteinPIPES1,4-piperazinediethanesulfonic acid. which are dynamic and regulated organelles derived from the endoplasmic reticulum (ER) (1Martin S. Parton R.G. Semin. Cell Dev. Biol. 2005; 16: 163-174Crossref PubMed Scopus (157) Google Scholar, 2Murphy D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (762) Google Scholar). LDs have been shown to undergo microtubule-based motility (3Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar, 4Targett-Adams P. Chambers D. Gledhill S. Hope R.G. Coy J.F. Girod A. McLauchlan J. J. Biol. Chem. 2003; 278: 15998-16007Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 5Valetti C. Wetzel D.M. Schrader M. Hasbani M.J. Gill S.R. Kreis T.E. Schroer T.A. Mol. Biol. Cell. 1999; 10: 4107-4120Crossref PubMed Scopus (234) Google Scholar) and to interact with a range of other organelles, including mitochondria, peroxisomes, and the ER (6Blanchette-Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226Abstract Full Text PDF PubMed Google Scholar, 7Vock R. Hoppeler H. Claassen H. Wu D.X. Billeter R. Weber J.M. Taylor C.R. Weibel E.R. J. Exp. Biol. 1996; 199: 1689-1697Crossref PubMed Google Scholar). Whereas LDs have been best described in adipocytes and steroidogenic cells of the testis, ovary, and adrenal gland, they are also present in a range of other cell types, and their formation can be induced in cultured cells by oleic acid treatment (3Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar), suggesting that all cells have the ability to generate LDs under conditions of elevated fatty acids. In recent years interest in the regulation of LDs in less specialized cell types has increased significantly, due in part to the observation that a dominant-negative truncation mutant of caveolin, Cav3DGV, is localized to the surface of LDs and induces a cholesterol imbalance in fibroblasts, in addition to inhibiting LD motility and catabolism (3Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar, 8Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (275) Google Scholar). Caveolins have been shown to bind cholesterol (9Murata M. Peranen J. Schreiner R. Wieland F. Kurzchalia T.V. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Crossref PubMed Scopus (767) Google Scholar) and fatty acids (10Trigatti B.L. Anderson R.G. Gerber G.E. Biochem. Biophys. Res. Commun. 1999; 255: 34-39Crossref PubMed Scopus (182) Google Scholar), and while predominantly localized to caveolar domains of the cell surface they can be redistributed to LDs upon fatty acid treatment (3Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar). In addition to the inhibitory effects of Cav3DGV on the LD, Cav3DGV also indirectly inhibits signaling from the cell surface through an effect on cholesterol, suggesting a link between the function of the LDs and functional maintenance of cell surface domains. lipid droplet endoplasmic reticulum tubulovesicular elements paraformaldehyde baby hamster kidney cells bovine serum albumin green fluorescent protein yellow fluorescent protein monomeric red fluorescent protein 1,4-piperazinediethanesulfonic acid. To begin to define the mechanisms regulating the formation and catabolism of the LD it is important to first identify the nature of the interaction of this organelle with other compartments within the cell. Several recent studies have undertaken proteomic analyses of LDs from a number of different cell types, under conditions of lipolysis or lipid deposition. These analyses identified numerous members of the Rab family of small GTPases associated with the LDs (11Brasaemle D.L. Dolios G. Shapiro L. Wang R. J. Biol. Chem. 2004; 279: 46835-46842Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 12Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. J. Biol. Chem. 2004; 279: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 13Fujimoto Y. Itabe H. Sakai J. Makita M. Noda J. Mori M. Higashi Y. Kojima S. Takano T. Biochim. Biophys. Acta. 2004; 1644: 47-59Crossref PubMed Scopus (275) Google Scholar, 14Umlauf E. Csaszar E. Moertelmaier M. Schuetz G.J. Parton R.G. Prohaska R. J. Biol. Chem. 2004; 279: 23699-23709Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The Rab family of proteins are essential regulators of vesicular traffic. Described as molecular switches, Rab proteins undergo conformational changes through cycles of GTP binding and hydrolysis (15Seabra M.C. Wasmeier C. Curr. Opin. Cell Biol. 2004; 16: 451-457Crossref PubMed Scopus (234) Google Scholar, 16Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2710) Google Scholar). The GTP-bound active form interacts directly with downstream effectors and indirectly with other components of the transport machinery controlling cargo selection, vesicle fusion, cytoskeletal transport, and integration of vesicle traffic with signal transduction pathways. Although the novel nature of the LD hemi-membrane makes it unlikely that proteins spanning the bilayer could associate with this organelle, this would not preclude association of Rab proteins whose attachment to membranes is regulated through prenylation at the C terminus, and protein-protein interactions (15Seabra M.C. Wasmeier C. Curr. Opin. Cell Biol. 2004; 16: 451-457Crossref PubMed Scopus (234) Google Scholar, 16Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2710) Google Scholar). Indeed, ten Rab GTPases have been found associated with LDs (11Brasaemle D.L. Dolios G. Shapiro L. Wang R. J. Biol. Chem. 2004; 279: 46835-46842Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 12Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. J. Biol. Chem. 2004; 279: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 13Fujimoto Y. Itabe H. Sakai J. Makita M. Noda J. Mori M. Higashi Y. Kojima S. Takano T. Biochim. Biophys. Acta. 2004; 1644: 47-59Crossref PubMed Scopus (275) Google Scholar, 14Umlauf E. Csaszar E. Moertelmaier M. Schuetz G.J. Parton R.G. Prohaska R. J. Biol. Chem. 2004; 279: 23699-23709Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), several of which have been previously localized to endocytic compartments. On the one hand, this complexity is not unusual, as several distinct Rab proteins can be associated with a single organelle undertaking multiple sorting functions, such as early endosomes and the Golgi complex (16Zerial M. McBride H. Nat. Rev. Mol. Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2710) Google Scholar). The predicted association of multiple Rab proteins with the lipid droplet suggests a dynamic interaction between this and other organelles in the cell. On the other hand, out of the Rab proteins associated with LDs, only five, i.e. Rab5c, Rab7, Rab10, Rab14, and Rab18, have been identified independently in at least two separate studies. In the present study we have analyzed the localization of Rab5, Rab7, Rab11, and Rab18, all previously identified in the endosomal system, with respect to LDs under conditions of neutral lipid synthesis. We have identified Rab18 as a major component of lipid droplets and further explored its role in lipid dynamics and lipid storage activities. Cell Culture—3T3-L1 fibroblasts (American Type Culture Collection, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mm l-glutamine, and differentiated using insulin, dexamethasone, biotin, and isobutyl-methylxanthine as described previously (17Shewan A.M. Marsh B.J. Melvin D.R. Martin S. Gould G.W. James D.E. Biochem. J. 2000; 350: 99-107Crossref PubMed Scopus (84) Google Scholar). Adipocytes were used between days 6 and 12 post-differentiation, or at 2-day intervals during the differentiation process as described in the results section. BHK-21 cells (baby hamster kidney cells) and Vero cells (African green monkey kidney epithelial cells) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) Serum-Supreme (BioWhittaker) and 2 mm l-glutamine. Antibodies, Plasmids, and Reagents—Mouse anti-GM130 (catalog no. 610823), monoclonal anti-caveolin-1 (catalog no. 610406), and monoclonal anti-caveolin-2 (catalog no. 610684) were obtained from BD Transduction Laboratories (BD Biosciences). Rabbit anti-perilipin A (catalog no. P1998) and mouse anti-α-tubulin (catalog no. T9026) were obtained from Sigma. Rabbit anti-Rab18 (18Lutcke A. Parton R.G. Murphy C. Olkkonen V.M. Dupree P. Valencia A. Simons K. Zerial M. J. Cell Sci. 1994; 107: 3437-3448Crossref PubMed Google Scholar) and rabbit anti-GFP (19Prior I.A. Harding A. Yan J. Sluimer J. Parton R.G. Hancock J.F. Nat. Cell Biol. 2001; 3: 368-375Crossref PubMed Scopus (457) Google Scholar) have been described previously. Alexa488- and Alexa594-conjugated secondary antibodies were obtained from Molecular Probes Inc. (Eugene, OR). Horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma. Oleic acid was obtained from Calbiochem and conjugated to fatty-acid free bovine serum albumin (Calbiochem) prior to use. Bodipy493/503 and Nile Red were obtained from Molecular Probes and prepared as saturated solutions in ethanol (working dilution, 1:200) and acetone (working dilution, 1:2000), respectively. All other chemicals were obtained from Sigma unless stated otherwise. GFP-Rab5 and YFP-Rab11 have been described previously (20Sonnichsen B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. Cell Biol. 2000; 149: 901-914Crossref PubMed Scopus (809) Google Scholar). GFP-Rab7 was obtained from Dr.Lucas Pelkmans, Max-Planck Institute, Dresden, Germany. mRFP-Cav3DGV was constructed using Cav3DGV-HA (21Luetterforst R. Stang E. Zorzi N. Carozzi A. Way M. Parton R.G. J. Cell Biol. 1999; 145: 1443-1459Crossref PubMed Scopus (109) Google Scholar) as a template to amplify a fragment using the following primers: 5′-GGGGTACCCGACGGTGTATGGAAGGTG-3′ and 5′-CGGGATCCTAGCCTTCCCTTCGCAG-3′. The PCR product was A-tailed and cloned into pGEM-T Easy (Promega, Madison, WI) and subsequently excised using BamHI and KpnI, and ligated into linearized mRFP-C3. mRFP-C3 was constructed from pRSETb-mRFP1 (supplied by Prof. Roger Tsien, Howard Hughes Medical Institute, University of California (22Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2009) Google Scholar)). To generate GFP-Rab18, the mouse Rab18 ORF was excised from myc-Rab18 using NdeI and BamHI (removing the myc tag), ligated into pSL1180 (Amersham Biosciences), and subsequently excised with BamHI and PstI and ligated into pEGFP-C1 (Clontech), resulting in an N-terminal GFP tag. All constructs were sequenced using ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA) in the Australian Genome Research Facility, University of Queensland. Indirect Immunofluorescence Microscopy and Real-time Video Microscopy—For immunofluorescence microscopy cells grown on glass coverslips were fixed with 4% paraformaldehyde (PFA) in PBS. Cells were permeabilized in 0.1% saponin (w/v) for 10 min, quenched for 10 min using 50 mm NHCl4, and blocked for 10 min using 0.2% bovine serum albumin/0.2% fish skin gelatin in PBS. Primary and secondary antibodies were diluted in blocking solution and incubated with the cells for 30 min at room temperature. Finally the coverslips were washed in PBS and mounted in Mowiol (Calbiochem). Labeling was analyzed using an Axiovert 200M SP LSM 510 META confocal laser scanning microscope (Zeiss) under oil, using either 100× or 63× oil immersion objectives. The data were processed using the LSM 510 Meta (Zeiss) software, and images were assembled using Photoshop 7.0 (Adobe Systems, Mountain View, CA). Quantitation of LD Rab18 labeling was performed on fluorescence images collected with identical settings, using ImageJ 1.33 to measure the mean pixel intensity of individual LDs. For each individual experiment between 20 and 70 LDs were analyzed. Cells for real-time microscopy were plated onto glass-bottom tissue culture dishes (MatTek Corp.) and transferred into CO2-independent medium supplemented with 0.1% fatty-acid free bovine serum albumin (Calbiochem) in the presence or absence of 100 μg/ml oleic acid. Time series were collected at 37 °C using an Axiovert 200M SP LSM 510 Meta confocal laser scanning microscope equipped with a heated stage and a 100× oil immersion objective. Cells were used for real-time data collection for a maximum of 1.5 h. Time series images were collected using a 488 nm excitation laser line at <20% maximum power using the Zeiss LSM510 Meta software. Images were converted to 8-bit TIFF files and further analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). QuickTime movies were assembled using ImageJ 1.33, and still images were compiled using Adobe Photoshop 7.0. LD Isolation—LDs were isolated using a modification of the procedure of Yu et al. (23Yu W. Cassara J. Weller P.F. Blood. 2000; 95: 1078-1085Crossref PubMed Google Scholar). Briefly, cells were scraped into dissociation buffer (25 mm Tris-HCl, pH 7.4, 100 mm KCl, 1 mm EDTA, 5 mm EGTA) containing a mixture of protease inhibitors (250 μm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin), and lysed by sonication for 10 s. LDs were isolated by sucrose density gradient centrifugation through 18.5%, 9%, and 4.1% sucrose steps and through top buffer (25 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm EGTA). Gradients were analyzed by Western blotting or by electron microscopy. Immunoprecipitation and Western Blotting—Immunoprecipitation was carried out essentially as described previously (24Morrow I.C. Rea S. Martin S. Prior I.A. Prohaska R. Hancock J.F. James D.E. Parton R.G. J. Biol. Chem. 2002; 277: 48834-48841Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Briefly, cells were lysed in 50 mm Tris, pH 7.4, 150 mm NaCl, 5 mm EDTA containing 1% Nonidet P-40, 0.1% SDS, and protease inhibitors. Equal volumes of lysates were immunoprecipitated using either Rab18 antiserum, GFP antiserum, or a non-immune rabbit serum and collected using protein A-Sepharose beads. Immunoprecipitated proteins were solubilized directly into Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting as described previously (25Martin S. Tellam J. Livingstone C. Slot J.W. Gould G.W. James D.E. J. Cell Biol. 1996; 134: 625-635Crossref PubMed Scopus (180) Google Scholar). Immunolabeled proteins were visualized using horseradish peroxidase-conjugated secondary antibodies and developed using the Supersignal ECL reagent (Pierce). Electron Microscopy—Immunoelectron microscopy of ultrathin cryosections was performed essentially as described previously (26Martin S. Ramm G. Lyttle C.T. Meerloo T. Stoorvogel W. James D.E. Traffic. 2000; 1: 652-660Crossref PubMed Scopus (41) Google Scholar, 27Martin S. Rice J.E. Gould G.W. Keller S.R. Slot J.W. James D.E. J. Cell Sci. 1997; 110: 2281-2291Crossref PubMed Google Scholar). Briefly, Vero cells transfected with GFP-Rab18 were incubated overnight in the presence of 100 μg/ml oleic acid and fixed in 2% paraformaldehyde/0.2% glutaraldehyde in 0.1 m PHEM buffer (60 mm PIPES, 25 mm HEPES, 2 mm MgCl2, 10 mm EGTA), pH 6.9, for 1 h at room temperature. Cells were embedded in 10% gelatin, cryoprotected using PVP-sucrose, and snap frozen onto specimen holders in liquid N2. Ultracryomicrotomy was performed by a slight modification of the Tokuyasu technique (28Tokuyasu K.T. Histochem. J. 1980; 12: 381-403Crossref PubMed Scopus (575) Google Scholar) as described previously (27Martin S. Rice J.E. Gould G.W. Keller S.R. Slot J.W. James D.E. J. Cell Sci. 1997; 110: 2281-2291Crossref PubMed Google Scholar), and sections were picked up with a 1:1 mixture of 2.3 m sucrose and 2% methyl cellulose (29Liou W. Geuze H.J. Slot J.W. Histochem. Cell Biol. 1996; 106: 41-58Crossref PubMed Scopus (437) Google Scholar). Grids were viewed using a Jeol 1010 transmission electron microscope. To perform immunoelectron microscopy on isolated LDs, BHK cells were transfected with GFP-Rab18 or GFP and subsequently incubated in 100 μg/ml oleic acid overnight. LDs were isolated using sucrose density gradient centrifugation as described above, and the top fractions, containing the LDs, were fixed in 4% PFA. Isolated LDs were applied to Formvar/carbon-coated copper grids and immunolabeled as described previously (25Martin S. Tellam J. Livingstone C. Slot J.W. Gould G.W. James D.E. J. Cell Biol. 1996; 134: 625-635Crossref PubMed Scopus (180) Google Scholar). Localization of GFP-Rab18 to Lipid Droplets and Apposition of Endosomal Compartments—To investigate the localization of Rab GTPases potentially involved in LD function in relation to LDs we expressed fluorescently tagged Rab5, Rab7, Rab11, and Rab18 in Vero cells. To increase LD formation, cells were incubated overnight in 100 μg/ml oleic acid conjugated to bovine serum albumin. Fatty acid concentrations higher than physiological levels have been used previously to induce the rapid formation of LDs in cultured cells (3Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar). Lower concentrations of oleic acid induced a similar formation over a longer period of time (results not shown). GFP-Rab5 and YFP-Rab11 were identified in punctate structures distributed throughout the cell (Fig. 1A) consistent with localization to early and recycling endosomes, respectively (20Sonnichsen B. De Renzis S. Nielsen E. Rietdorf J. Zerial M. J. Cell Biol. 2000; 149: 901-914Crossref PubMed Scopus (809) Google Scholar). In contrast, GFP-Rab7 was present in both small punctate vesicles and in larger endosomal vacuoles, consistent with localization to late endosomes (30Bucci C. Thomsen P. Nicoziani P. McCarthy J. van Deurs B. Mol. Biol. Cell. 2000; 11: 467-480Crossref PubMed Scopus (804) Google Scholar). All isoforms also showed varying levels of a cytosolic pool, frequently observed when Rab proteins are over-expressed (31Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Abstract Full Text PDF PubMed Scopus (888) Google Scholar). When cells were counterstained with Nile Red to identify lipid droplets, both GFP-Rab5- and YFP-Rab11-containing structures were occasionally identified in close apposition to Nile Red-positive structures, whereas GFP-Rab7-labeled endosomes were frequently observed in close apposition to LDs (Fig. 1A). However, Rab5, Rab7, and Rab11 were not observed to label the LD surface itself. In contrast, GFP-Rab18 showed specific and intense labeling of a subset of LDs (Fig. 1, B and C). In addition, GFP-Rab18 labeled the ER and small, possibly ER-associated, puncta distributed throughout the cells, as well as weak labeling in the region of the Golgi complex (Figs. 1B, 1C, and 4). In a small number of cells with very high levels of GFP-Rab18 expression there was a very strong labeling of the perinuclear region, and in these cells labeling for the Golgi marker GM130 suggested that the Golgi complex was disrupted in a similar manner to brefeldin A (results not shown). However, the predominant localization of GFP-Rab18 was to the LDs. GFP-Rab18 consistently labeled smaller sized LDs usually at the periphery of larger, unlabeled LDs, or a cluster of LDs (Fig. 1B). Intriguingly, in fixed cells GFP-Rab18 labeling was often observed to partially surround a LD, forming a crescent-shaped profile by fluorescence microscopy, suggestive of a partial enfolding of the LD surface by the Rab18 compartment (arrowheads, Fig. 1B). However, when live cells were imaged in real-time (Fig. 1C), the GFP-Rab18 profile was invariably ring-shaped, suggesting that PFA fixation altered the surface structure of the LDs. Differences between LD size between live and fixed, labeled cells have been described previously (4Targett-Adams P. Chambers D. Gledhill S. Hope R.G. Coy J.F. Girod A. McLauchlan J. J. Biol. Chem. 2003; 278: 15998-16007Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Interestingly, an additional observation in live cells, not detectable in fixed cells, was the presence of a single persistent brighter spot of Rab18 labeling occasionally observed on the LD surface, reminiscent of GFP-Rab5 on endosomal membranes (32Roberts R.L. Barbieri M.A. Pryse K.M. Chua M. Morisaki J.H. Stahl P.D. J. Cell Sci. 1999; 112: 3667-3675Crossref PubMed Google Scholar) (Fig. 1C).FIGURE 4Localization of GFP-Rab18 to LDs and the ER. Vero cells expressing GFP-Rab18 were incubated with 100 μg/ml oleic acid overnight and fixed for immunoelectron microscopy (A-C) or immunofluorescence microscopy (D and E). For immunoelectron microscopy, cryosections were labeled using anti-GFP antibodies and labeling detected using 10 nm protein A-gold. LDs were defined as large, electron-lucent structures that lacked a limiting membrane bilayer. GFP-Rab18 was highly localized to the surface of LDs (A and C), to the peripheral ER (A and D) and nuclear envelope (B), as well as groups of small, non-clathrin-coated tubulovesicular elements (TVE) adjacent to the LD surface (A) and distributed throughout the cell (B). C, in thicker cryosections Rab18 labeled a thin membrane layer adjacent to the ER, assumed to correspond to the surface of the LD itself (arrows). Localization of GFP-Rab18 to the ER was confirmed by immunofluorescent microscopic labeling for calnexin (D-E). Calnexin-positive structures were seen surrounding the GFP-Rab18-labeled LDs (D-E), and in a punctate reticular pattern throughout the cell, contiguous with, but not co-localizing with, a punctate reticular GFP-Rab18 labeling pattern (D). Note that as fluorescence intensity of GFP-Rab18 at the LD surface was frequently much brighter than the surrounding ER, simultaneous visualization of the two localizations was impracticable. ER, endoplasmic reticulum; LD, lipid droplets; NE, nuclear envelope; N, nucleus; TVE, tubulovesicular elements; arrowheads, plasma membrane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Localization of Endogenous Rab18 in Fibroblasts and Adipocytes—Although expressed GFP-Rab18, but not other tested Rab proteins, was clearly localized to LDs, we next sought to investigate the localization of the endogenous Rab18 protein, both in fibroblasts and in 3T3-L1 adipocytes, cells with a large number of active lipid droplets. A rabbit anti-serum raised against Rab18 (18Lutcke A. Parton R.G. Murphy C. Olkkonen V.M. Dupree P. Valencia A. Simons K. Zerial M. J. Cell Sci. 1994; 107: 3437-3448Crossref PubMed Google Scholar) was found to immunoprecipitate endogenous Rab18 and heterologously expressed GFP-Rab18 (Fig. 2, A and B), and to detect heterologously expressed GFP-Rab18 by immunofluorescence microscopy (results not shown). In addition, anti-Rab18 antiserum detected GFP-Rab18 immunoprecipitated using an anti-serum raised against GFP. Both endogenous Rab18 and heterologously expressed GFP-Rab18 often resolved as a doublet by Western blotting, presumably corresponding to both prenylated and non-prenylated forms. Fractionation of 3T3-L1 adipocyte cell lysates into membrane and cytosol fractions demonstrated that at steady state Rab18 was predominantly membrane associated (Fig. 2C). Expression of Rab18 was found to be higher in 3T3-L1 adipocyte lysates than in BHK or Vero cells by Western blotting (results not shown). We therefore hypothesized that expression could be directly related to LD formation. However, no change in expression of Rab18 was observed during differentiation of 3T3-L1 fibroblasts into adipocytes (results not shown). We next examined the localization of endogenous Rab18 in 3T3-L1 cells during differentiation to adipocytes. In 3T3-L1 fibroblasts, Rab18 labeling was only clearly detectable in a subset of cells containing endogenous LDs, where it was observed to localize to the LD surface (Fig. 3A). In addition, there was a low, dispersed, punctate labeling, not observed with a nonspecific anti-serum. During the differentiation process a more pronounced perinuclear labeling was detectable consistent with Golgi localization (Fig. 3A). Conversion of 3T3-L1 fibroblasts to an adipocyte phenotype, characterized by the accumulation of large amounts of neutral lipid, coincided with the localization of endogenous Rab18 to the LD surface. Rab18 was observed to label LDs with a distinct, punctate labeling pattern (Fig. 3A). A similar labeling pattern was observed in oleic acid-treated Vero cells, in which a small subset of LDs were labeled heavily for Rab18, whereas a large number of LDs had a single punctate dot of Rab18 labeling associated with the surface (Fig. 3A). Thus in both adipocytes and non-adipocyte cell lines endogenous Rab18 associates with a distinct subset of LDs. Rather than a spectrum of different labeling densities on different LDs, distinct LDs are either very strongly labeled or show negligible labeling. In conclusion, Rab18 was the only Rab protein that appeared to show specific localization to the surface of LDs as judged by light microscopy. Whether this repr" @default.
• W2117788306 created "2016-06-24" @default.
• W2117788306 creator A5012441246 @default.
• W2117788306 creator A5022150724 @default.
• W2117788306 creator A5029211283 @default.
• W2117788306 creator A5043736219 @default.
• W2117788306 creator A5078361771 @default.
• W2117788306 date "2005-12-01" @default.
• W2117788306 modified "2023-10-03" @default.
• W2117788306 title "Regulated Localization of Rab18 to Lipid Droplets" @default.
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