Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand*♦
2005; Elsevier BV; Volume: 280; Issue: 18 Linguagem: Inglês
10.1074/jbc.m500786200
ISSN1083-351X
AutoresJérôme N. Feige, Laurent Gelman, Cicerone Tudor, Yves Engelborghs, Walter Wahli, Béatrice Desvergne,
Tópico(s)Protein Degradation and Inhibitors
ResumoIn a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells. In a global approach combining fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET), we address the behavior in living cells of the peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors involved in lipid and glucose metabolism, inflammation control, and wound healing. We first demonstrate that unlike several other nuclear receptors, PPARs do not form speckles upon ligand activation. The subnuclear structures that may be observed under some experimental conditions result from overexpression of the protein and our immunolabeling experiments suggest that these structures are subjected to degradation by the proteasome. Interestingly and in contrast to a general assumption, PPARs readily heterodimerize with retinoid X receptor (RXR) in the absence of ligand in living cells. PPAR diffusion coefficients indicate that all the receptors are engaged in complexes of very high molecular masses and/or interact with relatively immobile nuclear components. PPARs are not immobilized by ligand binding. However, they exhibit a ligand-induced reduction of mobility, probably due to enhanced interactions with cofactors and/or chromatin. Our study draws attention to the limitations and pitfalls of fluorescent chimera imaging and demonstrates the usefulness of the combination of FCS, FRAP, and FRET to assess the behavior of nuclear receptors and their mode of action in living cells. The nucleus comprises different subdomains whose biological functions remain often very elusive, if known at all, and that differ fundamentally from cytoplasmic compartments in that they are not delineated by membranes (1Misteli T. Science. 2001; 291: 843-847Crossref PubMed Scopus (524) Google Scholar, 2Lamond A.I. Spector D.L. Nat. Rev. Mol. Cell. Biol. 2003; 4: 605-612Crossref PubMed Scopus (766) Google Scholar). Several proteins or complexes involved in processes as diverse as chromatin remodeling, transcription, and RNA processing form nuclear speckles throughout the nucleoplasm. These compartments would actually result from the temporary association of highly mobile components, which shuttle between these sites (3Pederson T. Cell. 2001; 104: 635-638Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Nuclear receptors are one of the most abundant classes of transcriptional regulators in metazoans. They compose a large family of ligand-activated proteins among which are the steroid hormone receptors, the thyroid hormone receptors (TRs), 1The abbreviations used are: TR, thyroid hormone receptor; VDR, vitamin D receptor; RAR, retinoic acid receptor; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXR, retinoid X receptor; FP, fluorescent protein; GFP, green FP; YFP, yellow FP; CFP, cyan FP; EYFP, enhanced YFP; ECFP, enhanced CFP; FRAP, fluorescence recovery after photobleaching; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; AR, androgen receptor; PR, progesterone receptor; MR, mineralocorticoid receptor; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole; RXR, retinoid X receptor. 1The abbreviations used are: TR, thyroid hormone receptor; VDR, vitamin D receptor; RAR, retinoic acid receptor; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXR, retinoid X receptor; FP, fluorescent protein; GFP, green FP; YFP, yellow FP; CFP, cyan FP; EYFP, enhanced YFP; ECFP, enhanced CFP; FRAP, fluorescence recovery after photobleaching; FCS, fluorescence correlation spectroscopy; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; AR, androgen receptor; PR, progesterone receptor; MR, mineralocorticoid receptor; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole; RXR, retinoid X receptor. the vitamin D receptor (VDR), the retinoic acid receptors (RARs), as well as the peroxisome proliferator-activated receptors (PPARs). It is generally assumed that, upon ligand binding, nuclear receptors either homodimerize or heterodimerize with the retinoid X receptor (RXR) and bind to specific responsive elements in the enhancer regions of their target genes. This promotes gene activation, after a complex array of events including both dissociation from and association with numerous cofactors (4Robyr D. Wolffe A.P. Wahli W. Mol. Endocrinol. 2000; 14: 329-347Crossref PubMed Scopus (325) Google Scholar). The three PPAR isotypes (named α, β/δ, and γ or NR1C1, NR1C2, and NR1C3 (5Committee Nuclear Receptors Nomenclature Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar)) are mainly involved in lipid and glucose homeostasis, regulation of food intake and body weight, and control of inflammation and wound healing (6Michalik L. Desvergne B. Wahli W. Nat. Rev. Cancer. 2004; 4: 61-70Crossref PubMed Scopus (503) Google Scholar, 7Escher P. Wahli W. Mutat. Res. 2000; 448: 121-138Crossref PubMed Scopus (405) Google Scholar). They are activated by naturally occurring or metabolized fatty acids derived from the diet or from intracellular signaling pathways and induce gene transcription as heterodimers with RXRs (8Hihi A.K. Michalik L. Wahli W. Cell Mol. Life Sci. 2002; 59: 790-798Crossref PubMed Scopus (268) Google Scholar). PPARs are also very important therapeutic targets for the treatment of hyperlipidemia and type 2 diabetes.Recent developments in live cell imaging have prompted investigations on the localization and mobility of nuclear receptors to decipher the molecular mechanisms underlying transcriptional activation. Genetically encoded fluorescent proteins (FPs) allow to monitor directly in living cells the behavior of proteins in their physiological context (9Lippincott-Schwartz J. Patterson G.H. Science. 2003; 300: 87-91Crossref PubMed Scopus (829) Google Scholar). For instance, it is possible to assess the dynamics of a protein by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) and to evaluate the interaction between two proteins by fluorescence resonance energy transfer (FRET).Classical confocal microscopy combined with the use of FPs has first allowed to address a particularly interesting issue: the modulation of nuclear receptor localization upon ligand binding. Ligand treatment induces a redistribution of the glucocorticoid receptor (GR) (10Htun H. Barsony J. Renyi I. Gould D.L. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4845-4850Crossref PubMed Scopus (326) Google Scholar), the androgen receptor (AR) (11Tyagi R.K. Lavrovsky Y. Ahn S.C. Song C.S. Chatterjee B. Roy A.K. Mol. Endocrinol. 2000; 14: 1162-1174Crossref PubMed Scopus (250) Google Scholar, 12Tomura A. Goto K. Morinaga H. Nomura M. Okabe T. Yanase T. Takayanagi R. Nawata H. J. Biol. 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Chem. 2000; 275: 41114-41123Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) are confined in the nucleus both in the presence and absence of ligand. Ligand binding promotes the formation of nuclear speckled patterns for ER, GR, AR, TR, RAR, and RXR (see Ref. 23DeFranco D.B. Mol. Endocrinol. 2002; 16: 1449-1455Crossref PubMed Scopus (86) Google Scholar for a review). These induced clusters only partially overlap with sites of transcription (24Grande M.A. van der Kraan I. de Jong L. van Driel R. J. Cell Sci. 1997; 110: 1781-1791Crossref PubMed Google Scholar), and their role in nuclear receptor function awaits further elucidation. The need for quantification for these events then lead to the use of more elaborated techniques, such as FRAP. Mobility studies have focused on the diffusion of receptors through the nucleoplasm on the one hand and at specific interaction sites on chromatin on the other (25McNally J.G. Muller W.G. Walker D. Wolford R. Hager G.L. 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A. 2004; 101: 2876-2881Crossref PubMed Scopus (124) Google Scholar), it is not clear at present which factors modulate and orientate nuclear receptor trafficking in the cell. The steps required to initiate transcription upon ligand binding (i.e. interaction with cofactors that allow chromatin remodeling and recruitment of the basic transcription machinery) also possibly influence nuclear receptor mobility. With that respect, FRET has emerged as a technique to characterize the mechanisms of cofactor recruitment following ligand binding in living cells (31Llopis J. Westin S. Ricote M. Wang Z. Cho C.Y. Kurokawa R. Mullen T.M. Rose D.W. Rosenfeld M.G. Tsien R.Y. Glass C.K. Wang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4363-4368Crossref PubMed Scopus (131) Google Scholar, 32Bai Y. Giguère V. Mol. Endocrinol. 2003; 17: 589-599Crossref PubMed Scopus (57) Google Scholar).The microscopy studies published so far point to two major issues: first, despite some common features, each nuclear receptor displays specific distributions and dynamics, justifying a detailed analysis of each member of this family. Second, each technique has its own spatial and temporal resolutions, which sometimes limit a comprehensive study. Our aim here was to study PPAR action in living cells by combining complementary microscopy techniques. As a first step, we characterize the function of enhanced yellow fluorescent protein (EYFP)-PPAR fluorescent chimeras. The presence and the physiological relevance of the fluorescent spots that may arise under certain experimental conditions was analyzed, and we demonstrate that these structures are non physiological and result from overexpression in individual cells. Regarding mobility, FRAP indicates that PPARs are highly mobile receptors that are not immobilized by ligand binding. A FCS study undertaken to circumvent FRAP time scale limitations gives two new major insights into PPAR behavior in living cells. First, in the absence of ligand, PPARs diffuse in the nucleus either in association with very big complexes and/or transiently interact with relatively immobile nuclear components. Second, ligand binding slows down PPARs, most probably by enhancing interactions with cofactors and chromatin. Moreover, FRET experiments demonstrate that PPARs readily form heterodimers with RXR in the absence of ligand. Finally, this study suggests guidelines for an appropriate examination of molecular behaviors in living cells.EXPERIMENTAL PROCEDURESMaterials—Rosiglitazone and 17β-estradiol were purchased from Sigma, Wy14,643 from Cayman Chemical Co. (Ann Arbor, MI), and 9-cis-retinoic acid from Biomol Laboratories (Plymouth Meeting, PA). L-165041 was synthesized in the laboratory by Marco Alves. The antibody directed against PPARα has been previously described (33Lemberger T. Saladin R. Vazquez M. Assimacopoulos F. Staels B. Desvergne B. Wahli W. Auwerx J. J. Biol. Chem. 1996; 271: 1764-1769Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), and those directed against PPARβ, PPARγ, and GFP were purchased from Affinity Bioreagents (Golden, CO), Wak-Chemie (Steinbach, Germany), and Roche Diagnostics (Rotkreuz, Switzerland), respectively. The antibodies against the α/β subunits of the 20 S proteasome and ubiquitylated proteins were from Affiniti Research Products (Mamhead, UK).Plasmid Constructs—cDNAs encoding mouse PPARα, PPARβ, and PPARγ1 as well as RXRα were subcloned after PCR amplification into the pEYFP-N1 and -C1, and pECFP-N1 and -C1 plasmids (Clontech) using XhoI/BamHI, BglII/SalI, XhoI/KpnI, and BglII/SalI restriction sites, respectively. Wild-type mPPARα was expressed from a cDNA cloned in the pBK-CMV vector. The pEYFPC1-ERα vector, the (PPRE)3-luciferase (Luc) reporter construct and the ECFP-DEVD-EYFP construct were kind gifts of Drs. H. Vogel (EPFL, Lausanne, Switzerland), R. M. Evans (Salk institute, San Diego, CA), and J. M. Tavaré (West-phalian Wilhelms University, Münster, Germany), respectively.Cell Culture and Transient Transfection Assays—COS-7 and MCF-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen). Penicillin and streptomycin (Invitrogen) were added to the medium at 100 units/ml and 100 μg/ml, respectively.Transient transfection assays were performed using Lipofectamine 2000 (Invitrogen), and luciferase activity assays were performed with the Promega dual reporter kit, according to the manufacturers' instructions. Renilla luciferase encoded by the normalization vector phRLTK (Promega) was used as an internal control for firefly luciferase normalization. Cells were plated in 12-well plates for luciferase assays or 4-well LabTek chambered coverglasses (Nunc) for microscopy studies. Unless otherwise stated, transfections were performed with 100 ng of vector per cm2 cell culture plate. After transfection, cells were left to recover in medium supplemented with 10% fetal calf serum for 5 h and grown in serum-free medium in the presence of PPAR ligands or vehicles for 18 h. Ligand concentrations were: 10–5 m for Wy14,643, 5.10–6m for l-165041, 10–6 m for rosiglitazone, 10–6m for 9-cis-retinoic acid, and 10–8 m for 17β-estradiol.Western Blots—Cells or tissues were lysed in ice-cold lysis buffer (20 mm Tris, 400 mm KCl, 2 mm dithiothreitol, 0.1 mm EDTA, glycerol 20%) supplemented with complete protease inhibitors (Roche Diagnostics) with a Dounce homogenizer. Protein extracts were resolved by SDS-PAGE as previously described (34Gelman L. Zhou G. Fajas L. Raspe E. Fruchart J.C. Auwerx J. J. Biol. Chem. 1999; 274: 7681-7688Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Primary antibodies against PPARα, PPARβ, and PPARγ were all used at dilutions of 1:2000 and the anti-GFP antibody was diluted 1:5000. Detection was performed using chemiluminescence (ECL advance, Amersham Biosciences).Pull-down Experiments—For DNA pull-downs, plates were coated with oligonucleotides containing the malic enzyme PPAR response element as previously described (35IJpenberg A. Tan N.S. Gelman L. Kersten S. Seydoux J. Xu J. Metzger D. Canaple L. Chambon P. Wahli W. Desvergne B. EMBO J. 2004; 23: 2083-2091Crossref PubMed Scopus (163) Google Scholar). For cofactor pull-downs, the GST-p3002–516 fusion protein was purified as described previously (34Gelman L. Zhou G. Fajas L. Raspe E. Fruchart J.C. Auwerx J. J. Biol. Chem. 1999; 274: 7681-7688Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). For both DNA and cofactor pull-downs, whole-cell extracts from transfected COS-7 cells were incubated 2.5 h at 4 °C in pull-down buffer in the oligonucleotide coated plates or with the GST-p3002–516 coated beads, and the appropriate PPAR ligands (Wy14,643 at 100 μm, L-165041 at 50 μm, and rosiglitazone at 10 μm). Plates or beads were washed four times with binding buffer and samples were boiled with 40 μl of 2× SDS-PAGE buffer and analyzed by Western blot.Immunofluorescence—Wounded skin was prepared as described previously (36Michalik L. Desvergne B. Tan N.S. Basu-Modak S. Escher P. Rieusset J. Peters J.M. Kaya G. Gonzalez F.J. Zakany J. Metzger D. Chambon P. Duboule D. Wahli W. J. Cell Biol. 2001; 154: 799-814Crossref PubMed Scopus (366) Google Scholar). Skin cryosections or transfected cells were fixed in 4% paraformaldehyde for 10 min. For the anti-PPARβ immunofluorescence, after an antigen-unmasking step (citrate buffer, pH 6, 100 °C, 10 min), the following processing was applied: wash with PBS, 0.1% Triton X-100 for 10 min; PBS for 5 min; 10% normal goat serum (Vector Laboratory) for 1 h; PBS, 2% BSA for 5 min. The primary antibody against PPARβ (Affinity Bioreagents) was applied overnight at 4 °C at 1/100th in PBS, 2% BSA, and slices were then incubated with a fluorescein isothiocyanate-coupled goat anti-rabbit IgG secondary antibody. Antibody incubations were followed by two 5-min washes in PBS, 2% BSA, and samples were mounted in DAPI containing-Vectashield mounting medium (Vector Laboratories). For the anti-20 S proteasome and anti-ubiquitin immunofluorescence, a classical protocol was applied using PBS, 2% BSA as a blocking agent and antibody dilutions of 1/1000 for 1 h and 1/500 overnight at 4 °C, respectively. Labeling was then performed with a chicken anti-mouse secondary antibody coupled to Alexa594 (Molecular Probes).Confocal Imaging—Live cells on LabTek chambered coverglasses were washed once with phenol red free Optimem medium (Invitrogen) containing ligands or their vehicles and observed in the same medium. For perfusion experiments, cells were grown on 18-mm round coverglasses, placed in a Ludin perfusion chamber (Life Imaging Service), and perfused at 200 μl/min. All observations were performed at 37 °C on a TCS SP2 AOBS confocal microscope (Leica) equipped with a whole-microscope incubator (Life Imaging Service). Acquisitions were performed with a 63×/NA 1.2 water immersion objective for live-cell imaging and a 63×/NA 1.4 oil immersion objective for immunofluorescence observation. Observation of EYFP fusion proteins was done by exciting at 514 nm and detecting emission between 525 and 575 nm. DAPI, fluorescein isothiocyanate, and Alexa594 were measured in the following respective settings: excitation 405 nm/emission 410–465 nm, excitation 488 nm/emission 500–600 nm, and excitation 594 nm/emission 620–660 nm. For quantitative analyses, background-corrected 8-bit images were acquired with identical settings (same laser power and detector gain) and quantified using the Leica Confocal Software version 2.4.FRET Experiments—Transfections were performed as described above, but expression levels of donor and acceptor proteins were adjusted to similar levels by Western blot. Fluorescence was recorded in three different settings: CFPex, 405 nm/CFPem, 465–485 nm; YFPex, 514 nm/YFPem, 525–545 nm; FRETex, 405 nm/FRETem, 525–545 nm. Laser power and detector gain were adjusted in the different channels to observe equimolar concentrations of CFP and YFP at equal intensities (equimolar concentrations of CFP and YFP were obtained by expressing a fusion protein of CFP and YFP spaced by 475 residues). Settings were kept unchanged for analysis of all samples. CFP and YFP spectral bleed-throughs (BT) in the FRET setting were determined on cells expressing CFP or YFP alone by calculating the intensity (I) ratios in the appropriate settings (IFRET/ICFP and IFRET/IYFP, respectively). FRET measured in co-expressing cells was then corrected for spectral bleed-throughs and normalized (NFRET) for expression levels according to the following formula (37Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). NFRET=IFRET−ICFP∗BTCFP−IYFP∗BTYFPICFP∗IYFP(Eq. 1) The image of FRET was generated with the "PixFRET" plug-in for the ImageJ software. This plug-in can be downloaded from www.unil.ch/cig/page16989.html.FRAP Experiments—FRAP experiments were performed on Leica TCS SP2 AOBS or Zeiss LSM510 confocal microscopes. The LSM510 microscope was equipped with an argon 488-nm laser and a 505–550-nm bandpass filter, and image analysis was performed with the Zeiss LSM software version 3.2. For qualitative analysis (images only), images were acquired with a 256 × 256-pixel resolution. However, for the quantitative analysis presented in Fig. 5 (recovery curves), the resolution was switched to 128 × 128 pixels, and forward-reverse scanning was applied allowing acquisition of images every 100 ms. Photobleaching was performed with maximal laser power of the 488 laser line, and bleaching time was minimized by analyzing bleaching efficiency on fixed cells (eight scans at 800 Hz on the TCS SP2 and 50 iterations on the LSM510). In Fig. 6 (LSM510), the bleached region was a thin strip (100 × 5 pixels) across the nucleus not exceeding 10% of the total nucleus area. Experiments were performed with a pinhole set to 2.5 airy units. Fluorescence intensities were calculated in the bleached zone and over the entire cell nucleus with NIH ImageJ version 1.30 or Zeiss LSM softwares. Fluorescence recovery was normalized to the bleaching of the entire nucleus as described previously (38Phair R.D. Misteli T. Nature. 2000; 404: 604-609Crossref PubMed Scopus (953) Google Scholar): recovery = (It/Io)*(To/Tt), where It is the average intensity in the region of interest in time point t, Io the average intensity in the region of interest during prebleach, Tt is the total nuclear intensity at time point t, and To is the average total nuclear intensity during prebleach. To extract half-recovery times and percentages of immobilization, the Origin 7.5 software (OriginLab, Northampton, MA) was used for nonlinear regression analysis. Recovery curves were fitted to the following equation. y(t)=y0+A1·(1−e−B1·t)+A2·(1−e−B2·t)(Eq. 2) Maximal recovery (Mr) was the sum of y0, A1, and A2, and half-recovery time the value of t when y = Mr/2.Fig. 6FRAP reveals that EYFP-PPARs are highly mobile both in ligand treated and untreated cells. Live COS-7 cells displaying a diffuse pattern for EYFP-PPARα (A), EYFP-ERα (B), EYFP-PPARβ (C), EYFP-PPARγ1 (D), EYFP-PPARα with wild-type RXRα (E) or EYFP-RXRα (F) were subjected to FRAP analysis on a Zeiss LSM510 microscope in the absence or presence of the respective ligands. For each condition, the normalized fluorescence recovery was averaged over 12 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FCS Experiments—FCS measurements were performed on a commercial LSM510 ConfoCor2 combination system (Zeiss, Jena, Germany) at room temperature. The 488-nm line of an argon-ion laser was focused through a Zeiss C-Apochromat 40×, NA 1.2 water immersion objective, and the fluorescence emission was recorded between 505 and 550 nm. The pinhole diameter was set to 90 μm, and detection was achieved using an avalanche photodiode. The 1/e2 lateral radii of the detection volume was determined to be ∼0.18 μm (488 nm excitation) from calibration measurements using standard dye (Rhodamine 6G, Molecular Probes). This value was used to convert diffusion times into diffusion coefficients. The setup was shown to be able to produce correct diffusion coefficients (39Krouglova T. Vercammen J. Engelborghs Y. Biophys. J. 2004; 87: 2635-2646Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Fluorescence intensity fluctuations were recorded at five spots in each nucleus. At each spot, measurements were performed over 25 s and repeated five times. On average, nine cells were analyzed. The autocorrelation curves were fitted to a one component model of free diffusion in three dimensions to derive the translational diffusion time through the confocal volume. Data were evaluated by Levenberg-Marquardt nonlinear least squares fitting to the appropriate model equations, using the ConfoCor2 or Origin software. The sizes of the EYFP-PPAR complexes were estimated by multiplying the molecular mass of EYFP by the factor (DEYFP/DEYFP-PPAR)3, assuming spherical symmetry of the complexes and normal diffusion in the viscous medium of the nucleus (i.e. EYFP experiences the same viscosity as the EYFP-PPAR complexes (40Seksek O. Biwersi J. Verkman A.S. J. Cell Biol. 1997; 138: 131-142Crossref PubMed Scopus (420) Google Scholar)).RESULTSCharacterization of EYFP-PPAR Chimeras—To monitor PPAR action in living cells, we constructed expression vectors for the different PPAR isotypes fused to EYFP either at their N or C terminus (EYFP-PPAR and PPAR-EYFP, respectively). Proper expression of the EYFP-PPARα, -β, and -γ1 fusion proteins in COS-7 cells was confirmed by Western blot (Fig. 1A). Expression levels were not affected by the position of the fluorescent tag (data not shown).Fig. 1Functional characterization of the EYFP-PPARs. A, Western blot analysis of EYFP-PPARα, EYFP-PPARβ, and EYFP-PPARγ expression in transfected COS-7 cells. Samples were resolved as described under "Experimental Procedures" with isotype-specific antibodies. B, independent DNA and cofactor interaction assays were performed using either a biotinylated double-stranded oligonucleotide containing a PPRE (DNA pull-down) or GST-p300 (cofactor pull-down). Interactions with PPARα, EYFP-PPARα, or PPARα-EYFP from COS-7 cell extracts were analyzed in the absence or presence of 100 μm of Wy14,643 and resolved with an anti-PPARα antibody. C, COS-7 cells were transfected with a (PPRE)3-Luc reporter constru
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