Real Time Analysis of STAT3 Nucleocytoplasmic Shuttling

The transcription factor STAT3 is most important for the signal transduction of interleukin-6 and related cytokines. Upon stimulation cytoplasmic STAT3 is phosphorylated at tyrosine 705, translocates into the nucleus, and induces target genes. Notably, STAT proteins are also detectable in the nuclei of unstimulated cells. In this report we introduce a new method for the real time analysis of STAT3 nucleocytoplasmic shuttling in living cells which is based on the recently established fluorescence localization after photobleaching (FLAP) approach. STAT3 was C-terminally fused with the cyan (CFP) and yellow (YFP) variants of the green fluorescent protein. In the resulting STAT3-CFP-YFP (STAT3-CY) fusion protein the YFP can be selectively bleached using the 514-nm laser of a confocal microscope. This setting allows studies on the dynamics of STAT3 nucleocytoplasmic transport by monitoring the subcellular distribution of fluorescently labeled and selectively bleached STAT3-CY. By this means we demonstrate that STAT3-CY shuttles continuously between the cytosol and the nucleus in unstimulated cells. This constitutive shuttling does not depend on the phosphorylation of tyrosine 705 because a STAT3(Y705F)-CY mutant shuttles to the same extent as STAT3-CY. Experiments with deletion mutants reveal that the N-terminal moiety of STAT3 is essential for shuttling. Further studies suggest that a decrease in STAT3 nuclear export contributes to the nuclear accumulation of STAT3 in response to cytokine stimulation. The new approach presented in this study is generally applicable to any protein of interest for analyzing nucleocytoplasmic transport mechanisms in real time. The transcription factor STAT3 is most important for the signal transduction of interleukin-6 and related cytokines. Upon stimulation cytoplasmic STAT3 is phosphorylated at tyrosine 705, translocates into the nucleus, and induces target genes. Notably, STAT proteins are also detectable in the nuclei of unstimulated cells. In this report we introduce a new method for the real time analysis of STAT3 nucleocytoplasmic shuttling in living cells which is based on the recently established fluorescence localization after photobleaching (FLAP) approach. STAT3 was C-terminally fused with the cyan (CFP) and yellow (YFP) variants of the green fluorescent protein. In the resulting STAT3-CFP-YFP (STAT3-CY) fusion protein the YFP can be selectively bleached using the 514-nm laser of a confocal microscope. This setting allows studies on the dynamics of STAT3 nucleocytoplasmic transport by monitoring the subcellular distribution of fluorescently labeled and selectively bleached STAT3-CY. By this means we demonstrate that STAT3-CY shuttles continuously between the cytosol and the nucleus in unstimulated cells. This constitutive shuttling does not depend on the phosphorylation of tyrosine 705 because a STAT3(Y705F)-CY mutant shuttles to the same extent as STAT3-CY. Experiments with deletion mutants reveal that the N-terminal moiety of STAT3 is essential for shuttling. Further studies suggest that a decrease in STAT3 nuclear export contributes to the nuclear accumulation of STAT3 in response to cytokine stimulation. The new approach presented in this study is generally applicable to any protein of interest for analyzing nucleocytoplasmic transport mechanisms in real time. The Janus kinase/STAT 1The abbreviations used are: STAT, signal transducer and activator of transcription; CFP, cyan fluorescent protein; FLAP, fluorescence localization after photobleaching; IL, interleukin; ROI, region of interest; SH2 domain, Src homology 2 domain; YFP, yellow fluorescent protein. pathway plays a major role in cytokine and growth factor signaling. In particular, the family members of the α-helix bundle cytokines comprising the hematopoietins and interferons exert their biological effects by the activation of STAT transcription factors (for review, see Ref. 1Levy D.E. Darnell Jr., J.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar). According to the canonical model (2Shuai K. Horvath C.M. Huang L.H. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (683) Google Scholar), signaling through the Janus kinase/STAT pathway is triggered by the binding of a cytokine to class I or class II cytokine receptors which leads to the activation of receptor-associated tyrosine kinases of the Janus kinase family. The activated Janus kinases phosphorylate the receptor on tyrosine residues followed by the recruitment of STAT monomers to these phosphorylated tyrosine motifs. While bound to the receptor, STATs are phosphorylated at a single tyrosine residue and subsequently form dimers by intermolecular phosphotyrosine-SH2 domain interactions. The STAT dimers freely diffuse through the cytosol (3Lillemeier B.F. Köster M. Kerr I.M. EMBO J. 2001; 20: 2508-2517Crossref PubMed Scopus (73) Google Scholar), associate with importin-α (4Sekimoto T. Imamoto N. Nakajima K. Hirano T. Yoneda Y. EMBO J. 1997; 16: 7067-7077Crossref PubMed Scopus (306) Google Scholar), and translocate via the nuclear pore complexes to the nuclear compartment to transactivate target genes. This canonical model has been challenged by accumulating data suggesting that STATs dimerize prior to activation. The preformation of STAT dimers seems to be independent of tyrosine phosphorylation (5Stancato L.F. David M. Carter-Su C. Larner A.C. Pratt W.B. J. Biol. Chem. 1996; 271: 4134-4137Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 6Novak U. Ji H. Kanagasundaram V. 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J. 2000; 345: 417-421Crossref PubMed Scopus (63) Google Scholar, 10Kretzschmar A.K. Dinger M.C. Henze C. Brocke-Heidrich K. Horn F. Biochem. J. 2004; 377: 289-297Crossref PubMed Scopus (89) Google Scholar, 11Chatterjee-Kishore M. Wright K.L. Ting J.P. Stark G.R. EMBO J. 2000; 19: 4111-4122Crossref PubMed Scopus (273) Google Scholar, 12Zeng R. Aoki Y. Yoshida M. Arai K. Watanabe S. J. Immunol. 2002; 168: 4567-4575Crossref PubMed Scopus (56) Google Scholar, 13Meyer T. Begitt A. Lodige I. van Rossum M. Vinkemeier U. EMBO J. 2002; 21: 344-354Crossref PubMed Scopus (144) Google Scholar, 14Meyer T. Gavenis K. Vinkemeier U. Exp. Cell Res. 2002; 272: 45-55Crossref PubMed Scopus (72) Google Scholar, 15Bhattacharya S. Schindler C. J. Clin. Invest. 2003; 111: 553-559Crossref PubMed Scopus (115) Google Scholar). The latter finding is either the result of a static subcellular distribution or a continuous dynamic shuttling of STAT molecules between the cytosol and the nucleus. STAT3 is one of the seven mammalian STAT proteins. It acts as a signal transducer for many cytokines and growth factors (16Levy D.E. Lee C.K. J. Clin. Invest. 2002; 109: 1143-1148Crossref PubMed Scopus (755) Google Scholar) and is of particular importance for the family of IL-6-type cytokines (17Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Biochem. J. 2003; 374: 1-20Crossref PubMed Scopus (2518) Google Scholar). STAT3 participates in a variety of biological processes such as the induction of acute phase protein synthesis in hepatocytes (18Wegenka U.M. Buschmann J. Lütticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (488) Google Scholar), the regulation of hematopoiesis and the immune response (16Levy D.E. Lee C.K. J. Clin. Invest. 2002; 109: 1143-1148Crossref PubMed Scopus (755) Google Scholar, 19Takeda K. Clausen B.E. Kaisho T. Tsujimura T. Terada N. Forster I. Akira S. Immunity. 1999; 10: 39-49Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar), embryo implantation (20Robb L. Li R. Hartley L. Nandurkar H.H. Koentgen F. Begley C.G. Nat. Med. 1998; 4: 303-308Crossref PubMed Scopus (417) Google Scholar, 21Stewart C.L. Kaspar P. Brunet L.J. Bhatt H. Gadi I. Kontgen F. Abbondanzo S.J. Nature. 1992; 359: 76-79Crossref PubMed Scopus (1775) Google Scholar), and development (22Takeda K. Noguchi K. Shi W. Tanaka T. Matsumoto M. Yoshida N. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3801-3804Crossref PubMed Scopus (1100) Google Scholar, 23Yoshida K. Taga T. Saito M. Suematsu S. Kumanogoh A. Tanaka T. Fujiwara H. Hirata M. Yamagami T. Nakahata T. Hirabayashi T. Yoneda Y. Tanaka K. Wang W.Z. Mori C. Shiota K. Yoshida N. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 407-411Crossref PubMed Scopus (573) Google Scholar). As a consequence of these diverse functions, STAT3 plays a crucial role in inflammatory, autoimmune, and certain neoplastic diseases (24Schindler C.W. J. Clin. Invest. 2002; 109: 1133-1137Crossref PubMed Google Scholar). Mice having a targeted deletion of STAT3 die early during embryonic development (22Takeda K. Noguchi K. Shi W. Tanaka T. Matsumoto M. Yoshida N. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3801-3804Crossref PubMed Scopus (1100) Google Scholar). STAT proteins consist of six domains: an N-terminal domain involved in cooperative DNA binding, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a C-terminal transactivation domain. The structure of a truncated, tyrosine-phosphorylated, dimeric STAT3 bound to DNA has been solved by x-ray crystallography (25Becker S. Groner B. Müller C.W. Nature. 1998; 394: 145-151Crossref PubMed Scopus (670) Google Scholar). The dimerization of STAT3 molecules is enforced by reciprocal binding of the phosphotyrosine-containing regions to the SH2 domains, which is a prerequisite for nuclear accumulation and DNA binding of STAT3. The activity of the C-terminal transactivation domain is modulated by phosphorylation at serine 727 (26Decker T. Kovarik P. Oncogene. 2000; 19: 2628-2637Crossref PubMed Scopus (710) Google Scholar). The major part of the data concerning the nucleocytoplasmic transport mechanisms of STAT proteins was obtained for STAT1 (27McBride K.M. Reich N.C. Science's STKE. 2003; http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/195/re13PubMed Google Scholar) and STAT5 (12Zeng R. Aoki Y. Yoshida M. Arai K. Watanabe S. J. Immunol. 2002; 168: 4567-4575Crossref PubMed Scopus (56) Google Scholar, 28Herrington J. Rui L. Luo G. Yu-Lee L.Y. Carter-Su C. J. Biol. Chem. 1999; 274: 5138-5145Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Only recently, some reports concentrated on the structural requirements for the nuclear translocation of STAT3. Several amino acid residues in the coiled coil domain and DNA binding domain were identified to be essential for nuclear translocation of STAT3 (29Ma J. Zhang T. Novotny-Diermayr V. Tan A.L. Cao X. J. Biol. Chem. 2003; 278: 29252-29260Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Furthermore, three export signals were defined in the C-terminal part of the coiled coil domain, the DNA binding domain, and the linker domain (15Bhattacharya S. Schindler C. J. Clin. Invest. 2003; 111: 553-559Crossref PubMed Scopus (115) Google Scholar). In the present paper, we focus on the nuclear translocation of STAT3 in unstimulated as well as in IL-6-stimulated HepG2 cells. We established a novel real time method for the analysis of nucleocytoplasmic shuttling in living cells which is based on the recently described FLAP (fluorescence localization after photobleaching) approach (30Dunn G.A. Dobbie I.M. Monypenny J. Holt M.R. Zicha D. J Microsc. 2002; 205: 109-112Crossref PubMed Scopus (53) Google Scholar, 31Zicha D. Dobbie I.M. Holt M.R. Monypenny J. Soong D.Y. Gray C. Dunn G.A. Science. 2003; 300: 142-145Crossref PubMed Scopus (141) Google Scholar). Our application involves a fluorescent fusion protein consisting of STAT3 connected to two independent fluorophores (STAT3-CFP-YFP) combined with pulse bleach techniques. The new method allowed us to investigate the dynamics of the nucleocytoplasmic transport of STAT3 by monitoring the general subcellular distribution of fluorescently labeled STAT3 and synchronous tracing a selectively bleached subpopulation. Applying this novel approach we found that (i) STAT3 constitutively shuttles between the cytoplasmic and nuclear compartments in unstimulated cells; (ii) constitutive shuttling of STAT3 is independent of tyrosine phosphorylation; (iii) balanced shuttling of STAT3 requires the interplay of export and import signals; and (iv) reduced nuclear export contributes to IL-6-induced nuclear accumulation of STAT3. All experiments were carried out using the human hepatoma cell line HepG2 and the simian monkey kidney cell line COS7 (both purchased from ATCC, Rockville, MD). Dulbecco's modified Eagle's medium/Ham's F-12 1:1 mix with 15 mm Hepes and l-glutamine (Cambrex Corp., East Rutherford, NJ) supplemented with 10% heat-inactivated fetal calf serum was employed for culturing of HepG2 cells, whereas COS7 cells were grown in Dulbecco's modified Eagle's medium with Glutamax-I (Invitrogen) containing 10% fetal calf serum. For starvation conditions cells were cultured in pure serum-free Dulbecco's modified Eagle's medium with Glutamax-I, sodium pyruvate, 4.5 g/liter glucose, and pyridoxine (Invitrogen). Transient transfection of plasmids into HepG2 cells was performed by using FuGENE 6 (Roche Applied Science) according to the instructions of the manufacturer. COS7 cells were transiently transfected utilizing the DEAE-dextran method. pSVL-STAT3-CY was cloned on the basis of pSVLΔEcoRI gp130-ET-CFP-YFP and pSVLΔEcoRI STAT3-YFP (32Herrmann A. Sommer U. Pranada A.L. Giese B. Küster A. Haan S. Becker W. Heinrich P.C. Müller-Newen G. J. Cell Sci. 2004; 117: 339-349Crossref PubMed Scopus (49) Google Scholar). These plasmids encode fluorescent fusion proteins that consist of the N-terminal signal protein STAT3 or gp130 and the C-terminal fluorescent YFP or CFP-YFP tag, respectively. All of these plasmids contain a single XhoI and a single BstEII restriction site. The XhoI site is located in the multiple cloning site upstream of the start codon. The BstEII site connects the STAT3 or gp130 encoding part with the part that encodes the fluorescent tag. Thus, the basic plasmids were cut by using the restriction enzymes XhoI and BstEII (Roche Applied Science). Afterwards the fragment encoding STAT3 was ligated into the pSVLΔEcoRI vector that included the DNA sequence for the CFP-YFP tag. Cloning of STAT3(1–320)-CY—A fragment corresponding to amino acids 113–320 of STAT3 was amplified by PCR introducing a BstEII site with the antisense primer (5′-cggtgaccag gcactcttca ttaagtttct g-3′). After cloning into the TOPO-vector (Invitrogen) the DNA was sequenced. The verified plasmid was digested with XbaI (Roche Applied Science) and BstEII and cloned into pSVL-STAT3-CY cut with the same enzymes. Cloning of STAT3(321–771)-CY—A fragment encoding amino acids 321–610 of STAT3 was generated by PCR introducing an XhoI site and a start codon with the sense primer (5′-ctcgagatgt tcgtggtgga gcggcag-3′). The product was analyzed as described above, cut with XhoI and XmaI (New England BioLabs), and cloned into pSVL-STAT3-CY digested with the same enzymes. Cloning of STAT3(Y705F)-CY—Using pCAG GS Neo HA STAT3(Y705F) (33Nakajima K. Yamanaka Y. Nakae K. Kojima H. Ichiba M. Kiuchi N. Kitaoka T. Fukada T. Hibi M. Hirano T. EMBO J. 1996; 15: 3651-3658Crossref PubMed Scopus (521) Google Scholar) as a template, a fragment corresponding to amino acids 596–771 of STAT3(Y705F) was created by PCR introducing a BstEII site at the C terminus with the antisense primer (5′-gtgtgaggtg accacatggg ggaggt-3′). The product was analyzed as described above, digested with XmaI and BstEII, and ligated into pSVL-STAT3-CY cut with the same enzymes. Antibodies against STAT3 (BD Biosciences), phosphorylated STAT3 (Sigma), and GFP (Rockland Immunochemicals, Gilbertsville, PA) were used for immunoprecipitation (3 μl/ml lysate) and for Western blot analysis (1:1,000 dilution). Secondary antibodies were purchased from DAKO (Hamburg, Germany). HepG2 cells were cultured on 10-ml dishes, starved for 16 h, stimulated with 20 ng/ml recombinant human IL-6 for the indicated periods of time, and lysed by treatment with radioimmune precipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0,5% Nonidet P-40, 1 mm NaF, 15% glycerol, 20 mm β-glycerophosphate, 1 mm Na3VO4, 0,25 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 1 μg/ml leupeptin). The lysates were incubated with the respective specific antibody for 16 h at 4 °C, which was previously immobilized to protein A-Sepharose for immunoprecipitation. After two washing steps with ice-cold lysis buffer the proteins were eluted with 4× Laemmli buffer, separated by SDS-PAGE, and subsequently transferred to a polyvinylidene difluoride membrane by semidry blot technique. For detection the membrane was blocked with 10% bovine serum albumin in TBS-N (20 mm Tris-HCl, pH 7.6, 137 mm NaCl, 0,1% Nonidet P-40) for 1 h, rinsed three times with TBS-N, incubated with the appropriate antibody (1:1,000 dilution in TBS-N) for 16 h at 4 °C, washed twice with TBS-N for 10 min, treated with a suitable horseradish peroxidase-conjugated secondary antibody (1:2,000 dilution) for 30 min, again washed twice with TBS-N for 10 min, and finally exposed to enhanced chemiluminescence (Amersham Biosciences) followed by fluorography. The membrane was stripped in stripping buffer (2% SDS, 62.5 mm Tris-HCl, pH 6.7, 78 μlof β-mercaptoethanol/10 ml) for 35 min at 70 °C, washed twice with TBS-N for 10 min, and treated with the primary and secondary antibodies as indicated in the figure. HepG2 cells were cultured on 6-well plates (9.6 cm2/well), transiently transfected with pGL3 α2 M Luc (luciferase construct with α2-macro-globulin promoter), pCR™3 LacZ (galactosidase construct with a constitutively active promoter) (Amersham Biosciences) and the indicated plasmid by using FuGENE 6 (Roche Applied Science), starved with serum-free medium for 16 h, and stimulated with 20 ng/ml IL-6 for 16 h. The preparation of the lysates and the luciferase measurements were carried out according to the instructions that were supplied with the luciferase kit (Promega, Madison, WI). Luciferase activity values were normalized to the transfection efficiency that was measured as β-galactosidase activity. The experiments were done in triplicate, and the mean ± S.D. values were calculated. The imaging and bleaching studies were performed by using a Zeiss LSM 510 (Carl Zeiss, Jena, Germany) equipped with an argon and a helium-neon laser, a 63× water-corrected objective, and a perfusion chamber (manufactured in house) in which living adherent cells could be studied microscopically. HepG2 cells were transiently transfected with the indicated plasmid and grown on glass coverslips for later transfer to the perfusion chamber. Prior to the experiments the cells were starved for 16 h. The perfusion chamber was loaded with the coverslip and filled with cell culture medium. A constant temperature of 37 °C was maintained by a thermostat. For real time imaging studies cyan fluorescence was excited at λ = 458 nm and detected by using a bandpass filter BP 470–490. Yellow fluorescence was excited at λ = 514 nm and a bandpass filter BP 530-600 was used. The experiments were carried out by stimulation with 20 ng/ml IL-6 at time point 0, and thereafter pictures were taken every 30 s or at the indicated time points. For the pulse bleach experiments in living cells the microscope settings are shown in detail in Tables I and II.Table IMicroscope settings for the pulse bleach experiments presented in Fig. 3Laser configurationArgon laser output100%Multi-track modeTrack 1 (YFP)Track 2 (CFP)Excitation514 nm458 nmTransmission0.05%5.0%HFT458/514458/514FilterBPaBP, bandpass 530-600BP 470-490Scan settingsResolution256 × 256Data depth12 bitScan speed10Zoom5.0Scan directionOne wayModeLineNumber1MethodMeanYFP channelCFP channelPinhole262 μm262 μmDetector gain7001000Amplifier offset0.00.0Amplifier gain1.01.0ROI and bleach settingsDetection ROI diameter20 pixelBleach ROI diameter40 pixelBleach laser line514 nmTransmission100%Bleach iterations First bleach pulse100 Continuous pulse1 iteration every 3.3 s bleachinguntil time point 120 sor 180 s (as indicated)Time series settingsMeasurement interval3.3 sa BP, bandpass Open table in a new tab Table IIMicroscope settings for the pulse bleach experiments presented in Fig. 4Laser configurationArgon laser output35%Multi-track modeTrack 1 (YFP)Track 2 (CFP)Excitation514 nm458 nmTransmission0.1%5.0%HFT458/514458/514FilterBPaBP, bandpass 530-600BP 470-490Scan settingsResolution256 × 256Data depth12 bitScan speedMaxZoom5.0Scan directionOne wayModeLineNumber1MethodMeanYFP channelCFP channelPinhole262 μm262 μmDetector gain7501000Amplifier offset-0.025-0.05Amplifier gain1.01.0ROI and bleach settingsDetection ROI diameter20 pixelBleach ROI diameter30 pixelBleach laser line514 nmTransmission100%Bleach iterations First bleach pulse345 or 1,300 (as indicated) Continuous pulse30 iterations every 10 s bleachingTime series settingsMeasurement interval3 sa BP, bandpass Open table in a new tab Characterization of STAT3-CY Fluorescent Fusion Proteins—The subcellular concentrations of fluorescent fusion proteins and their variations over time can be monitored using confocal laser-scanning microscopy. In terms of studying transport processes it is helpful to label a population of fluorescent signal proteins using a bleaching approach as done in fluorescence loss in photobleaching experiments (34Lippincott-Schwartz J. Snapp E. Kenworthy A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 444-456Crossref PubMed Scopus (980) Google Scholar). The problem with fluorescent fusion proteins containing a single fluorophore is that as soon as bleaching has been performed it is impossible to follow their concentration because they have lost their fluorescence. To overcome this problem, the protein of interest can be fused to two distinct fluorophores as in the recently described FLAP method (30Dunn G.A. Dobbie I.M. Monypenny J. Holt M.R. Zicha D. J Microsc. 2002; 205: 109-112Crossref PubMed Scopus (53) Google Scholar, 31Zicha D. Dobbie I.M. Holt M.R. Monypenny J. Soong D.Y. Gray C. Dunn G.A. Science. 2003; 300: 142-145Crossref PubMed Scopus (141) Google Scholar). As a modification of this approach, we have constructed a STAT3-CFP-YFP fusion protein (STAT3-CY) as a functional variant of STAT3 with two different fluorescent tags (Fig. 1A). The STAT3 part consisting of 771 amino acids is identical with wild type murine STAT3α. A linker of 3 amino acids connects STAT3 with the C-terminal fluorophores CFP and YFP. For further studies, we have generated three STAT3 variants as CFP-YFP fusion proteins. In STAT3(Y705F)-CY tyrosine 705 has been mutated to phenylalanine. STAT3(1–320)-CY contains the N-terminal moiety, STAT3(321–771)-CY the C-terminal moiety of STAT3 (Fig. 1A). COS7 cells were transfected with STAT3, STAT3-CY, or the deletion mutants. After lysis of stimulated and unstimulated cells immunoprecipitations were performed with a GFP antibody that also recognizes YFP and CFP. Subsequently, the recombinant proteins were detected in Western blot with the GFP antibody (Fig. 1B, top panel). The mobilities of the fusion proteins in SDS-PAGE correspond well with the calculated molecular masses of 149, 93, and 110 kDa for STAT3-CY, STAT3(1–320)-CY, and STAT3(321–771)-CY, respectively. As expected, an antibody directed against an N-terminal epitope of STAT3 recognizes STAT3-CY and STAT3(1–320)-CY but not STAT3(321–771)-CY (Fig. 1B, middle panel). Tyrosine phosphorylation in response to cytokine stimulation is observed only for STAT3-CY and not for the deletion mutants (Fig. 1B, bottom panel). An unspecific band appears in all lanes at the position of STAT3-CY. Nevertheless, stimulation-induced phosphorylation of STAT3-CY is clearly detectable. Nontagged STAT3 is not detected because it has not been precipitated by the GFP antibody. The following experiments were carried out to demonstrate that STAT3-CY is as functional as wild type STAT3 with respect to transactivation and nuclear translocation. The transactivation capability of STAT3-CY was tested in a reporter gene assay (Fig. 1C). IL-6 stimulation of mock-transfected HepG2 cells leads to a pronounced induction of the α2-macroglobulin promoter mediated by endogenous STAT3. A significantly enhanced induction was observed in cells transfected with wild type STAT3. Transfection of HepG2 cells with the fluorescent constructs STAT3-YFP and STAT3-CY led to results similar to those shown for wild type STAT3. Real time imaging studies with IL-6-stimulated HepG2 cells that were transfected with STAT3-CY (Fig. 2A) demonstrate identical kinetics with regard to nuclear translocation as observed in studies with wild type STAT3 and single-tagged STAT3-YFP (32Herrmann A. Sommer U. Pranada A.L. Giese B. Küster A. Haan S. Becker W. Heinrich P.C. Müller-Newen G. J. Cell Sci. 2004; 117: 339-349Crossref PubMed Scopus (49) Google Scholar). The highest concentrations of fluorescent STAT3 within the nucleus were found between 15 and 40 min after IL-6 stimulation. From these findings we conclude that the fluorescent tags do not interfere with the basic functions of STAT3. IL-6 stimulation did not affect the subcellular distribution of STAT3(Y705F)-CY (Fig. 2B) or the deletion mutants (not shown). In unstimulated cells the distribution of STAT3 (Y705F)-CY was indistinguishable from STAT3-CY. Notably, the subcellular distributions of the two deletion mutants differed markedly. Whereas STAT3(1–320)-CY was almost equally distributed between the cytoplasm and the nucleus, STAT3(321–771)-CY was largely excluded from the nucleus (Fig. 2C). The YFP Fluorophore in STAT3-CY Can Be Bleached Selectively—STAT3-CY was designed to have two independent fluorophores that can be bleached selectively. The yellow fluorescent fluorophore is bleached efficiently and selectively using intense laser light at a wavelength of 514 nm. According to the excitation spectrum of CFP the cyan fluorophore should not be affected. This prediction has been tested by bleaching STAT3-CY nuclear foci that are often formed upon IL-6 stimulation of transfected HepG2 cells (Fig. 2, A and D). The formation of these subnuclear structures can also be observed in the case of wild type STAT3 and single-tagged STAT3-YFP (32Herrmann A. Sommer U. Pranada A.L. Giese B. Küster A. Haan S. Becker W. Heinrich P.C. Müller-Newen G. J. Cell Sci. 2004; 117: 339-349Crossref PubMed Scopus (49) Google Scholar). Application of a 514-nm laser pulse to a single dot (Fig. 2D, upper panels, arrows) by using the laser of the confocal microscope leads to a strong decrease in YFP fluorescence, whereas CFP fluorescence remains largely unaffected (Fig. 2D, lower panels, arrows). The selective bleaching of YFP enabled marking of a subpopulation of STAT3-CY and following its fate by determining the ratio of YFP to CFP fluorescence. Fig. 3A shows a general setup of the pulse bleach experiments in which we studied the nuclear import and export processes of STAT3-CY in living cells. For this purpose one "bleach ROI" and three "detection ROIs" were defined. Within the area of the bleach ROI the YFP moiety of STAT3-CY can be bleached with 514-nm laser pulses. The detection ROIs monitored cytosolic, nuclear, and background fluorescence intensities. In the following figures the data are presented in diagrams that show background-corrected fluorescence intensities of CFP and YFP within the indicated cytosolic or nuclear ROIs. A Dynamic Equilibrium between Nuclear Import and Export Results in Constant Subcellular Concentrations of STAT3-CY in Unstimulated Cells—Fig. 3A and the first image in Fig. 2A show the subcellular distribution of STAT3-CY in unstimulated HepG2 cells. Interestingly, STAT3-CY is detectable within the nucleus. Monitoring of fluorescence intensities within the cytosolic and nuclear detection ROIs without any bleaching proves that subcellular concentrations of STAT3-CY remain constant in unstimulated cells for at least 20 min (Fig. 3B. Slight intensity variations are caused by random cellular movements, small shifts in the focus plane, or little bleaching because of data recording. We asked whether the observed constant subcellular distribution of STAT3-CY is caused by the lack of any transport between cytosol and nucleus or whether it is a result of a dynamic equilibrium between nuclear import and export processes. For this reason we examined the nuclear transport process by pulse bleach experiments. In addition to the three detection ROIs, a bleach ROI was defined in the cytosol as outlined above. Yellow and cyan fluorescence intensities in the cytosol and the nucleus were measured for 20 min. At time point 30 s a strong bleach pulse at a wavelength of 514 nm was applied to th