The cDNA construct encoding N-terminally YFP-tagged AR was generated by replacing EGFP in pGFP-(GA)6
-AR (Farla et al., 2004
) by EYFP-C1 (CLONTECH Laboratories, Inc.). The C-terminally CFP-tagged AR was generated by replacing EGFP by ECFP-N3 (CLONTECH Laboratories, Inc.) in pAR-(GA)6
-EGFP in which two AR fragments from pcDNA-AR0mcs (lacking the AR stop codon; Sui et al., 1999
) and pAR0 (Brinkmann et al., 1989
), respectively, were sequentially inserted in EGFP-N3 (CLONTECH Laboratories, Inc.) followed by the introduction of a spacer sequence coding for a (Gly-Ala)6
stretch. The construct coding for double-tagged AR (pYFP-[GA]6
-CFP) was generated by combining a fragment of N-terminally YFP-tagged AR pYFP-(GA)6
-AR with a fragment of C-terminally CFP-tagged AR pAR-(GA)6
-CFP. The F23,27A/L26A variants were generated by QuikChange (Stratagene) mutagenesis using primers 5′-ACCTACCGAGGAGCTGCACAGAATGCTGCACAGAGCGTGCGCGAA-3′ and 5′-TTCGCGCACGCTCTGTGCAGCATTCTGTGCAGCTCCTCGGTAGGT-3′. To generate the A573D variants, the AR DBDs of pYFP-AR-CFP and pAR-(GA)6
-CFP were replaced by a pGFP-AR(A573D) (Farla et al., 2004
) fragment containing the AR DBD (A573D) mutation. EYFP in pYFP-(GA)6
-AR was replaced by an ECFP-EYFP fusion to obtain pCFP-YFP-(GA)6-AR. The YFP-tagged ARA54 peptide construct was obtained by annealing the primers 5′-GATCGACCCTGGTTCACCATGTTTTAACCGGCTGTTTTATGCTGTGGATGTTG-3′ and 5′-AATTCAACATCCACAGCATAAAACAGCCGGTTAAAACATGGTGAACCAGGGTC-3′ containing the FNRLF motif and inserting the fragment in pEYFP-C2 (CLONTECH Laboratories, Inc.). Structures of novel constructs were verified by appropriate restriction digestions and by sequencing. Sizes of expressed proteins were verified by Western blotting. pCYFP encoding the ECFP-EYFP fusion was provided by C. Gazin (Hôpital Saint-Louis, Paris, France). The (ARE)2
-TATA Luc reporter was a gift from G. Jenster (Josephine Nefkens Institute, Rotterdam, Netherlands).
Cell culture, transfections, and luciferase assay
2 d before microscopic analyses, Hep3B cells were grown on glass coverslips in 6-well plates in α-MEM (Cambrex) supplemented with 5% FBS (HyClone), 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. At least 4 h before transfection, the medium was substituted by medium containing 5% dextran charcoal stripped FBS. Transfections were performed with 1 μg/well AR or CFP-YFP expression constructs or 0.5 μg/well empty vector in FuGENE6 (Roche) transfection medium. In the indicated experiments, YFP-tagged ARA54 peptide expression constructs (0.5 μg/well) were added. 4 h after transfection, the medium was replaced by medium with 5% dextran charcoal stripped FBS with or without 100 nM R1881. Hep3B cells stably expressing AR constructs were subjected to the same medium-replacement schedule.
For the AR transactivation experiments, Hep3B cells were cultured in 24-well plates on α-MEM supplemented with 5% dextran charcoal stripped FBS in the presence or absence of 100 nM R1881 and transfected using 50 ng AR expression construct and 100 ng (ARE)2TATA Luc reporter. 24 h after transfection, cells were lysed and luciferase activity was measured in a luminometer (Fluoroscan Ascent FL; Labsystems Oy). Light emission was recorded during 5 s, after a delay of 2 s.
Western blot analysis
Hep3B cells were cultured and transfected in 6-well plates. 24 h after transfection, cells were washed twice in ice-cold PBS and lysed in 200 μl Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 10 mM DTT, and 0.001% Bromophenol blue). After boiling for 5 min, a 5-μl sample was separated on a 10% SDS-polyacrylamide gel and blotted to Nitrocellulose Transfer Membrane (Protran; Schleicher and Schuell). Blots were incubated with anti-AR (1:2,000; mouse monoclonal F34.4.1) or anti–β-actin (1:10,000; mouse monoclonal anti–β-actin [Sigma-Aldrich]) and subsequently incubated with HRP-conjugated goat anti–mouse antibody (DakoCytomation). Proteins were visualized using Super Signal West Pico Luminol solution (Pierce Chemical Co.), followed by exposure to x-ray film.
Confocal imaging and FRET acceptor photobleaching
Live-cell and immunofluorescence imaging was performed using a confocal laser-scanning microscope (LSM510; Carl Zeiss MicroImaging, Inc.) equipped with a Plan-Neofluar 40×/1.3 NA oil objective (Carl Zeiss MicroImaging, Inc.) at a lateral resolution of 100 nm (FRET acceptor bleaching) or 70 nm (immunofluorescence). An argon laser was used for excitation of CFP, GFP, and YFP at 458, 488, and 514 nm, respectively, and a He/Ne laser was used to excite Cy3 at 543 nm.
Interactions between either the N- and C-terminal domain of the YFP-AR-CFP or between AR-CFP and YFP-ARA54 were assessed using acceptor photobleaching. For this, YFP and CFP images were collected sequentially before photobleaching of the acceptor. CFP was excited at 458 nm at moderate laser power, and emission was detected using a 470–500 nm bandpass emission filter. YFP was excited at 514 nm at moderate laser power, and emission was detected using a 560-nm longpass emission filter. After image collection, YFP in the nucleus was bleached by scanning a nuclear region of ~100 μm2 25 times at 514 nm at high laser power, covering the largest part of the nucleus. After photobleaching, a second YFP and CFP image pair was collected. Apparent FRET efficiency was estimated (correcting for the amount of YFP bleached) using the equation abFRET = ([CFPafter − CFPbefore] × YFPbefore)/([YFPbefore − YFPafter] × CFPafter), where CFPbefore and YFPbefore are the mean prebleach fluorescence intensities of CFP and YFP, respectively, in the area to be bleached (after background subtraction), and CFPafter and YFPafter are the mean postbleach fluorescence intensities of CFP and YFP, respectively, in the bleached area. The apparent FRET efficiency was finally expressed relative to control measurements in cells expressing either free CFP and YFP (abFRET0) or the CFP-YFP fusion protein (abFRETCFP-YFP fusion): apparent FRET efficiency = (abFRET − abFRET0)/(abFRETCFP-YFP fusion − abFRET0). For statistical analysis, the abFRET datasets were tested for normality using the Kolmogorov-Smirnov test, and datasets were compared using the one-tailed t test.
For high-resolution immunofluorescent imaging of BrUTP incorporated into nascent RNA, Cy3 was excited at 543 nm at moderate laser power and emission was detected using a 560-nm longpass emission filter. GFP-AR was excited at 488 nm at moderate laser power, and emission was detected using a 505–530-nm bandpass emission filter. Cy3 and GFP images were recorded sequentially to avoid cross talk.
Spectroscopic analysis of crude cell lysates of cells expressing YFP-AR-CFP was performed on a fluorescence spectrophotometer (F-4500; Hitachi) by recording spectra at 425 nm excitation. The apparent FRET efficiency was calculated as the ratio of the emission intensities at 525 and 475 nm. Background fluorescence of lysates of cells not expressing YFP-AR-CFP prepared in the same way was negligible. Spectra were recorded of lysates in the absence and presence of 300 μM of synthesized peptides containing an FQNLF or LQNLL motif, respectively.
Simultaneous FRAP and FRET
To study the mobility of interacting proteins, a narrow strip spanning the nucleus was scanned at 458 nm excitation with short intervals (100 ms) at low laser power (YFP is sufficiently excited at this wavelength; Fig. S4 A). Fluorescence intensities of the donor (CFP) and acceptor (YFP) were recorded simultaneously using 470–500-nm bandpass and 560-nm longpass filters, respectively. After 40 scans, a high-intensity, 100-ms bleach pulse at 514 nm was applied to specifically photobleach YFPs inside the strip (CFP was not bleached by the bleach pulse; Fig. S4 B). Subsequently, scanning of the bleached strip was continued at 458 nm at low laser intensity. The curves are either normalized by calculating Inorm = (Iraw − Ibg)/(Ipre − Ibg) or to compare donor-FRAP and acceptor-FRAP curves by calculating Inorm = (Iraw − I0)/(Ifinal − I0), where Ipre, I0, and Ifinal are the fluorescent intensities before, immediately after, the bleach and after complete recovery, respectively, and Ibg is the background intensity.
YFP/CFP ratio imaging
Because YFP and CFP are present in exactly the same quantity in cells expressing YFP-AR-CFP, ratio imaging can be applied to study the spatial distribution of ARs with and without N/C interaction. Local differences in YFP/CFP ratio within the nucleus of cells expressing YFP-AR-CFP will only be observed if the ratio between N/C-interacting ARs, showing a relatively high YFP/CFP ratio, and non–N/C-interacting ARs, showing a relatively low YFP/CFP ratio, are different. For high-resolution YFP/CFP ratio imaging, YFP and CFP were imaged simultaneously using a moderate excitation at 458 nm and a 470–500-nm bandpass emission filter for CFP and a 560-nm longpass emission filter for YFP. To reduce noise, eight times line averaging was used. Images were analyzed using the KS-400 image analysis package (Carl Zeiss MicroImaging, Inc.). Ratio images were obtained by calculating for each pixel (IYFP − Ibg)/(ICFP − Ibg), where IYFP and ICFP are the intensities of the YFP and CFP emission, respectively, and Ibg is the background intensity. To obtain regions representing successive relative intensity ranges (), the mean of IYFP and ICFP was calculated for each pixel as Imean = (IYFP + ICFP)/2. The mean Imean of each nucleus (termed μ in ) and the standard deviation, σ, were then calculated after (manual) selection of the nuclear area and exclusion of the nucleoli (). The mean ratio in areas with pixel intensities Imean < μ + σ, μ + σ < Imean < μ + 2σ and Imean > μ + 2σ were then first calculated for CFP-YFP-AR expressing cells. Because these molecules emit at a fixed YFP/CFP ratio irrespective of their conformation or local concentration, any difference in ratio in the three selected areas is due to imaging artifacts. Indeed, CFP/YFP ratio increased in CFP-YFP-AR expressing cells with low intensity and decreased in cells with high intensities probably because of the nonlinearity of the detectors. Therefore, data obtained from each cell expressing YFP-AR-CFP and the non–DNA-binding mutant YFP-AR(A573D)-CFP were expressed relative to the mean ratio measured in corresponding areas in seven cells expressing CFP-YFP-AR with similar expression level. For statistical analysis, the YFP/CFP ratio imaging datasets were tested for normality using the Kolmogorov-Smirnov test, and datasets were compared using the t test.
Immunofluorescent labeling of nascent RNA
Nascent RNA was detected by BrUTP incorporation in permeabilized living Hep3B cells stably expressing GFP-AR (Farla et al., 2004
) according to Wansink et al. (1993)
. Cells were grown overnight on coverslips in medium containing 5% dextran charcoal stripped FBS in the presence of 100 nM R1881. The procedure of BrUTP incorporation has been previously described (Wansink et al., 1993
). Cells were permeabilized in glycerol buffer (20 mM Tris HCl, 0.5 mM MgCl2
, 0.5 mM EGTA, 25% glycerol, and 1 mM PMSF) supplemented with 0.05% Triton X-100 and 10 U/ml RNAsin for 3 min. To allow BrUTP incorporation, permeabilized cells were incubated for 30 min at RT in synthesis buffer (100 nM Tris HCl, 5 nM MgCl2
, 0.5 mM EGTA, 200 mM KCl, 50% glycerol, 0.05 mM SAM, 20 U/ml RNAsin, and 0.5 mM PMSF) supplemented with 0.5 mM ATP, CTP, GTP, and BrUTP (or UTP as control; Sigma-Aldrich). Next, cells were fixed in 2% formaldehyde in PBS, incubated in 0.5% Triton X-100/PBS for 5 min and in 100 nM glycin/PBS for 10 min, each step followed by two PBS washes. After blocking with PBG (0.05% gelatin and 0.5% BSA in PBS), incorporated BrUTP was immunolabeled overnight with a rat anti-BrdU mAb (Seralab) diluted 1:500 in PBS at 4°C. After four washes with PBG, cells were incubated for 90 min at RT with biotin-conjugated sheep anti–rat IgG (Jackson ImmunoResearch Laboratories) 1:200 in PBS followed by four washes with PBG. The biotinylated antibody was then visualized with Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories) 1:250 in PBS for 30 min at RT. After extensive washing with PBG and PBS, cells were embedded in Vectashield containing DAPI.
Online supplemental material
Fig. S1 shows YFP-AR-CFP expression analysis of cells used in the acceptor photobleaching FRET experiments and in the simultaneous FRAP and FRET measurements. Fig. S2 presents the validation of FRET measurements by acceptor photobleaching (abFRET) and shows the hormone dependency of FRET measured in cells expressing YFP-AR-CFP. Fig. S3 shows the minimal YFP/CFP ratio change after the addition of R1881 in cells expressing YFP-AR(F23,27A/L26A)-CFP variant. Fig. S4 presents the control experiments for donor-FRAP and acceptor-FRAP on cells expressing YFP-AR and AR-CFP. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200609178/DC1