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Endocrinology Vol. 144, No. 9 4070-4079
Copyright © 2003 by The Endocrine Society

Yellow Fluorescent Protein-Tagged and Cyan Fluorescent Protein-Tagged Imaging Analysis of Glucocorticoid Receptor and Importins in Single Living Cells

Masayuki Tanaka, Mayumi Nishi, Masafumi Morimoto, Tohru Sugimoto and Mitsuhiro Kawata

Departments of Anatomy and Neurobiology (M.T., M.N., M.K.), and Pediatrics (M.T., M.M., T.S.), Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan

Address all correspondence and requests for reprints to: Dr. Mitsuhiro Kawata, Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid receptor (GR) acts as a ligand-dependent transcription factor after nuclear transport from the cytoplasm in the liganded state. Importins are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway. To investigate the spatial and temporal relation between GR and importins, we analyzed the subcellular distribution of GR and importins in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP-) GR was translocated from the cytoplasm to the nucleus, showing a similar time course as FP-importin-{alpha} in the coexpressed cells with the fusion proteins. In contrast to FP-importin-{alpha}, the distribution of FP-importin-ß was little changed upon ligand treatment in the coexpressed cells with FP-GR and FP-importin-ß. Analysis using fluorescence resonance energy transfer proved that GR directly interacted with importin-{alpha} in the whole area of the cytoplasm upon ligand treatment and detached importin-{alpha} shortly after nuclear import. However, direct interaction between GR and importin-ß was not detected. These studies showed visual evidence of the nuclear importing of GR in association with importin-{alpha} in single living cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS are indispensable not only for the maintenance of metabolic homeostasis, but also for treatment of a wide variety of human disorders. Glucocorticoids exert hormone action in target tissues via binding to the glucocorticoid receptor (GR). The GR, as a hormone-dependent transcription factor, belongs to the nuclear receptor superfamily (1, 2, 3, 4). It is generally accepted that the unliganded GR predominantly resides in the cytoplasm and that hormone activation leads to the translocation of GR from the cytoplasm to the nucleus (5, 6, 7, 8, 9). From the viewpoint of signal transduction, the glucocorticoid signal that finally influences gene expression must be transmitted to the nucleus via receptor translocation (10). Therefore, nuclear import of GR is one of the key processes in the regulation of glucocorticoid hormone action.

Whereas molecules less than 20–40 kDa in size can passively diffuse through aqueous channels that span the nuclear pore complex (NPC) from the cytoplasm to the nucleus, most macromolecules, greater than 40 kDa are transported through gated channels of the NPC by active mechanisms (11, 12). The best characterized active nuclear protein import is mediated by a nuclear localization signal (NLS), referred to as classical NLS, containing one or more clusters of basic amino acids (13). The import of substrates with classical NLS, such as simian virus 40 large T antigen, is initiated by the formation of an NLS-dependent complex with importin-{alpha} and importin-ß (14). Importin-{alpha} and importin-ß are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway across the NPC (15). Importin-{alpha} recognizes the NLS and binds to importin-ß via its N-terminal sequences, which are rich in basic amino acids, referred to as the importin-ß-binding domain (14). Importin-ß accounts for the targeting of NPC (16). In addition to this import pathway, recent studies have identified several novel types of pathways in which importin-ß binds directly to their cognate cargoes without importin-{alpha} (17, 18, 19), and nuclear protein itself moves into the nucleus without importin-{alpha} and importin-ß (20, 21).

GR has two NLSs: one is NL1 consisting of a stretch of basic amino acids at the immediate C-terminal end of the receptor DNA binding domain, and another is NL2 in the ligand binding domain (22). It has been reported that NL1 is involved in rapid nuclear import of agonist-treated GR and regulates the nuclear import of GR associated with importin-{alpha} (23). In contrast, NL2-mediated nuclear transfer of GR is slower and independent of importin-{alpha} (23). However, the spatial and temporal interactions between GR and importins has been less studied due to the limitations of previous methods. Recently, green fluorescent protein (GFP) chimeras of GR were used to study dynamic receptor trafficking in living cells (24, 25, 26, 27). To elucidate the spatio-temporal relation between GR and importins in response to corticosterone (CORT), we analyzed time-lapse images of the cells coexpressing cyan fluorescent protein-tagged (CFP-) GR and either yellow fluorescent protein-tagged (YFP-) importin-{alpha} or YFP-importin-ß upon CORT treatment. Multicolor variants of GFP have enabled us to perform simultaneous imaging of at least two different molecules in single living cells.

Current developments of multicolor fluorescent protein variants also allow visualization of protein-protein interaction in living cells by the fluorescence resonance energy transfer (FRET) technique (28). FRET can only occur when the distance separating the two fluorophores is less than 100 Å (28). Thus, FRET is a sensitive method for determining the relative proximity of labeled protein partners. FRET measures the proximity of two molecules as a consequence of the degree to which the fluorescence energy excited in a donor fluorophore, linked to one factor, is not emitted and instead is nonradiatively transferred to an acceptor fluorophore, linked to another factor (29, 30, 31). Using the FRET technique, we can determine when and where specific protein partners associate with one another in a living material at the cellular level. We applied a microscope-based assay using FRET to confirm whether direct interaction between GR and importins occurs in response to CORT in living cells.

Here, we report for the first time the results of visualization of subcellular distribution of CFP-GR and YFP-importins in response to CORT in cells coexpressed with the fusion proteins. Furthermore, the FRET technique proved the direct interaction between GR and importin-{alpha} upon CORT treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction
CFP-GR was constructed as previously reported (32). The 6RGR vector (provided by Dr. K. R. Yamamoto, University of California, San Francisco, CA) containing rat liver GR cDNA was digested at the BamHI sites and subcloned into the pGEM-4 vector (Promega Corp., Madison, WI), resulting in pGEM-4-GR. The GR cDNA with a truncated 5'-coding region was isolated from pGEM-4-GR by EaeI-BamHI digestion and ligated in-frame into the multiple cloning site (Bsp121I and BamHI) of pECFPC1 (Clontech Laboratories, Inc., Palo Alto, CA) using a ligation kit (TaKaRa, Kusatsu, Japan). To generate YFP-importin-{alpha} construct, a cDNA fragment containing the entire coding region of the mouse importin-{alpha} gene was obtained by introducing a Sal site just upstream of the first ATG in the genes that cloned into the pGEX-2T-PTAC58 vector (provided by Dr. Yoshihiro Yoneda, Graduate School of Medicine, Osaka University, Suita, Japan) with a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). After cutting with Sal, the gene was subcloned into the pEYFPC1 vector (Clontech Laboratories, Inc.) cut with the same restriction enzyme. To generate YFP-importin-ß construct, a Kpn restriction enzyme site was inserted just downstream of the first ATG in a cDNA fragment containing mouse importin-ß that cloned into pGEX-2T-PTAC97 vector (provided by Dr. Y. Yoneda). After cutting with Kpn, the gene was subcloned into the pEYFPC1 vector cut with the same restriction enzyme. To create a CFP-GR impaired nuclear localization (CFP-GRNL1-), we introduced point mutations in the NL1 of GR at amino acids 513–515 with a QuikChange Site-Directed Mutagenesis Kit. CFP-GRNL1- consisted of mutating amino acids 513KKK515 of wild-type rat GR to 513NNN515 as previously reported (23).

Cell culture and transfection
COS-1 cells were obtained and grown as described previously (27). Cell cultures were maintained in DMEM (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma-Aldrich Corp.). The day before transfection, cells were reseeded in a four-well multidish, 16 mm in diameter (Nunc, Roskilde, Denmark), at an initial plating density of 2 x 104 cells/well in 400 µl medium in a humidified atmosphere at 37 C with 5% CO2/95% air. Plasmid DNA (200 ng/well) was transiently transfected into cells by a liposome-mediated method using Lipofectamine Plus (Life Technologies, Inc.) according to the manufacturer’s instructions. Before analyzing, the cells were washed five times with 500 µl PBS and then cultured again in a serum-free medium (OptiMEM, Life Technologies, Inc.) for at least 15 h to remove any effects of the remaining steroid hormones.

For ligand stimulation, cells were treated with 10-6 or 10-9 M CORT (Sigma-Aldrich Corp.) at 37 C. To investigate the effect of 90-kDa heat shock protein (hsp90) on the interaction between GR and importin-{alpha}, cultured COS-1 cells were treated with 10-6 M geldanamycin (Sigma-Aldrich Corp.) for 3 h before CORT treatment.

Immunoblotting
COS-1 cells plated on a 35-mm dish were transfected with the expression plasmids, CFP-GR, CFP-GRNL1-, YFP-importin-{alpha}, or YFP-importin-ß, and then cultured overnight before being lysed in 1x Laemmli sample buffer. Proteins from the cell lysates were separated by 10% SDS-PAGE. Samples were electroblotted to polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp., Bedford, MA) using a semidry blotting apparatus (Transblot-SD, Bio-Rad Laboratories, Inc., Hercules, CA). The membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline and 0.05% Tween (TBST), and then incubated with an anti-GFP polyclonal antibody (1:100 dilution; StressGen Biotechnologies Corp., Victoria, Canada), which cross-reacts with CFP and YFP; an anti-Rch-1 antibody (1:2500 dilution; Transduction Laboratories, Lexington, KY); or antikaryopherin-ß antibody (1:1000 dilution; Transduction Laboratories) overnight at 4 C. After washing with TBST, the membrane was incubated with horseradish peroxidase-conjugated goat antirabbit IgG secondary antibody (1:5,000 dilution; Bio-Rad Laboratories, Inc.) or horseradish peroxidase-conjugated goat antimouse IgG secondary antibody (1:5,000 dilution; Bio-Rad Laboratories, Inc.) at 25 C for 1 h, and then once more washed in TBST. Signals were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).

Confocal laser scanning
COS-1 cells (1 x 104) plated on a poly-L-lysine-coated, 35-mm glass-bottomed dishes (Matsunami Glass, Inc., Ltd., Kishiwada, Japan) were transfected with either 200 ng YFP-importin-{alpha} or YFP-importin-ß expression plasmids using Lipofectamine Plus before being cultured in OptiMEM for 24 h. The transfected cells were observed with a x63 oil immersion lens (Carl Zeiss, Jena, Germany). Images were collected with a confocal laser scanning microscope (LSM510, Carl Zeiss) using a filter set with 514-nm excitation and 530- to 600-nm emission, and a 458/514-nm dichroic mirror for the YFP signal.

Time-lapse image acquisition and analysis
The living cell image acquisition was performed in a temperature-controlled room at 37 C. Images were acquired using a Quantix high resolution, cooled, charge-coupled device camera (Photometrics, Tucson, AZ) attached to a microscope (IXL70, Olympus Corp., Tokyo, Japan) equipped with an epifluorescence attachment. Cells were observed with a x40 objective lens. YFP fluorescence was viewed using a filter set with 500AF25 excitation and 545AF35 emission, and a 525DRLP dichroic mirror (Omega Optical, Inc., Brattleboro, VT), and CFP fluorescence was viewed using with a filter set with 440AF21 excitation and 480AF30 emission, and a 455DRLP dichroic mirror. Time-lapse image capturing and data evaluation were performed using the image analysis software program, MetaMorph (Universal Imaging Corp., West Chester, PA). For high resolution analysis, an image deconvolution procedure, Nearest Neighbor Estimate, was applied to z-series focal plane images.

FRET
COS-1 cells were reseeded in a four-well multidish (16-mm diameter; Nunc) at an initial plating density of 2 x 104 cells/well in 400 µl medium in a humidified atmosphere at 37 C with 5% CO2/95% air. Plasmid DNA (200 ng/well) was transiently transfected into cells using Lipofectamine Plua according to the manufacturer’s instructions. Before analyzing, the cells were washed five times with 500 µl PBS and then cultured again in a serum-free medium (OptiMEM) for at least 15 h to remove any effects of the remaining steroid hormones. For ligand stimulation, cells were treated with 10-6 or 10-9 M CORT at 37 C. The DNA ratios of the two expression vectors were adjusted to obtain approximately equal amounts of the pair of interacting proteins. At least 15 h after transfection, cells were imaged with a charge-coupled device camera (Photometrics). YFP and CFP fluorescence was detected using the filter sets described above. FRET fluorescence was detected using a filter set with 440AF21 excitation and 535AF26 emission, and a 455DRPL dichroic mirror (Omega Optical, Inc.). The ratio of FRET/cyan fluorescence was then calculated to obtain ratio images using MetaMorph software (Universal Imaging Corp.) after appropriate background subtraction. Concretely, background fluorescence was measured in a space (i.e. where no cell was present). Total fluorescence was then subtracted from background fluorescence. Ratio images were pseudocolored where the red range indicated a high ratio and the blue range indicated a low range.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of fusion proteins and their functional verification
Sequencing of the ligated fragments in CFP-GR, CFP-GRNL1-, YFP-importin-{alpha}, and YFP-importin-ß showed that the correct reading frames were constructed. Protein expression and transcriptional activity of CFP-GR were previously evaluated by Western blot analysis and luciferase reporter assays, showing that the tagged GR proteins retained ability similar to that of the wild-type GR to induce transactivation (32). Protein expression of CFP-GRNL1- was evaluated by Western blotting. The expressed CFP-GRNL1- was detected with the band at the same molecular weight as CFP-GR (data not shown). A cDNA fragment containing the sequence of mouse importin-{alpha} or mouse importin-ß was ligated in-frame to the 3'-end of enhanced YFP of cytomegalovirus promoter-driven expression vectors. These plasmids were transiently transfected into COS-1 cells. Lysates of the transfected cells were analyzed by Western blotting. The expected sizes of fusion proteins of importin-{alpha} (58 kDa) and importin-ß (97 kDa) with the 27-kDa GFP color variants YFP are 85 and 124 kDa, respectively. The expressed YFP-importin-{alpha} and YFP-importin-ß were detected with bands at the expected molecular weights (Fig. 1Go, A–C). To characterize the fusion proteins in detail, we compared the distributions of the fusion proteins in living cells with those of untagged importin-{alpha} and importin-ß examined by immunocytochemical studies (33). To evaluate the subcellular distribution of YFP-importin-{alpha} and YFP-importin-ß, the constructs were transiently transfected in COS-1 cells. Confocal laser scanning microscopy showed the distinct subcellular localization pattern of YFP-importin-{alpha} (Fig. 2AGo) and YFP-importin-ß (Fig. 2BGo). The z-sections imaged throughout the thickness of the cell indicated that YFP-importin-{alpha} was diffusely distributed in the cytoplasm. In contrast, YFP-importin-ß was present in the perinuclear region with a dot-like pattern. These results were consistent with those of previous studies using immunocytochemistry (33). Many findings have suggested that importin-ß binds to NPCs and shuttles between the nucleus and the cytoplasm through the NPC (16, 34, 35). The dot-like distribution of YFP-importin-ß presumably corresponds to the site of NPCs. These results demonstrated that YFP-importin-{alpha} and YFP-importin-ß fusion proteins were appropriate to our present study.



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  		FIG. 1. A, Immunoblotting of YFP-importin-{alpha}-expressing (lane 1) and YFP-importin-ß-expressing (lane 2) COS-1 cells detected with anti-GFP antibody. Lane 1, Cells expressing YFP-importin-{alpha} showed GFP staining at the predicted molecular mass of 85 kDa; lane 2, cells expressing YFP-importin-ß exhibited GFP staining at the predicted molecular mass of 124 kDa. B, Immunoblotting of YFP-importin-{alpha}- or YFP-importin-ß-expressing COS-1 cells detected with anti-Rch-1 antibody. Lane 1, Cells expressing YFP-importin-{alpha} showed Rch-1 staining at the predicted molecular mass of 85 kDa; lane 2, cells expressing YFP-importin-ß showed no staining. C, Immunoblotting of YFP-importin-{alpha}- or YFP-importin-ß-expressing COS-1 cells detected with antikaryopherin-ß antibody. Lane 1, Cells expressing YFP-importin-{alpha} showed no staining; lane 2, cells expressing YFP-importin-ß exhibited karyopherin-ß staining at the predicted molecular mass of 124 kDa.

 


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  		FIG. 2. The distribution of YFP-importin-{alpha} and YFP-importin-ß in living COS-1 cells. COS-1 cells transfected with YFP-importin-{alpha} or YFP-importin-ß expression plasmids were cultured in the absence of serum for at least 15 h before observation. All images were captured using confocal laser scanning microscopy. The z-axis observation is shown from a confocal laser scanning microscope from the bottom to the upper aspects of COS-1 cells that transiently expressed YFP-importin-{alpha} or YFP-importin-ß. A, Confocal microscopy showed diffuse YFP-importin-{alpha} fluorescence in the cytoplasm of the transiently expressing COS-1 cell (a, bottom; b, middle; c, upper). B, YFP-importin-ß was distributed in the perinuclear region with a dot-like pattern (a, bottom; b, middle; c, upper). A Nomarski differential interference contrast image of the cell (Nom) is shown. Bar, 20 µm.

 
Taken together, our studies with sequencing, transactivation assays, Western blotting, and microscopic analysis demonstrated that CFP-GR, YFP-importin-{alpha}, and YFP-importin-ß encode functional proteins that distribute in the cell in the same way as the corresponding endogenous proteins.

Dynamics of CFP-GR and YFP-importin-{alpha} in response to CORT in the cotransfected cells
YFP and CFP, well characterized spectral variants of GFP, have been used for double imaging of two different molecules because it is easy to distinguish each fluorescence using specific filters. When the CFP-GR construct was singly transfected to COS-1 cells, we could observe CFP fluorescence using a filter set for CFP imaging, but only faint autofluorescence was noted using a filter set for YFP imaging despite a 10-fold longer capture time (Fig. 3AGo). In contrast, when the YFP-importin-{alpha} construct was singly transfected, YFP fluorescence was observed using a filter set for YFP imaging. However, only faint autofluorescence was noted using a filter set for CFP imaging despite a 10-fold longer capture time (Fig. 3BGo). Thus, CFP fluorescence and YFP fluorescence could be separately observed without significant cross-over of the fluorescence signal using the filter sets employed in this study. To investigate how CFP-GR and YFP-importin-{alpha} were distributed when the proteins were coexpressed in a single living cell, expression plasmids of CFP-GR and YFP-importin-{alpha} were cotransfected to COS-1 cells. Fluorescent images of the fusion proteins were captured before and after the addition of CORT (0, 10, 20, and 30 min). To visualize the intracellular area where CFP-GR and YFP-importin-{alpha} were localized, the images were pseudocolored with cyan and yellow, respectively. Before the addition of CORT, CFP-GR was predominantly localized in the cytoplasm, and YFP-importin-{alpha} was localized in both the cytoplasm and the nucleus. With regard to YFP-importin-{alpha}, the fluorescence intensity in the cytoplasm was higher than that in the nucleus. Time-lapse images recorded in the coexpressed COS-1 cells revealed that cytoplasmic CFP-GR and YFP-importin-{alpha} simultaneously translocated from the cytoplasm to the nucleus upon CORT treatment. Nuclear translocation of the fusion proteins started within 10 min after the addition of 10-6 M CORT and was completed within 30 min; it was excluded from nucleolar regions. The fluorescence of CFP-GR was completely translocated to the nucleus from the cytoplasm. However, some of the fluorescence of YFP-importin-{alpha} remained in the cytoplasm even after 30 min (Fig. 4AGo). When the coexpressed cells were treated with 10-9 M CORT, cytoplasmic CFP-GR and YFP-importin-{alpha} simultaneously translocated from the cytoplasm to the nucleus. However, both proteins accumulated in the nuclear region slower than these proteins treated with 10-6 M CORT, and some of the fluorescence of the proteins in the cytoplasm was still observed even after 30 min (Fig. 4BGo). As a control, the construct of CFP-GRNL1- and YFP-importin-{alpha} were cotransfected to COS-1 cells. We introduced point mutations in the NL1 of GR at amino acids 513–515, GRNL1- (Fig. 4CGo). This was created by mutating amino acids 513KKK515 of wild-type rat GR to 513NNN515 as previously reported (23). The distribution of CFP-GRNL1- was similar to that of CFP-GR without CORT. Before CORT addition, CFP-GRNL1- and YFP-importin-{alpha} were predominantly localized in the cytoplasm. Upon CORT treatment, the distribution of CFP-GRNL1- and YFP-importin-{alpha} was little changed (Fig. 4DGo).



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  		FIG. 3. Representative fluorescence images of a singly expressed cell with CFP-GR, YFP-importin-{alpha}, or YFP-importin-ß with filter sets of both CFP and YFP. COS-1 cells were singly expressed with CFP-GR (A), YFP-importin-{alpha} (B), or YFP-importin-ß (C). Fluorescent images are shown of CFP-GR-expressing cell that was captured for 300 msec with a filter set for CFP and captured for 3000 msec with a filter set for YFP. Fluorescent images of YFP-importin-{alpha} or YFP-importin-ß-expressing cells were captured for 3000 msec with a filter set of CFP and for 300 msec with a filter set of YFP. Bar, 20 µm.

 


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  		FIG. 4. Dual color imaging of GR and importin-{alpha} with GFP spectral variants in single COS-1 cells. COS-1 cells were transiently coexpressed with either CFP-GR or CFP-GRNL1- and YFP-importin-{alpha}. Coexpressed living COS-1 cells were cultured in the absence of serum and steroids for at least 15 h before observation. A, Fluorescent images of CFP-GR (upper) and YFP-importin-{alpha} (lower) were captured before (0 min) and after the addition of 10-6 M CORT at 10-min intervals. Images of CFP-GR were pseudocolored with cyan, and images of YFP-importin-{alpha} were colored yellow. B, Fluorescent images of CFP-GR (upper) and YFP-importin-{alpha} (lower) were captured before (0 min) and after the addition of 10-9 M CORT at 10-min intervals. C, Schematic representation of the structure for wild-type GR (WTGR) and mutant GR (GRNL1-). NL1 of rat GR overlaps with the receptor DNA binding domain (DBD). Site-directed mutagenesis was used to convert lysines 513–515 to asparagines (lowercase) in GRNL1-. D, COS-1 cells were transiently coexpressed with CFP-GRNL1- and YFP-importin-{alpha}. Fluorescent images of CFP-GRNL1- (upper) and YFP-importin-{alpha} (lower) were captured before (0 min) and 30 min after the addition of 10-6 M CORT. Images of CFP-GRNL1- were pseudocolored with cyan, and images of YFP-importin-{alpha} were colored yellow. All images were shown after applying a deconvolution procedure. Bar, 20 µm.

 
Dynamics of CFP-GR and YFP-importin-ß in response to CORT in the cotransfected cells
When the YFP-importin-ß construct was singly transfected into COS-1 cells, YFP fluorescence was observed using a filter set for YFP imaging, but only faint autofluorescence was noted using a filter set for CFP imaging despite a 10-fold longer capture time (Fig. 3CGo). Both YFP-importin-ß and either CFP-GR or CFP-GRNL1- were coexpressed in single living COS-1 cells. Before the addition of CORT, CFP-GR and CFP-GRNL1- were predominantly localized in the cytoplasm, and YFP-importin-ß mainly resided in the perinuclear region. In the coexpressed cells with CFP-GR and YFP-importin-ß, there was no detectable difference in the localization of YFP-importin-ß upon CORT treatment, whereas CFP-GR translocated into the nucleus (Fig. 5AGo). In the cotransfected cells with the constructs of CFP-GRNL1- and YFP-importin-ß, the distribution of the fusion proteins was little changed upon CORT treatment (Fig. 5BGo).



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  		FIG. 5. Dual color imaging of GR and importin-ß with GFP spectral variants in single COS-1 cells. COS-1 cells were transiently coexpressed with either CFP-GR or CFP-GRNL1- and YFP-importin-ß. Coexpressed living COS-1 cells were cultured in the absence of serum and steroids for at least 15 h before observation. A, Fluorescent images of CFP-GR (upper) and YFP-importin-ß (lower) were captured before (0 min) and 30 min after the addition of 10-6 M CORT. Images of CFP-GR were pseudocolored with cyan, and images of YFP-importin-ß were colored yellow. B, Fluorescent images of CFP-GRNL1- (upper) and YFP-importin-ß (lower) were captured before (0 min) and 30 min after the addition of 10-6 M CORT. All images were shown after applying a deconvolution procedure. Bar, 20 µm.

 
Ratio images of the cells coexpressing CFP-GR and YFP-importin-{alpha} monitored by living cell FRET
The low level of CFP and YFP bleed-through enabled us to selectively and accurately measure the amounts of CFP-GR and YFP-importin-{alpha} coexpressed in the same cell (Fig. 3Go, A and B). To elucidate direct interaction between GR and importin-{alpha} upon CORT treatment, we used FRET analysis based on microscopy. We used CFP-GR for the donor and YFP-importin-{alpha} for the acceptor, which were coexpressed in COS-1 cells. Therefore, direct interaction was confirmed as an increase in the ratio of FRET/cyan fluorescence from the cells coexpressing CFP-GR and YFP-importin-{alpha} upon CORT treatment relative to the ratio of FRET/cyan fluorescence emitted from coexpression with the fusion proteins before CORT addition. If no interaction occurs between GR and importin-{alpha} upon CORT treatment, the ratio of FRET/cyan fluorescence should be little changed before vs. after CORT treatment. In the FRET study we observed the cells coexpressing similar levels of the fusion proteins on the basis of their specific fluorescence intensities. In this study we applied the color table to show the ratio of FRET/cyan fluorescence. The ratio images were pseudocolored; the red range indicated a high ratio, and the blue range indicated a low ratio. As shown in Fig. 4Go, CFP-GR was predominantly localized in the cytoplasm, and YFP-importin-{alpha} was localized in both the cytoplasm and the nucleus before the addition of CORT. With regard to YFP-importin-{alpha}, the fluorescence intensity in the cytoplasm was higher than that in the nucleus. The nuclear translocation of the fusion proteins started within 10 min and was completed within 30 min after CORT treatment. With appropriate filters, the resulting images showed that there was a low ratio of FRET/cyan fluorescence in the cells coexpressing the fusion proteins before CORT treatment. Ten minutes after 10-6 M CORT treatment, a high ratio of FRET/cyan fluorescence was observed in the whole area of the cytoplasm, and a low ratio of FRET/cyan fluorescence was detected in the nucleus. Thirty minutes after 10-6 M CORT treatment, a low ratio of FRET/cyan fluorescence was observed in the nucleus (Fig. 6AGo). In the presence of 10-9 M CORT, ratio images were almost the same as those in treatment with 10-6 M CORT (data not shown).



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  		FIG. 6. Ratio images of the cell coexpressing CFP-GR and YFP-importin-{alpha} monitored by living cell FRET. A, COS-1 cells were coexpressed with CFP-GR and YFP-importin-{alpha}. Coexpressed living COS-1 cells were cultured in the absence of serum and steroids for at least 15 h before observation. Fluorescent images of CFP-GR and YFP-importin-{alpha}-expressing cells were captured using a filter set for CFP or YFP, respectively. FRET fluorescence was detected using a filter set with 440AF21 excitation and 535AF26 emission, and 455DRPL dichroic mirror at 0, 10, and 30 min after 10-6 M CORT treatment. The ratio of the FRET/cyan fluorescence was calculated to obtain the ratio images using MetaMorph software. The ratio images were pseudocolored; the red range indicated a high ratio, and the blue range indicated a low ratio. A Nomarski differential interference contrast image of the cell (Nom) is shown together. B, The ratio images of COS-1 cells coexpressing CFP-GRNL1- and YFP-importin-{alpha} before (0 min) and 10 min after the addition of 10-6 M CORT. C, The ratio images of COS-1 cells, treated with 10-6 M geldanamycin, coexpressing CFP-GR and YFP-importin-{alpha} before (0 min) and 10 min after the addition of 10-6 M CORT. Bar, 20 µm.

 
Control ratio images were generated from the cells coexpressing CFP-GRNL1- and YFP-importin-{alpha}. Even 10 min after CORT treatment, little change in the ratio of FRET/cyan fluorescence in the coexpressed cells was observed compared with the ratio before CORT treatment (Fig. 6BGo). These FRET experiments demonstrated that GR directly interacted with importin-{alpha} through the NL1 domain of GR in the whole area of the cytoplasm and detached importin-{alpha} shortly after nuclear import of the proteins in living cells.

We investigated the interaction between the GR-hsp90 complex and importin-{alpha} using geldanamycin. COS-1 cells coexpressing CFP-GR and YFP-importin-{alpha} were pretreated with 10-6 M geldanamycin for 3 h and then exposed to CORT. The ratio images of FRET/cyan fluorescence were not greatly changed before and after CORT treatment (Fig. 6CGo).

Ratio images of the cells coexpressing CFP-GR and YFP-importin-ß monitored by living cell FRET
To elucidate the direct interaction between GR and importin-ß upon CORT treatment, we used FRET analysis based on microscopy. We used CFP-GR for the donor and YFP-importin-ß for the acceptor, which were coexpressed in COS-1 cells. A low ratio of FRET/cyan fluorescence was continuously observed in cells coexpressing the fusion proteins upon CORT treatment (Fig. 7Go).



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  		FIG. 7. Ratio images of the cell coexpressing CFP-GR and YFP-importin-ß monitored by living cell FRET. COS-1 cells were coexpressed with CFP-GR and YFP-importin-ß. Coexpressed living COS-1 cells were cultured in the absence of serum and steroids for at least 15 h before observation. Fluorescent images of CFP-GR and YFP-importin-ß-expressing cells were captured using a filter set for CFP or YFP, respectively. FRET fluorescence was detected using a filter set with 440AF21 excitation and 535AF26 emission, and a 455DRPL dichroic mirror at 0, 10, and 30 min after 10-6 M CORT treatment. The ratio of the FRET/cyan fluorescence was then calculated to obtain the ratio images using MetaMorph software. The ratio images were pseudocolored; the red range indicated a high ratio, and the blue range indicated a low ratio. A Nomarski differential interference contrast image of the cell (Nom) is shown together. Bar, 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been reported that GR translocates to the nucleus from the cytoplasm upon ligand treatment via binding to importin-{alpha} (23). However, the spatio-temporal relation between GR and importins is still not clear. Therefore, further investigations are needed to understand the nuclear import of GR.

Our experiments with yellow and cyan fluorescent chimeras of the proteins provided the first evidence of the spatio-temporal relation between GR and importins upon CORT treatment in single living cells. At first, we generated N-terminal-tagged CFP-GR, YFP-importin-{alpha}, and YFP-importin-ß expression plasmids. The transcriptional activity of CFP-GR was confirmed by luciferase reporter assays, and the expression of the correct size protein was confirmed by Western blot analysis. Characterization of YFP-importin-{alpha} and YFP-importin-ß was achieved by Western blot analysis. In addition, we confirmed that the distribution of the fusion proteins in living cells was compatible with that of importins observed by previous immunocytochemical studies (33).

We and others used alternate GFP chimeras of GR to show hormone-dependent translocation from the cytoplasm to the nucleus (26, 27, 32). In the present study we focused on the relation between GR and importins during GR translocation from the cytoplasm to the nucleus upon hormone treatment. Although recent studies used comparable GFP-imaging systems for steroid receptor trafficking in living cells, no study has been performed in the coexpression cells of fluorescent protein-tagged (FP)-GR and FP-importins. Multicolor fluorescent protein tagging and advanced microscopy methods allowed us to visualize the relation between GR and importins in single living cells.

Before the addition of CORT, CFP-GR was predominantly localized in the cytoplasm, and YFP-importin-{alpha} was localized both in the cytoplasm and in the nucleus. With regard to YFP-importin-{alpha}, the fluorescence intensity in the cytoplasm was higher than that in the nucleus. YFP-importin-ß, however, resided in the perinuclear region in the absence of CORT. It is possible that the subcellular localization of GR and importins is altered depending on the cell cycle. As COS-1 cells used in the present study were maintained in the absence of serum for at least 15 h before observation, most of the cells might be synchronized in the G1 phase (32). The cell cycle of COS-1 cells was evaluated using bromodeoxyuridine incorporation for S phase and lamin A/C or cyclin C1 antibody for G1 phase markers. We observed that approximately 70–80% of the cells were in the G1 state, and about 20–30% of cells were in S phase (32). With regard to GR, previous studies revealed that GR tagged with appropriate fluorescent markers resided in the cytoplasm in the hormone-free condition (24, 25, 26, 27). Also, the distribution of YFP-importin-{alpha} was compatible with the previous finding, which proved that importin-{alpha} was found in the cytoplasm during the interphase (36).

A previous study using glutathione-S-transferase pull-down assay reported that GR interacts with importin-{alpha} (23). However, no study has been performed to visualize the spatio-temporal relation between GR and importins in real-time imaging analysis. When CFP-GR was coexpressed with YFP-importin-{alpha} into COS-1 cells, the fusion proteins translocated from the cytoplasm to the nucleus at mostly the same speed upon CORT treatment. CFP-GR completely moved into the nucleus from the cytoplasm within 30 min after 10-6 M CORT treatment. However, some of the fluorescence of YFP-importin-{alpha} remained in the cytoplasm 30 min after the addition of 10-6 M CORT. There are possible explanations for why YFP-importin-{alpha} did not completely move into the nucleus in living COS-1 cells. One is that importin-{alpha} combined with not only GR, but also various other nuclear proteins. Thus, all YFP-importin-{alpha} molecules might not necessarily move into the nucleus together with CFP-GR. Another possibility is that endogenous importin-{alpha} in COS-1 cells recognized CFP-GR, and the proteins moved together into the nucleus. We confirmed that COS-1 cells expressed endogenous importin-{alpha} by immunocytochemistry (data not shown). In contrast, little distributional change in YFP-importin-ß was observed upon CORT treatment in the coexpression cells with CFP-GR and YFP-importin-ß despite importin-ß being a shuttling protein through the NPC (16). During GR translocation to the nucleus from the cytoplasm upon CORT treatment, YFP-importin-ß continuously localized around the perinuclear region, suggesting that importin-ß worked in a very limited area of the perinuclear region in which the present microscope did not capture these tiny changes.

Previous studies suggest that overexpression of proteins can cause predominantly nuclear mislocalization (37). To rule out the possibility that nuclear localization of CFP-GR and YFP-importin-{alpha} upon CORT treatment was due to protein overexpression, we performed several experiments. In singly expressed cells with CFP-GR, the cytoplasmic CFP-GR did not translocate from the cytoplasm to the nucleus during 60-min observation before CORT treatment (data not shown). In the cells expressing CFP-GRNL1-, the distribution of the fusion protein changed little upon CORT treatment (data not shown). Also, in singly expressed cells with YFP-importin-{alpha}, the cytoplasmic YFP-importin-{alpha} was little translocated from the cytoplasm to the nucleus during 60-min observation with or without CORT treatment (data not shown). In the coexpressed cells with CFP-GR and YFP-importin-{alpha}, the distributional change in the proteins was not observed during 60-min observation before CORT treatment (data not shown). In the coexpressed cells with CFP-GRNL1- and YFP-importin-{alpha}, the fusion proteins did not move into the nucleus from the cytoplasm upon CORT treatment (Fig. 4DGo). These results proved that overexpression of proteins did not contribute to nuclear accumulation in our study.

Only recently has FRET been used to measure protein-protein interaction in living cells (38, 39, 40). Some of the spectral variants of GFP were shown to be suitable as donors and acceptors for FRET microscopy. Previous studies reported a direct transfer of excitation energy from CFP to YFP within living cells (39, 41, 42, 43, 44). No study has been performed to visualize the direct interaction between GR and importins. We used FRET microscopy to visualize the protein-protein interaction in the living cells coexpressing CFP-GR and either YFP-importin-{alpha} or YFP-importin-ß. The ratio of FRET/cyan fluorescence in the cells coexpressing CFP-GR and YFP-importin-{alpha} increased upon CORT treatment. This required that the fluorophores attached to GR and importin-{alpha} be separated by less than approximately 100 Å. The interaction between two proteins tagged with appropriate fluorescent markers can be detected by measuring the emission intensities of the acceptor fluorophore after exciting the donor fluorophore. We used CFP-GR for the donor and either YFP-importin-{alpha} or YFP-importin-ß for the acceptor. In the coexpressed cells with CFP-GR and YFP-importin-{alpha}, a high ratio of FRET/cyan fluorescence was observed in the whole area of the cytoplasm at 10 min after CORT treatment, and a low ratio of FRET/cyan fluorescence in the nucleus was detected throughout the FRET experiment. With regard to ratio images of FRET/cyan fluorescence in the nucleus, it is true that a low ratio in the nucleus was detected throughout the experiment, but the ratio captured 10 min after CORT treatment slightly increased compared with those at 0 and 30 min. Concretely, the green range was detected 10 min after CORT treatment compared with the images captured at 0 and 30 min. These results suggest that GR directly interacted with importin-{alpha} in the whole area of the cytoplasmic region and detached importin-{alpha} shortly after nuclear import of the proteins. As a control, we observed cells coexpressing CFP-GRNL1- and YFP-importin-{alpha}. The ratio images of the cells coexpressing the proteins changed little before and after CORT treatment, suggesting that there was no interaction between GRNL1- and importin-{alpha}.

GR is known to exist as a large heteroprotein complex that contains, among its multiple components, the hsp90 (45, 46). To explore hsp90’s role in the formation of the GR-importin-{alpha} complex, we performed a FRET study using geldanamycin. As geldanamycin binds to hsp90 (47), which inhibits nuclear translocation of GR (32), it may be a good tool with which to analyze the effect of hsp90 on the interaction between GR-hsp90 complex and importin-{alpha}. In this study we observed no significant differences in the ratio images of FRET/cyan fluorescence with geldanamycin before and after CORT treatment. This FRET experiment using geldanamycin revealed that importin-{alpha} cannot interact with the GR-hsp90 complex. Previous reports have indicated that hsp90 is required to maintain the GR protein in a conformation capable of binding hormone. After hormone binding, however, the GR is no longer found associated with hsp90, and it is this form of the receptor that translocates to the nucleus (48, 49, 50, 51). This result suggests that the conformational change in GR due to the dissociation of GR and hsp90 is required to form the GR-importin-{alpha} complex.

Compared with importin-{alpha}, when FRET microscopy was used to visualize the protein-protein interaction between GR and importin-ß, there was no difference in the ratio of FRET/cyan fluorescence before and after CORT treatment. This suggests that there was no direct interaction between GR and importin-ß.

In conclusion, the use of CFP-GR, CFP-GRNL1-, YFP-importin-{alpha}, and YFP-importin-ß allowed real-time visualization of GR translocation in association with importin-{alpha} and importin-ß in single living cells. This is the first report to demonstrate the different actions of importins during nuclear translocation of GR in single living cells by employing dual color imaging with YFP and CFP. Furthermore, FRET analysis revealed the relation of GR to importins and hsp90. The system should be quite useful for clarifying dynamic changes in the subcellular localization and interaction of functional molecules that cannot be detected in fixed cells.


   Acknowledgments
 
We thank Dr. Y. Yoneda for supplying the importin-{alpha} and importin-ß cDNAs.


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (Japan) and Special Coordination Fund for Promoting Science and Technology.

Abbreviations: CFP-, Cyan fluorescent protein-tagged; CORT, corticosterone; FP-, fluorescent protein-tagged; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GR, glucocorticoid receptor; hsp, heat shock protein; NL, nuclear localization; NLS, nuclear localization signal; NPC, nuclear pore complex; TBST, Tris-buffered saline and 0.05% Tween; YFP-, yellow fluorescent protein-tagged.

Received March 10, 2003.

Accepted for publication May 27, 2003.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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