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• W2042070545 abstract "To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes. To examine the oligomeric state and trafficking of the dopamine transporter (DAT) in different compartments of living cells, human DAT was fused to yellow (YFP) or cyan fluorescent protein (CFP). YFP-DAT and CFP-DAT were transiently and stably expressed in porcine aortic endothelial (PAE) cells, human embryonic kidney (HEK) 293 cells, and an immortalized dopaminergic cell line 1RB3AN27. Fluorescence microscopic imaging of cells co-expressing YFP-DAT and CFP-DAT revealed fluorescence resonance energy transfer (FRET) between CFP and YFP, which is consistent with an intermolecular interaction of DAT fusion proteins. FRET signals were detected between CFP- and YFP-DAT located at the plasma membrane and in intracellular membrane compartments. Phorbol esters or amphetamine induced the endocytosis of YFP/CFP-DAT to early and recycling endosomes, identified by Rab5, Rab11, Hrs and EEA.1 proteins. Interestingly, however, DAT was mainly excluded from Rab5- and Hrs-containing microdomains within the endosomes. The strongest FRET signals were measured in endosomes, indicative of efficient oligomerization of internalized DAT. The intermolecular DAT interactions were confirmed by co-immunoprecipitation. A DAT mutant that was retained in the endoplasmic reticulum (ER) after biosynthesis was used to show that DAT is oligomeric in the ER. Moreover, co-expression of an ER-retained DAT mutant and wild-type DAT resulted in the retention of wild-type DAT in the ER. These data suggest that DAT oligomers are formed in the ER and then are constitutively maintained both at the cell surface and during trafficking between the plasma membrane and endosomes. Plasma membrane transporters belonging to the family of Na+/Cl–-dependent neurotransmitter transporters play an important role in terminating the activity of the monoamine neurotransmitters and of γ-aminobutyric acid (see Ref. 1Zahniser N.R. Doolen S. Pharmacol. Ther. 2001; 92: 21-55Crossref PubMed Scopus (240) Google Scholar). Thus, reuptake of dopamine (DA) 1The abbreviations used are: DA, dopamine; DAT, dopamine transporter; GFP, YFP, and CFP, green, yellow, and cyan fluorescent proteins; FRET, fluorescence resonance energy transfer; FRETC, corrected FRET; PAE, porcine aortic endothelial; ER, endoplasmic reticulum; PMA, phorbol 12-myristate 13-acetate; TM, predicted transmembrane domains; sulfo-NHS-S-S-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; SERT, serotonin transporter; GAT-1, γ-aminobutyric acid transporter; EGFR, epidermal growth factor receptor; CMF-PBS, Ca2+,Mg2+-free phosphate-buffered saline; DTT, dithiothreitol; FRETN, normalized sensitized FRET.1The abbreviations used are: DA, dopamine; DAT, dopamine transporter; GFP, YFP, and CFP, green, yellow, and cyan fluorescent proteins; FRET, fluorescence resonance energy transfer; FRETC, corrected FRET; PAE, porcine aortic endothelial; ER, endoplasmic reticulum; PMA, phorbol 12-myristate 13-acetate; TM, predicted transmembrane domains; sulfo-NHS-S-S-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; SERT, serotonin transporter; GAT-1, γ-aminobutyric acid transporter; EGFR, epidermal growth factor receptor; CMF-PBS, Ca2+,Mg2+-free phosphate-buffered saline; DTT, dithiothreitol; FRETN, normalized sensitized FRET. from the synaptic cleft by the dopamine transporter (DAT) serves as the major mechanism for terminating dopaminergic neurotransmission in the brain (2Giros B. Jaber M. Jones S.R. Wightman R.M. Caron M.G. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2040) Google Scholar). Because the efficiency of DA removal depends on the number of DAT molecules expressed at the plasma membrane, trafficking processes that control transporter distribution in the cell represent a potentially important mechanism by which neurotransmission could be regulated. Newly synthesized DAT acquires glycosylation in the endoplasmic reticulum (ER) and Golgi complex and is then trafficked to the plasma membrane. Typically, a large pool of mature DAT molecules are found at the cell surface of dopaminergic neurons and when DAT is heterologously expressed in tissue culture cells. However, DAT localization can be altered rapidly. Acute exposure of cells to either phorbol esters or substrates reduces the number of plasma membrane DATs and thus DAT function, and this reduction is due to acceleration of DAT endocytosis through a dynamin-dependent mechanism (3Daniels G.M. Amara S.G. J. Biol. Chem. 1999; 274: 35794-35801Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 4Gulley J.M. Kosobud A.E. Rebec G.V. Neurosci Lett. 2002; 322: 165-168Crossref PubMed Scopus (8) Google Scholar, 5Melikian H.E. Buckley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar, 6Saunders C. Ferrer J.V. Shi L. Chen J. Merrill G. Lamb M.E. Leeb-Lundberg L.M. Carvelli L. Javitch J.A. Galli A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6850-6855Crossref PubMed Scopus (322) Google Scholar). Recently, it has been shown that DAT can interact with the anchoring protein PICK 1 and the adaptor protein Hic-5 (7Torres G.E. Yao W.D. Mohn A.R. Quan H. Kim K.M. Levey A.I. Staudinger J. Caron M.G. Neuron. 2001; 30: 121-134Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 8Carneiro A.M. Ingram S.L. Beaulieu J.M. Sweeney A. Amara S.G. Thomas S.M. Caron M.G. Torres G.E. J. Neurosci. 2002; 22: 7045-7054Crossref PubMed Google Scholar). However, in general, the molecular mechanisms controlling DAT trafficking are not yet well understood. The DAT molecule is predicted to have 12 membrane-spanning sequences with both amino and carboxyl termini oriented intracellularly. The specific function of DAT transmembrane (TM) motifs and termini are not well established. TM domains may play a role in intra- and intermolecular interactions. Many membrane receptors and other integral membrane proteins require dimerization or higher oligomerization for their activity. Several lines of evidence have suggested that monoamine transporters are also dimers or oligomers. Results with dominant negative forms of the serotonin transporter (SERT) and norepinephrine transporter were consistent with this idea (9Chang A.S. Starnes D.M. Chang S.M. Biochem. Biophys. Res. Commun. 1998; 249: 416-421Crossref PubMed Scopus (36) Google Scholar, 10Kitayama S. Ikeda T. Mitsuhata C. Sato T. Morita K. Dohi T. J. Biol. Chem. 1999; 274: 10731-10736Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Oligomerization of SERT was demonstrated directly by co-immunoprecipitation (11Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (188) Google Scholar). The results of fluorescence resonance energy transfer (FRET) studies further confirmed that SERT and a γ-aminobutyric acid transporter (GAT-1) are homo-oligomers (12Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 13Scholze P. Freissmuth M. Sitte H.H. J. Biol. Chem. 2002; 277: 43682-43690Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Early radiation inactivation studies also suggested that DAT exists as a dimer or oligomer (14Berger S.P. Farrell K. Conant D. Kempner E.S. Paul S.M. Mol. Pharmacol. 1994; 46: 726-731PubMed Google Scholar, 15Milner H.E. Beliveau R. Jarvis S.M. Biochim. Biophys. Acta. 1994; 1190: 185-187Crossref PubMed Scopus (46) Google Scholar). Recently, the potential for DAT to exist as dimer or higher-order oligomer in the plasma membrane has been demonstrated using chemical cross-linking (16Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (171) Google Scholar). More recently, Torres and co-workers (17Torres G.E. Carneiro A. Seamans K. Fiorentini C. Sweeney A. Yao W.D. Caron M.G. J. Biol. Chem. 2003; 278: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) reported detection of DAT oligomerization by co-immunoprecipitation. In general, the mechanisms and functional roles of oligomerization of monoamine transporters remain to be defined. However, it has been proposed that SERT oligomerization may be important for its transport activity (9Chang A.S. Starnes D.M. Chang S.M. Biochem. Biophys. Res. Commun. 1998; 249: 416-421Crossref PubMed Scopus (36) Google Scholar). Oligomerization may be also required for proper trafficking of the newly synthesized transporter to the plasma membrane (13Scholze P. Freissmuth M. Sitte H.H. J. Biol. Chem. 2002; 277: 43682-43690Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17Torres G.E. Carneiro A. Seamans K. Fiorentini C. Sweeney A. Yao W.D. Caron M.G. J. Biol. Chem. 2003; 278: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Hence, we generated fluorescent human DAT fusion proteins, which allow visualization of DAT interactions during trafficking in living cells, and then used these proteins in conjunction with high resolution FRET microscopy to examine DAT oligomerization in various cellular compartments. The effective energy transfer between cyan (CFP) and yellow fluorescent protein (YFP) occurs when two proteins are 1–5 nm apart. This close proximity typically requires direct interaction of proteins tagged with YFP and CFP. Measurements of sensitized FRET signals (fluorescence of the acceptor due to energy transfer from excited donor) on a pixel-by-pixel basis are designed to visualize protein-protein interactions in individual compartments of single living cells. Using this approach, we found that DAT is oligomerized in all cellular compartments in which it is localized, including post-synthetic compartments, plasma membrane, and endosomes, where DAT is accumulated when endocytosis is stimulated by phorbol esters or the DAT substrate amphetamine. Chemicals and Antibodies—Phorbol 12-myristate 13-acetate (PMA), d-amphetamine sulfate, DA, pargyline, and catechol were purchased from Sigma (St. Louis, MO). (–)-Cocaine HCL was obtained from National Institute on Drug Abuse/RTI International (Research Triangle Park, NC). Polyclonal rabbit antibody to GFP ab290 was purchased from Abcam Ltd. (Cambridge, UK); monoclonal mouse antibody to GFP was from Zymed Laboratories (South San Francisco, CA); monoclonal rat antibody to the amino terminus of DAT was from Chemicon, Inc. (Temecula, CA); and monoclonal mouse antibody to EEA.1 was from Transduction Laboratories, Inc. (San Diego, CA). [3H]DA (3,4-[ring-2,5,6-3H]dihydroxyphenylethylamine hydrochloride; specific activity, 60.0 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Plasmids—To generate the fusion proteins of human DAT (18Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar) with YFP (YFP-DAT) or CFP (CFP-DAT), a DNA fragment encoding the full-length DAT was obtained by PCR using Pfu polymerase (Stratagene Cloning Systems, La Jolla, CA), with a KpnI restriction site introduced into the 5′-end and a SmaI site into the 3′-end, and cloned into the pEYFP-C1 vector or pECFP-C1 vector (Clontech, Palo Alto, CA), respectively. To generate YFP-DAT′C615, a stop codon was created at the position corresponding to amino acid residue 616 in the full-length DAT/pEYFP-C1 using a QuikChange site-directed mutagenesis kit according to the manufacture's protocol (Stratagene). To generate YFP-ΔN-DAT lacking amino-terminal cytoplasmic portion, a DNA fragment encoding the DAT sequence corresponding to amino acid residues 66–620 was obtained by PCR using Pfu polymerase with a KpnI restriction site introduced into the 5′-end and a SmaI site into the 3′-end, and cloned into the pEYFP-C1 vector. Human Rab5a and Rab11a cDNAs were obtained from Guthrie cDNA Resource Center (Guthrie Research Institute, Sayre, PA). To generate YFP fusion proteins, full-length Rab5a and Rab11a were amplified by PCR and ligated into pEYFP-C1 by using the BglII and XhoI restriction sites. All constructs were verified by dideoxynucleotide sequencing. The construct EGFR-CFP was previously described (19Sorkin A. McClure M. Huang F. Carter R. Curr. Biol. 2000; 10: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). YFP-Hrs (hepatocyte-growth factor receptor substrate) was provided by Dr. H. Stenmark (Radium Institute, Oslo, Norway). Cell Culture and Transfections—Porcine aortic endothelial (PAE) cells were grown in F12 medium containing 10% fetal bovine serum, antibiotics, and glutamine. HEK293T cells (further referred to as HEK293) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, antibiotics, and glutamine. Cells were grown to 50–80% confluency and transfected with appropriate plasmids using Effectine (Qiagen, Hilden, Germany). In transient transfection experiments, the cells were split 1 day after transfection onto glass coverslips and used for experiments on the second or third day at about 50% confluency. For biochemical immunoprecipitation and biotinylation experiments the cells were plated into 100- or 35-mm dishes, respectively, and used for experiments on the second day at about 90% confluency. Entactin-Collagen IV-Laminin coating (Upstate Biotechnology, Lake Placid, NY) was applied to 35-mm dishes to increase HEK293 cell adherence. The immortalized dopaminergic cell line 1RB3AN27 was kindly provided by Dr. K. Prasad (University of Colorado Health Sciences Center) (20Clarkson E.D. Rosa F.G. Edwards-Prasad J. Weiland D.A. Witta S.E. Freed C.R. Prasad K.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1265-1270Crossref PubMed Scopus (51) Google Scholar). These cells were grown in RPMI medium supplemented with 10% fetal bovine serum and antibiotics. For selection of stable clones, PAE cells transfected with CFP-DAT, YFP-DAT, DAT, or YFP-ΔN-DAT were grown with G418 (400 μg/ml) to obtain a pool of constitutively expressing cells. The single cell clone selection was then carried out by the limited dilution method in 96-well dishes. PAE cells stably expressing DAT were transfected with a mixture of YFP-ΔN-DAT and pSUMS-Hygro constructs (10:1) and grown with G418 and hygromycin (0.2 μg/ml) to obtain clones stably expressing both DAT and YFP-ΔN-DAT. Uptake Assays—PAE cells expressing hDAT, CFP-DAT, or YFP-DAT were grown in 12-well plates for 2–3 days. The cells were rinsed and then assayed in Krebs-Ringer HEPES buffer (KRH; 120 mm NaCl, 4.7 mm KCl, 2.2. mm CaCl2, 1.2 mm Mg SO4, 1.2 mm KH2PO4, 10 mm glucose, 10 mm HEPES, pH 7.4) supplemented with 10 μm pargyline, 10 μm ascorbic acid, and 10 μm catechol. Assays (1 ml) included 50 nm [3H]DA plus increasing amounts of unlabeled DA (final concentrations 50 nm to 50 μm). Nonspecific [3H]DA accumulation was determined in the presence of 1 mm cocaine and averaged 2.0 ± 0.2% of total. After 10 min of incubation at 37 °C, uptake was terminated by quickly washing the cells three times with 1 ml of ice-cold KRH. Cells were then solubilized in 0.5 ml of 3% trichloroacetic acid for 30 min with gently shaking. Accumulated [3H]DA was determined by liquid scintillation counting. Data were analyzed using GraphPad Prism (San Diego, CA). Immunoprecipitation—PAE cells stably expressing DAT and/or YFP-ΔN-DAT grown on 100-mm dishes to about 90% confluency were washed three times with Ca2+,Mg2+-free phosphate-buffered saline (CMF-PBS). The cells were then solubilized by scraping with a rubber policeman in lysis buffer (50 mm NaCl, 2 mm MgCl2, 20 mm HEPES, pH 7.2, 10% glycerol, 10 mm dithiothreitol (DTT), 1 mm EGTA, 1 mm EDTA, 10 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 544 μm iodoacetamide, 10 μg/ml aprotinin, 1% Triton X-100, 1% sodium deoxycholate) and by further incubating for 10 min at 4 °C. The lysates were then cleared by centrifugation for 10 min at 16,000 × g and incubated with polyclonal antibodies to GFP (18 μg per dish) overnight at 4 °C. Under these conditions, at least 95% of YFP-ΔN-DAT was immunoprecipitated. The precipitates were washed twice with lysis buffer supplemented with 100 mm NaCl, washed once without NaCl, and then denatured by heating in sample buffer for 5 min at 95 °C. The immunoprecipitates and the aliquots of cell lysates were resolved on 7.5% SDS-PAGE, and the proteins were transferred to the membrane. Western blotting was performed with monoclonal antibodies to GFP or DAT followed by species-specific secondary antibodies conjugated with horseradish peroxidase. The enhanced chemiluminescence kit was purchased from Pierce (Rockford, IL). Surface Biotinylation—PAE or HEK293T cells were grown in 35-mm dishes and used at 100% confluency. Cells were washed twice with cold phosphate-buffered saline containing 0.1 mm CaCl2 and 1 mm MgCl2 (PBS) and incubated for 20 min on ice with 1 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (EZ-Link™ sulfo-NHS-S-S-biotin, Pierce) in PBS, followed by a second incubation with fresh NHS-S-S-biotin. After biotinylation, the cells were washed twice with cold PBS, incubated 20 min on ice with 0.1 m glycine in PBS, washed with PBS again, and solubilized in lysis buffer (see above) supplemented with 10 mm Tris-HCl (pH 6.8) in which DTT was omitted. Cell lysates were precleared by centrifugation at 16,000 × g; the biotinylated proteins were precipitated with NeutrAvidin™ beads (Pierce), washed five times with lysis buffer, incubated in sample buffer supplemented with 100 mm DTT for 10 min to cleave disulfide bonds, and denatured by heating the beads in sample buffer at 95 °C for 5 min. Supernatants from the NeutrAvidin precipitation and the aliquots of cell lysates were further subjected to immunoprecipitation with rat anti-DAT or polyclonal anti-GFP. NeutrAvidin beads and immunoprecipitates were analyzed by electrophoresis and Western blotting. Immunofluorescence Staining—PAE cells expressing CFP-DAT were grown on glass coverslips and treated with Me2SO, PMA, or amphetamine at 37 °C. The cells were then washed with CMF-PBS and fixed with freshly prepared 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 15 min at room temperature and mildly permeabilized using a 3-min incubation in CMF-PBS containing 0.1% Triton X-100 and 0.5% bovine serum albumin at room temperature. Cells were then incubated in CMF-PBS containing 0.5% bovine serum albumin at room temperature for 1 h with a monoclonal antibody to EEA.1 and then incubated for 30 min with a secondary donkey anti-mouse IgG labeled with Cy3 (Jackson Laboratories, West-Glove, PA). Both primary and secondary antibody solutions were precleared by centrifugation at 100,000 × g for 10 min. After staining, the coverslips were mounted in Fluoromount-G (Southern Biotech Inc., Birmingham, AL) containing 1 mg/ml para-phenylenediamine. To obtain high resolution three-dimensional images of cells, the fluorescence imaging workstation consisted of a Nikon inverted microscope equipped with a 100× oil immersion objective lens, cooled charge-coupled device Sensi-Cam QE 16 MHz (Cooke, Germany), z-step motor, dual filter wheels, and a xenon 175-watt light source, all controlled by SlideBook 3.0 software (Intelligent Imaging Innovation, Denver, CO). Typically, 25–40 serial two-dimensional images were recorded at 100- to 200-nm intervals. A Z-stack of images obtained was deconvoluted using a modification of the constrained iteration method. Final arrangement of all images was performed using Adobe Photoshop. Live Cell Microscopy and FRET Imaging—PAE or HEK293 cells transiently or stably co-expressing various YFP/CFP-tagged proteins were grown on glass coverslips. The coverslips were mounted in a microscope chamber and placed on a microscope stage. To visualize CFP and YFP, consecutive images were acquired through corresponding filter channels. The method of sensitized FRET measurement has been described previously (19Sorkin A. McClure M. Huang F. Carter R. Curr. Biol. 2000; 10: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 21Jiang X. Sorkin A. Mol. Biol. Cell. 2002; 13: 1522-1535Crossref PubMed Scopus (170) Google Scholar). We previously validated this technique by measuring FRET intensities in various control interaction pairs and by using the donor fluorescence recovery method (21Jiang X. Sorkin A. Mol. Biol. Cell. 2002; 13: 1522-1535Crossref PubMed Scopus (170) Google Scholar). Briefly, images were acquired sequentially through YFP, CFP, and FRET filter channels. Filter sets used were: YFP (excitation, 500/20 nm; emission, 535/30 nm), CFP (excitation, 436/10 nm; emission, 470/30 nm), and FRET (excitation, 436/10 nm; emission, 535/30 nm). An 86004BS dichroic mirror (Chroma, Inc.) was utilized. Binning 2 × 2 modes and 100- to 250-ms integration times were used. The background images were subtracted from the raw images prior to carrying out FRET calculations. Corrected FRET (FRETC) was calculated on a pixel-by-pixel basis for the entire image using Equation 1: FRETC = FRET – (0.50 × CFP) – (0.02 × YFP), where FRET, CFP, and YFP correspond to background-subtracted images of cells co-expressing CFP and YFP acquired through the FRET, CFP, and YFP channels, respectively. 0.50 and 0.02 are the fractions of bleed-through of CFP and YFP fluorescence, respectively, through the FRET filter channel. The negative FRETC values obtained in some control experiments are due to slight overestimation of the bleed-through coefficients. FRETC values were calculated from the mean fluorescence intensities for each selected sub-region of the image containing individual ruffles, phylopodia and lamellipodia (plasma membrane), diffuse fluorescence of the plasma membrane, endosomes, and tubular-vesicular ER structures according to Equation 1. Normalized sensitized FRET (FRETN) values for individual cellular compartments were calculated according to Equation 2: FRETN = FRETC/YFP × CFP, where FRETC, CFP, and YFP are the mean intensities of FRETC, CFP, and YFP fluorescence in the selected sub-region. In these calculations, the fluorescence intensity of CFP was underestimated because of donor fluorescence quenching due to FRET. The decrease in donor fluorescence was calculated from FRETC values using the conversion coefficient G (3.2 in all experiments) as described (22Gordon G.W. Berry G. Liang X.H. Levine B. Herman B. Biophys. J. 1998; 74: 2702-2713Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar) and was found to be about 5–10% of CFP fluorescence intensities. Therefore, the underestimation did not significantly affect FRETN values. Because FRETN displays a non-linear dependence when donor or acceptor are in a significant molar excess over each other, FRETN values were calculated in the sub-regions of the cell in which the amount of the donor did not exceed the amount of the acceptor more than 2-fold and vice versa. FRETC images are presented in pseudocolor mode. FRETC intensity is displayed stretched between the low and high renormalization values, according to a temperature-based lookup table with blue (cold) indicating low values and red (hot) indicating hot values. To eliminate the distracting data from regions outside of cells, the YFP channel was used as a saturation channel, and the FRETC images are displayed as YFP intensity-modulated images. In these images, data with YFP values greater than the high threshold of the saturation channel are displayed at full saturation, whereas data values below the low threshold are displayed with no saturation (i.e. black). All calculations were performed using the Channel Math and FRET modules of the SlideBook software. Characterization of CFP-DAT and YFP-DAT—To visualize DAT in living cells, fusion proteins of full-length DAT with YFP or CFP were generated, in which YFP or CFP was fused to the amino terminus of DAT (Fig. 1A). Previous studies reported that the fusion of GFP or YFP to the amino terminus of DAT did not affect functional properties of the transporter (3Daniels G.M. Amara S.G. J. Biol. Chem. 1999; 274: 35794-35801Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 23Carvelli L. Moron J.A. Kahlig K.M. Ferrer J.V. Sen N. Lechleiter J.D. Leeb-Lundberg L.M. Merrill G. Lafer E.M. Ballou L.M. Shippenberg T.S. Javitch J.A. Lin R.Z. Galli A. J. Neurochem. 2002; 81: 859-869Crossref PubMed Scopus (174) Google Scholar). YFP-DAT and CFP-DAT were transiently or stably expressed in PAE cells. PAE cells have been our preferred cell line for fluorescence microscopic analyses, because these cells are flattened and have minimal background autofluorescence, which allows clear visualization of organelles in living cells by wide-field microscopy. The presence of functional transporters in PAE cells stably expressing YFP/CFP-DAT was confirmed by measurements of [3H]DA uptake. All cell lines exhibited specific [3H]DA uptake (Fig. 1B), defined as the difference between [3H]DA uptake in the absence and presence of 1 mm cocaine. Kinetic analysis confirmed that each cell line contained high affinity, cocaine-sensitive [3H]DA uptake with K m values in the range of 2–16 μm. CFP-DAT was expressed in PAE cells at higher levels than YFP or untagged DAT and thus tended to yield higher K m values. Importantly, however, K m values measured in PAE cells expressing YFP-DAT, CFP-DAT, or untagged DAT were not consistently different from each other (Fig. 1B) and were similar to values measured in our previous work with DAT expressed in Xenopus oocytes (24Doolen S. Zahniser N.R. J. Pharmacol. Exp. Ther. 2001; 296: 931-938PubMed Google Scholar). The plasma membrane expression of DAT fusion proteins in PAE cells was confirmed by surface biotinylation assays using a membrane-impermeable derivative of biotin. Biotinylated untagged DAT and YFP-DAT migrated on SDS-PAGE as smeared bands of ∼73–75 kDa and 103–105 kDa (the difference corresponds to ∼26 kDa of YFP), respectively (Fig. 1C). Minor biotinylated bands of ∼180–190 kDa for DAT and ∼250 kDa for YFP-DAT were also detected. These forms may represent SDS-resistant dimers of DAT and YFP-DAT, correspondingly. Several species of DAT and YFP-DAT were also detected in the supernatants from the NeutrAvidin affinity column. The non-biotinylated forms, which migrated similar to the biotinylated forms, are likely to correspond to surface-exposed DAT (especially, in the case of YFP-DAT) that were not biotinylated due to the limited efficiency of biotinylation or internalized, endosomal DAT. Other forms with molecular weights different from the surface forms probably represent monomeric and dimeric species of DAT and YFP-DAT located intracellularly. A 75- to 80-kDa band of YFP-DAT (IC-1) recognized by antibodies to DAT (Fig. 1C) and GFP (data not shown) represents non-glycosylated YFP-DAT. The corresponding form of DAT (50 kDa) was poorly detected because of the overlap with IgG, but was clearly detectable in cell lysates (data not shown). A 180- to 190-kDa form (IC-2) may correspond to SDS-resistant dimers of IC-1 (Fig. 1C). That YFP- and CFP-DAT were efficiently transported along the biosynthetic pathway to the cell surface was also demonstrated by fluorescence microscopy. Fig. 2 shows that CFP-DAT and YFP-DAT co-expressed in PAE cells were mainly localized in the plasma membrane, often concentrating in ruffle- and phylopodia-like structures (Fig. 2). In addition, a pool of fluorescent DAT could be also seen in the perinuclear and Golgi area of many cells. These transporters probably represent newly synthesized YFP/CFP-DAT located in the ER and Golgi complex. FRET between CFP-DAT and YFP-DAT—DA uptake and cell surface biotinylation experiments validated the use of YFP-DAT and CFP-DAT expressed in PAE cells to examine the oligomeric state of these proteins in various cellular compartments. In previous studies we described a three-filter method of FRET microscopic analysis in living cells (19Sorkin A. McClure M. Huang F. Carter R. Curr. Biol. 2000; 10: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). This method allows rapid measurement of sensitized FRET signals with high spatial resolution. Fig. 2A shows that positive CFP → YFP FRETC signals were detecte" @default.
• W2042070545 created "2016-06-24" @default.
• W2042070545 creator A5022117254 @default.
• W2042070545 creator A5033899179 @default.
• W2042070545 creator A5058902481 @default.
• W2042070545 creator A5073726679 @default.
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• W2042070545 date "2003-07-01" @default.
• W2042070545 modified "2023-09-28" @default.
• W2042070545 title "Oligomerization of Dopamine Transporters Visualized in Living Cells by Fluorescence Resonance Energy Transfer Microscopy" @default.
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