Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4845-4850, May 1996 Cell Biology

Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera (steroid receptor/confocal laser scanning microscopy/mouse mammary tumor virus/transcription factor/intracellular calcium) HAN HTUN*, JULIA BARSONYt, ISTVAN RENYIt, DANIEL L. GOULDt, AND GORDON L. HAGER*t *Laboratory of Molecular Virology, National Cancer Institute, Bethesda, MD 20892-5055; and tLaboratory of Cellular Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by William J. Rutter, University of California, San Francisco, CA, December 28, 1995 (received for review October 21, 1995)

ABSTRACT A highly fluorescent mutant form of the tion on target genes. Furthermore, we have observed what green fluorescent protein (GFP) has been fused to the rat appears to be direct localization of GR on target genes. Finally, glucocorticoid receptor (GR). When GFP-GR is expressed in three-dimensional reconstruction of nuclear GR binding sites living mouse cells, it is competent for normal transactivation suggests a reproducible nuclear organization of target sites. of the GR-responsive mouse mammary tumor virus promoter. These findings show that GFP-GR fusions provide a powerful The unliganded GFP-GR resides in the cytoplasm and trans- approach to the study of receptor function and indicate that locates to the nucleus in a hormone-dependent manner with ligand-dependent changes in receptor activity can now be ligand specificity similar to that of the native GR receptor. addressed in single cells over extended time periods to follow Due to the resistance of the mutant GFP to photobleaching, receptor activation, translocation, target site selection, inacti- the translocation process can be studied by time-lapse video vation, and cycling between the nucleus and cytoplasm. In microscopy. Confocal laser scanning microscopy showed nu- addition, it is clear the GFP-GR will be invaluable in exploring clear accumulation in a discrete series of foci, excluding the organization and architecture of the interphase nucleus. nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although con- AND METHODS centration into nuclear foci is not observed. This reproducible MATERIALS pattern of transactivation-competent GR reveals a previously Cell Lines and Plasmids. Cell line 1471.1 contains multiple undescribed intranuclear architecture of GR target sites. copies of a bovine papilloma virus-mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-chloramphenicol Steroid receptors are hormone-dependent activators of gene acetyltransferase (CAT) fusion in the murine adenocarcinoma expression. It is generally accepted that the unliganded glu- C127 cell (11). pCI-nGFP-C656G was derived from pCI- cocorticoid receptor (GR) resides in the cytoplasm and that nH6HA-C656G and pZA69. pCI-nH6HA-C656G DNA ex- hormone activation leads to both nuclear accumulation and presses rat GR with the C656G mutation from the cytomeg- gene activation (see refs. 1-6 and references therein). How- alovirus promoter/enhancer, and is tagged at the N terminus ever, the mechanisms involved in nuclear translocation and with (His)6 and hemagglutinin epitope (12). A 768-bp GFP targeting of steroid receptors to regulatory sites in chromatin cDNA fragment with the S65T mutation was inserted between are poorly understood. It has been difficult to discriminate the two tags and GR. Other plasmids used in this study are as between the ability of a given receptor mutant, or a given follows: pLTRLuc (full-length MMTV LTR driving the ex- receptor-ligand combination, to participate in the separate pression of a luciferase gene) (13), pCMVIL2R [(interleukin processes of receptor activation, nuclear translocation, se- 2 receptor (IL-2 R) expression plasmid] (14), and pUC18 (Life quence-specific DNA binding, and promoter activation. Technologies, Grand Island, NY). The paucity of information on these issues stems in part Transfection. Plasmid DNA was transiently introduced into from the lack of appropriate technology to study the various 1471.1 cells either by calcium phosphate coprecipitation using stages in nuclear targeting. Because knowledge of these steps a N N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES)- is essential for understanding the mechanism of steroid hor- based buffer (15) or by electroporation. For calcium phosphate mone action, we have taken the approach of tagging GR with coprecipitation, semiconfluent cells were resuspended at 7 x a chromophore, allowing us to visualize in vivo, with the least 104 cells per ml in DMEM supplemented with charcoal/ perturbation, the changes in receptor subcellular localization dextran-treated fetal bovine serum (HyClone), dispensed as 1 upon exposure to activating ligand. ml into a 2 x 2 cm2 Lab-Tek Chamber Slide (Nunc) or as 10 Recent characterization of a chromophore, the green fluo- ml into a 100-mm Petri dish layered with 24.5-mm diameter rescent protein (GFP), provides a general method to label coverslips. Cells were grown at 37°C overnight, medium was proteins in living cells. Chimeras formed with a highly efficient replaced in the morning, and in the afternoon, cells were variant of GFP (7) afford a unique opportunity to examine the transfected with 1 ml of transfection mixture containing 20 jig organization of proteins of interest within various cellular of plasmid DNA per 10 ml of cells. Cells were left 12-16 hr, compartments (8-10). We have prepared a fusion between medium was replaced, and cells were allowed to recover for 2 GFP and the rat GR. This chimera functions normally in hr before further treatment and imaging. For calcium deple- cytoplasmic/nuclear translocation and gene activation. Using this reagent, we have monitored subcellular trafficking of GR Abbreviations: GFP, green fluorescent protein; GR, glucocorticoid in living cells. We find that nuclear translocation can be receptor; IL-2 R, interleukin-2 receptor; MMTV, mouse mammary characterized as a process separate and distinct from localiza- tumor virus; CAT, chloramphenicol acetyltransferase; LTR, long terminal repeat. Data deposition: The sequence of the GFP-GR expression plasmid The publication costs of this article were defrayed in part by page charge pCI-nGFP-C656G has been deposited in the GenBank data base payment. This article must therefore be hereby marked "advertisement" in (accession no. U53602). accordance with 18 U.S.C. §1734 solely to indicate this fact. To whom reprint requests should be addressed.

4845 Downloaded by guest on September 26, 2021 4846 Cell Biology: Htun et al. Proc. Natl. Acad. Sci. USA 93 (1996)

tion experiments, cells were electroporated with 5-20 ,g of ColEl pCI-nGFP-C656G DNA for 2 x 107 cells in 0.2 ml of cold DMEM at 250 V and 800 ,uF in a 0.4-cm electrode gap >( ~~~Promoter ATG electroporation chamber, left to recover on ice for 5 min, and Chimeric Intron-4 (His)6 then diluted in DMEM supplemented with dextran/charcoal- T7 Promoter treated fetal bovine serum before plating. Cells were then Beta- Hitope grown for 12-16 hr, and medium was replaced before treat- sreen Mutation ment and imaging. Enrichment of Transfected Cells and Analysis of Cytosolic I I _ ~~~~~~Protein Extracts. Cells that took up exogenous DNA were enriched by pCl-nGFP-C656G (Gly cotransfection with pCMVIL2R, an IL-2 R expression plas- mid, and selection for IL-2 RI cells using magnetic beads \ J /; S F~~~~~~~~Ala)5 (Dynal, Lake Success, NY) coated with mouse anti-human 9 hage GFP-GR/ / IL-2 R antibody (Boehringer Mannheim, clone 3G10), as \fl ori Fusio/ described (14). Extracts from the IL-2 RI and IL-2 R- cells \% // ~~~~C656G were assayed for the amount of luciferase activity in a Mi- lucocorticoid SV40 Late _ ReceptorcDNA croLumat LB96P as recommended by the manufacturer (EG PolyA & G/Berthold/Wallac, Gaithersburg, MD) and for CAT ac- Polylinker C656G Mutation tivity as described (16). B) Determination of Intracellular Calcium. Intracellular free calcium concentrations were determined in single cells by C) 12 measuring the signal from the calcium-sensitive indicator o Fura-2, according to Tsien and Harootunian (17). Intracellular c 10 x 0 0 calcium content was measured in three independent experi- 'oW c ments-at least 20 cells in each experiment. Ratio imaging was 0 8 performed using IMAGE 1 software (Universal Imaging, West Co0 0 8- c_ c Chester, PA), using 340-nm and 380-nm excitation, 510-nm .c- a Zeiss Photomi- emission, and 490-nm dichroic barrier filters, z croscope III microscope, enclosed in a temperature-controlled 4 CaL incubator, and an intensified (Videoscope, Dallas) charge- coupled device camera (Dage, model 72, Michigan City, IN), 20~ :da _ -GFP-GR and optical disc recorder (Panasonic). Intracellular free cal- ~~~- G4--R cium concentrations in cells with calcium-supplemented buffer 0 .2 2 0 were 350 + 183 nM, whereas in calcium-free buffer, the ug pCI-nGFP-C656G Activating Ligand concentrations were 60 ± 11 nM. Transfected and For time course cells Image Acquisition Analysis. studies, FIG. 1. Construction and characterization of GFP-GR. (A) Plas- were placed into a Dvorak-Stotler chamber and perfused at 37°C mid pCI-nGFP-C656G contains the GFP fused to the C656G mutant with assay buffer for 3 min, then with the same buffer containing GR. (B) Dexamethasone (dex)-dependent stimulation of MMTV- 1 nM dexamethasone (dex) for 2 hr at 10 ml/hr flow rates (using pLTRLuc (13) is shown for GFP-GR transfected cells. Solid bars a perfusion system from Adams & List, Westbury, NY). Samples represent the IL-2 R+-selected population activated for 2 hr with 1 nM were evaluated using a Zeiss Axiovert 10 microscope surrounded dexamethasone, and the open bar depicts activation ofthe endogenous by an incubator and equipped for epifluorescence with illumina- receptor with 100 nM dexamethasone. The Western blot below the tion from Gixenon burner, 480-nm excitation and 535-nm emis- panel shows detection of endogenous GR and transfected GFP-GR sion, and 505-nm dichromatic barrier filters (Chroma Technol- present in extracts from parallel transfections with the BuGR 2 were 15 sec with a monoclonal antibody (18) by enhanced chemiluminescence (ECL; ogy, Brattleboro, VT). Images acquired every Amersham). (C) Ligand specificity is presented for activation of Photometrics (Tucson, AZ) high-resolution, cooled charge- endogenous MMTV LTR-CAT sequences present in the 1471.1 cells coupled device camera (model P200) on a Silicon Graphics (11). Cells were treated for 4 hr with the indicated ligand, then (Mountain View, CA) workstation (model 4D310-VGX), using harvested, and levels of CAT activity were determined. custom software and incorporating functions from a vendor- supplied library (G.W. Hannaway & Associates, Boulder, CO). which makes the resulting chromophore 6-fold more fluores- Experiments requiring real-time image acquisition were per- cent than the wild-type GFP (7). formed on the imaging system described for the intracellular Since GR is ubiquitously present in all mouse cells, we used calcium measurements. a variant having a higher affinity for its ligand than the Confocal laser scanning microscopy was carried out on a endogenous receptor. S65T GFP was fused to a rat GR that Nikon Optiphot microscope equipped with Bio-Rad MRC-600 contains a cysteine-to-glycine mutation at position 656 of the confocal laser scanning unit, with krypton-argon laser 488-nm steroid-binding domain (19). This point mutation, C656G, fluorescent excitation, using a Fluor 100/1.3 oil-phase objec- increases the affinity of the receptor 9-fold for its ligand. The tive. From living cells expressing GFP-GR, serial 0.5-,um of this mutation selective of the optical sections were collected. Three-dimensional image ren- presence permits a¢tivation dering, analysis, and reconstruction were carried out with the transfected chimeric receptor without activation of the endog- ANALYZE software from the Mayo Clinic, Rochester, MN. enous receptor. Transcriptional Competence ofGFP-GR. When the GFP-GR chimera is introduced into cultured mouse cells, a fusion polypep- RESULTS tide with the predicted molecular mass of 118 kDa is produced Tagging of GR with GFP. To develop a highly efficient, (Fig. IB). When these cells are stimulated with 1 nM dexameth- fluorescent version of GR, we generated a GFP-GR chimera asone, a cotransfected reporter construct (pLTRLuc) containing with a 27-kDa GFP variant fused in frame to the second amino the luciferase reporter gene under the control of MMTV LTR is acid of rat GR (see Materials and Methods) (Fig. 1A). The GFP activated (Fig. 1B), and accumulation of luciferase activity is variant, from the jellyfishAequorea victoria, contains a serine- dependent on the amount of GFP-GR expression plasmid used. to-threonine substitution at amino acid 65 (S65T mutation), No significant luciferase activity accumulated in the 1 nM dex- Downloaded by guest on September 26, 2021 Cell Biology: Htun et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4847 amethasone-treated cells without added GFP-GR, indicating scanning microscopy, we examined transfected samples for that 1 nM dexamethasone activated the GFP-GR chimeric subcellular localization of the chimeric GFP-GR protein (Fig. protein but not the endogenous GR. With increasing amount of 2). We observed significant fluorescence in the cytoplasm of the GFP-GR expression plasmid, luciferase activity in the 1 nM -10% of total cells, approximately the fraction that typically dexamethasone-treated IL-2 RI (seeMaterials and Methods) cells acquires transfected DNA. Thus, the GFP was functional as a reaches the same level as that in 100 nM dexamethasone-treated chromophore in essentially all of the expressing cells in this IL-2 RI cells lacking GFP-GR expression plasmid (compare 2 mammalian system. ,tg of pCI-nGFP-C656G/1 nM dexamethasone with 0 ,tg pCI- Upon exposure to dexamethasone, translocation of GFP-GR nGFP-C656G/100 nM dexamethasone). Because the 100 nM occurs in 100% of fluorescing cells, with the rate of cytoplasm- dexamethasone treatment gives complete activation of the en- to-nuclear translocation dependent on the concentration of hor- dogenous GR in the latter case, we conclude that the GFP-GR mone. At 10 nM, complete translocation was induced within 10 chimeric receptor is fully functional in dexamethasone-mediated min at 37°C, with half-maximal nuclear accumulation at 5 min; transcriptional activation of the transiently introduced reporter this rate is consistent with previous findings (23). The rate of plasmid DNA. translocation is decreased with 1 nM dexamethasone (complete When activated by dexamethasone, GFP-GR is competent translocation over 30 min with half maximum at 9-10 min) and to induce not only the transiently introduced MMTV LTR- further reduced with 0.1 nM dexamethasone (complete translo- luciferase reporter DNA, as mentioned above (Fig. 1B), but cation within 2 hr with half maximum at 1 hr). also the multicopy MMTV LTR-CAT reporter genes present Analysis of a time-lapse series revealed that GFP-GR in 1471.1 cells (1 nM and 10 nM dexamethasone, Fig. 1C). In accumulated along fibrillar structures (Fig. 2A and 3A) and in the perinuclear region very rapidly after hormone addition contrast, treatment with 10 nM RU486, an antagonist with within With real-time or a poor (Fig. 2B), probably seconds. imaging, little GR agonist activity, progesterone, agonist, perinuclear accumulation was observed in a pulsatile pattern results in little activation of the MMTV LTR-CAT reporter; with 1 to 2-sec intervals between brightness changes. After 3 17f3-estradiol, a steroid that shows no affinity for GR, fails to min with 1 nM dexamethasone, GFP-GR was noticeably activate the LTR. Thus, transcriptional activation of the present in the nucleus, but not in the nucleoli (Fig. 2C). When MMTV LTR target genes by GFP-GR maintains the ligand approximately one-third of the protein had been translocated specificity characteristic of GR (refs. 19-22; also see refer- (9-10 min), a punctate pattern appeared (Fig. 2D), and ences in ref. 2). translocation was complete after 30 min (Fig. 2F). During Visualization of GFP-GR Cytoplasm-to-Nucleus Translo- translocation, the cells frequently became rounded and moved cation in a Single Metabolically Active Cell. Because the S65T along the long axis of the cell. We observed reduction of the variant of the GFP chromophore is resistant to photobleaching cell surface, as well as the nuclear volume, during the trans- (7), it was possible to use confocal and time-lapse video location. One hour after hormone treatment, the cells reattach microscopy to observe GFP-GR over extended periods. Using and regain a more flattened shape. computer-controlled high-resolution video and confocal laser Ligand Specificity of Cytoplasm-to-Nucleus Translocation. Whereas complete translocation of GFP-GR was observed in all fluorescing cells treated with dexamethasone (Fig. 3C), other classes of steroid hormones induced GFP-GR translocation to various extent reflective of the affinity for GR. The glucocorti- coid antagonist RU486, known to have a high affinity for GR (19), was as potent as dexamethasone for induction of translo- cation (Fig. 3D). Progesterone, a weak GR agonist, required a concentration 100-fold higher than dexamethasone for translo- cation; however, approximately one-half of the GFP-GR re- mained in the cytoplasm (data not shown). In contrast, 17,B- estradiol, a steroid hormone that does not bind GR, did not cause intranuclear GFP-GR accumulation (10 nM, Fig. 3B). Thus, GFP-GR maintained ligand-dependent cytoplasm-to-nuclear translocation, with analog specificity identical to that for the untagged GR with the C656G point mutation (19). Furthermore, whereas ligand binding may suffice to trigger efficient cytoplasm- to-nucleus translocation, the receptor, once in the nucleus, shows differing degrees in its ability to activate the LTR (compare Fig. 3 with Fig. 1C). Role of Intracellular Free Calcium and Energy in GFP-GR Translocation. We also addressed two important issues concern- ing the nuclear import of proteins, the role of Ca2+ and the energy requirement for translocation. Intracellular stores of Ca>2 were depleted by incubating the cells with the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and the calcium ionophore ionomycin in calcium-free media [intracellular free calcium con- tent was measured with ratio imaging in Fura-2-loaded cells (see Materials andMethods). The cytoplasmic pattern ofGFP-GR was FIG. 2. Time-dependent translocation of GFP-GR. Murine ade- not significantly altered by calcium depletion (Fig. 4A). When nocarcinoma cells (1471.1) were cultured on coverslips and transfected Ca2+-depleted cells were subsequently exposed to dexametha- with GFP-GR fusion chimera 1 day before microscopy. Cells were sone in Ca2+-free media, the hormone induced complete trans- placed into a Dvorak-Stotler chamber and perfused at 37°C with assay buffer for 3 min (A), then with buffer containing 1 nM dexamethasone location of GFP-GR (Fig. 4B). were for 2 hr. Perinuclear accumulation was evident at 1 min (B), nuclear To study the energy dependence of ligand binding, cells entry at 3 min (C), target site binding at 7 min (D), half-maximal exposed to dexamethasone at 4°C (Fig. 4C); then hormone was accumulation at 9 min (E), and complete translocation at 30 min (F). removed and the cells were warmed to 37°C under continuous (Bar = 10 ,im.) monitoring with video microscopy. At 4°C, translocation was Downloaded by guest on September 26, 2021 4848 Cell Biology: Htun et al. Proc. Natl. Acad. Sci. USA 93 (1996)

FIG. 3. Ligand specificity of nuclear accumulation. Cells (1471.1) were treated with buffer (A), 10 nM 1713-estradiol (B), 10 nM dexamethasone (C), or 10 nM RU486 (D) for 30 min at 37°C. At the end of hormone treatment, images (A-D Left) from living cells express- ing GFP-GR were visualized with confocal laser scan- ning microscope. Arrows on the phase-contrast images (A-D Right) point to the nuclei of GFP-GR-expressing cells. (Bar = 10 ,um.) completely arrested. Rewarming led to complete translocation activated GFP-GR into intranuclear foci immediately suggests and reappearance of the focal GFP-GR localization (Fig. 4D). that a specific architecture may underlie this distribution. This experiment indicates that hormone binding to GR in Organized Architecture ofInterphase Nuclei As Revealed by living cells does not require energy, in contrast to the energy- GFP-GR. Three-dimensional image analysis of the points of dependent step of translocation. GR accumulation in dexamethasone-treated cells reveals a Focal Accumulation of Nuclear GFP-GR Correlates with nonrandom distribution of GFP-GR accumulation. Most Transcriptional Activation. When the intranuclear accumula- strikingly, comparison of adjacent cells demonstrates a repro- tion of GFP-GR is examined in detail, it is readily apparent ducible pattern of intranuclear structure for GFP-GR accu- that the receptor localizes most prominently at specific foci mulation. This intranuclear architecture is shown in Fig. 5 within the nucleus (note the bright spots in Figs. 3C, 4B, and (Upper) for a typical pair of nuclei. A predominance of is a low level of accumulation in a GFP-GR-accumulating foci is observed in the quadrant of the 4D). In addition, there nucleus adjacent to the glass attachment surface of the cell, diffuse reticular pattern, forming the basis for the nuclear whereas a group of large patches of GFP-GR-containing foci background fluorescence (Fig. 3C). The 'presence of these is observed in the top half. Nucleolar structures were always focal accumulations is unique to dexamethasone-treated cells devoid of GFP-GR. The nuclear pattern of RU486-treated and is not observed in 17,3-estradiol- (Fig. 3B) or progesterone- cells was again strikingly different from dexamethasone- exposed cells (data not shown). In RU486-treated cells, al- treated cells (Fig. 5, Lower; note also Fig. 3D). Although though a few focal points may be present, they are not readily essentially all of the GFP-GR is translocated, intranuclear discernible. Instead, GFP-GR accumulates in a diffuse pattern RU486-liganded receptor is distributed throughout the nu- with regions of condensation in a reticular pattern (Fig. 3D). cleus in a reticular pattern that excludes nucleoli. Depleting intracellular Ca2+ did not affect the dexametha- sone-mediated formation of intranuclear foci (Fig. 4B), but it decreased the amount of diffuse GFP-GR in the nucleus. The DISCUSSION ability of agonist to induce focal accumulation of GFP-GR In the present study, we have shown the usefulness of GFP in correlated strongly with its ability to activate transcription monitoring the activity of a steriod hormone receptor. The (Fig. 1C). The striking accumulation of dexamethasone- ability to directly observe living cells has allowed us to follow Downloaded by guest on September 26, 2021 Cell Biology: Htun et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4849

FIG. 4. Ca2+ and energy requirements for GFP-GR translocation. Cells were preincubated in calcium-free medium with 5 ,uM thapsi- gargin, 2 ,LM ionomycin, and 5 mM EGTA for 1 hr. Measurement of intracellular free Ca2+ with Fura-2 and ratio imaging showed that this treatment effectively depleted the cells of calcium. Then, cells were placed in calcium-free medium without hormone (A), or with 10 nM dexamethasone in the same buffer for 30 min at 37°C (B). To study the effect of temperature on ligand binding and translocation, cells were exposed to 10 nM dexamethasone for 30 min at 4°C (C). Following this treatment, hormone was removed and cells were warmed to 37°C (D). Images were taken from living cells with a confocal laser scanning microscope (A and B) or with a cooled charge-coupled device camera from the Axiovert 10 microscope system (C and D) as described. Bar = 10 Am. in real time the process of cytoplasm-to nucleus translocation, and it has revealed differences in GR intranuclear accumula- tion pattern dependent on the type of activating ligand. Furthermore, the patterns of GR accumulation are remark- FIG. 5. Three-dimensional architecture of GFP-GR nuclear target ably similar between adjacent cells, suggesting an order in the sites. Serial 0.5-,um sections of nuclei from dexamethasone-treated organization of the interphase nucleus. cells were collected with a confocal laser scanning fluorescent micro- Because the use of GFP as a tag involves fusing a rather large scope, digitized images were imported into a Silicon Graphics Indigo protein (27 kDa) to GR, we thought it essential to examine 2 workstation, and three-dimensional image segmentation, rendering, whether any GR activity has been compromised by GFP. To analysis, and reconstruction were carried out with the ANALYZE software. Two nuclei from cells treated with dexamethasone are shown this end, we fused GFP to a rat GR with the C656G point (Upper), and two nuclei from RU486-treated cells are presented mutation, which can be selectively activated without activating (Lower). GFP-GR distributions in the nuclei are displayed as pseudo- the endogenous receptor. By a number of criteria, GFP-GR colored, voxel-gradient-shaded, three-dimensional projections. The functions very much like GR. In particular, the tagged receptor nuclear distributions are also mapped onto the side walls of the boxes resides in the cytoplasm until activated by a ligand, then it as orthogonal summed voxel projections. (Box width = 30 ,um.) translocates into the nucleus at a rate comparable to that previously reported (23). The rate and extent of GFP-GR bition of import by chilling indicates that this transport is translocation show a dependence on the concentration of the facilitated. Accumulation of GFP-GR along fibrillar struc- activating ligand as well as a ligand specificity reflective of the tures before dexamethasone addition (Fig. 2A and 3A) and in native receptor (19). Furthermore, because both dexametha- the perinuclear region after dexamethasone addition, suggests sone treatment and RU486 treatment lead to complete trans- that the cytoskeleton is involved in the transport process (Fig. location of GFP-GR from the cytoplasm to the nucleus in all 2B; refs. 6 and 24-26). Finally, the pulsatile brightness changes cells, essentially all of the GFP-GR molecules exist in a in the perinuclear region support an energy- and microtubule- conformation competent for both ligand binding and nuclear dependent active transport process. translocation (Fig. 3C). Once in the nucleus, GFP-GR's ability In our system, depleting endoplasmic reticulum calcium to activate the transcription of a MMTV LTR reporter gene stores and removing extracellular calcium did not prevent depends on the type of activating ligand, consistent with GFP-GR import into the nucleus. Previous studies, using previous results for GR (summarized in ref. 2). In the case of isolated nuclei or permeabilized cell systems, indicated that a potent agonist, dexamethasone, less GFP-GR is required for calcium is required for both passive diffusion and signal- activation of transiently introduced MMTV LTR-luciferase mediated transport through the nuclear pore (27, 28). Whereas reporter gene than for the endogenous GR, indicating that we cannot exclude the possibility that other intracellular even with respect to transactivation potential, the presence of calcium pools exist that may contribute to the GFP-GR GFP has not altered the transcriptional potency ascribed to the translocation, differences could also be attributed to protein- C656G point mutation (19). Thus, in all aspects, the presence specific transport mechanisms or to the involvement of soluble of GFP appears not to have affected normal GR function. cytoplasmic factors. Because the S65T variant of GFP used here is highly The rate of translocation of GFP-GR was dependent on excitable at 488-nm wavelength and is resistant to photo- hormone concentration, reflecting the dose and time depen- bleaching, we have been able to follow the course of cytoplasm- dence of GR action. This suggests that the rate of translocation to-nucleus translocation of GR in a single living cell for an contributes significantly to GR function. Recently, immu- extended period of time (Fig. 2). Upon binding to dexameth- nophilin ligand FK506 was shown to enhance both the rate of asone, GFP-GR moves vectorially toward the nucleus. Inhi- translocation and extent of transactivation by GR (29). Our Downloaded by guest on September 26, 2021 4850 Cell Biology: Htun et al. Proc. Natl. Acad. Sci. USA 93 (1996) studies indicate that GFP-GR will be a useful model to study 1. Gasc, J.-M. & Baulieu, E. E. (1987) in Steroid Hormone Receptors: reagents that modify rates of nuclear translocation, in addition Their Intracellular Localisation, ed. Clark, C. R. (Ellis Horwood, to those that affect rate of nuclear export (30). Chichester, England), pp. 233-250. Shortly after GFP-GR enters the nucleus, foci of bright 2. Beato, M. (1989) Cell 56, 335-344. 3. Carson-Jurica, M. A., Schrader, W. T. & O'Malley, B. W. (1990) fluorescence appear for the dexamethasone-induced cells (Fig. Endocr. Rev. 11, 201-220. 2D). These bright foci of dexamethasone-induced GFP-GR 4. Gronemeyer, H. (1993) in Steroid HormoneAction, ed. Parker, M. appear to be organized in a reproducible three-dimensional G. (Oxford University Press, New York), pp. 94-117. architecture. Because the presence of these localized foci 5. Tsai, M. J. & O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, correlates with transcriptional activation of the amplified 451-486. MMTV LTR-CAT fusion chimeras present in this cell, we 6. Akner, G., Wikstrom, A. C. & Gustafsson, J. A. (1995) J. Steroid argue that the foci of bright fluorescence reflect the activation Biochem. Mol. Biol. 52, 1-16. of target genes by the dexamethasone-activated GFP-GR; this 7. Heim, R., Cubitt, A. B. & Tsien, R. Y. (1995) Nature (London) hypothesis can now be subjected to direct test. The focal 373, 663-664. 8. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. accumulation of GFP-GR in these centers introduces obvious C. (1994) Science 263, 802-805. opportunities to study colocalization of other activities needed 9. Wang, S. & Hazelrigg, T. (1994) Nature (London) 369, 400-403. for GR activation from target genes organized in chromatin. 10. Prasher, D. C. (1995) Trends Genet. 11, 320-323. In contrast to dexamethasone, RU486-activated GFP-GR 11. Archer, T. K., Cordingley, M. G., Marsaud, V., Richard-Foy, H. accumulates almost exclusively in a reticular pattern found & Hager, G. L. (1989) in Proceedings: Second International CBT throughout the nucleus but excluding nucleoli; this pattern Symposium on the Steroid/Thyroid Receptor Family and Gene could result from association with the nuclear matrix (31, 32). Regulation, eds. Gustafsson, J. A., Eriksson, H. & Carlstedt- Using immunofluorescence in fixed cells, van Steensel et al. Duke, J. (Birkhauser, Berlin), pp. 221-238. recently reported (33) that GR accumulates in clusters within 12. Niman, H. L., Houghten, R. A., Walker, L. E., Reisfeld, R. A., Wilson, I. A., Hogle, J. M. & Lerner, R. A. (1983) Proc. Natl. the nucleus, but they found no difference between agonist- and Acad. Sci. USA 80, 4949-4953. RU486-activated receptor; furthermore, they suggested that 13. Lefebvre, P., Berard, D. S., Cordingley, M. G. & Hager, G. L. these clusters are not directly involved in active transcription. (1991) Mol. Cell. Biol. 11, 2529-2537. In contrast, hepatocyte nuclear factor 4 (HNF-4) was shown to 14. Giordano, T., Howard, T. H., Coleman, J., Sakamoto, K. & be generally distributed in nuclei when dephosphorylated, but Howard, B. H. (1991) Exp. Cell Res. 192, 193-197. localized in subnuclear domains when expressed in the phos- 15. Chen, C. & Okayama, H. (1987) Mol. Cell Biol. 7, 2745-2752. phorylated, transcriptionally competent form (34). These lat- 16. Gorman, C. M., Moffat, L. F. & Howard, B. H. (1982) Mol. Cell ter findings are in agreement with our observation that only Biol. 2, 1044-1051. transcriptionally competent GR localizes in the nuclear foci. 17. Tsien, R. Y. & Harootunian, A. T. (1990) Cell Calcium 11, 93-109. Finally, it has been argued that DNA in the nucleus is localized 18. Gametchu, B. & Harrison, R. W. (1984) Endocrinology 114, in a nonrandom fashion (35-38). The reproducible pattern of 274-279. agonist-induced GFP-GR accumulation may reflect an inherent 19. Chakraborti, P. K., Garabedian, M. J., Yamamoto, K. R. & order of the interphase nucleus; we observed the asymmetric Simons, S. S., Jr. (1991) J. Biol. Chem. 266, 22075-22078. nuclear accumulation not only in nuclei from neighboring cells 20. Becker, P. B., Gloss, B., Schmid, W., Strahle, U. & Schutz, G. but also in individual random cells. We note that Lawrence and (1986) Nature (London) 324, 686-688. coworkers (39) reported an asymmetric distribution of poly(A)- 21. Otten, A. D., Sanders, M. M. & McKnight, G. S. (1988) Mol. associated transcription domains, also in the lower portion of Endocrinol. 2, 143-147. nuclei of human diploid fibroblasts. The availability of functional 22. Lanz, R. B. & Rusconi, S. (1994) Endocrinology 135, 2183-2195. 23. Picard, D. & Yamamoto, K. R. (1987) EMBO J. 6, 3333-3340. GFP fusions should advance our ability to evaluate the potential 24. Perrot-Applanat, M., Lescop, P. & Milgrom, E. (1992) J. Cell existence of a nuclear architecture. Biol. 119, 337-348. In conclusion, the use of GFP-GR allowed us to visualize 25. Scherrer, L. C. & Pratt, W. B. (1992)J. Steroid Biochem. Mol. Biol. steroid receptor translocation in real time in living cells. It has 41, 719-721. led to the discovery of an intranuclear architecture of GR 26. Pirollet, F., Job, D., Margolis, R. L. & Garel, J. R. (1987) EMBO target sites. The GFP-GR should serve as an invaluable tool J. 6, 3247-3252. for understanding further details of receptor activation, the 27. Pruschy, M., Ju, Y., Spitz, L., Carafoli, E. & Goldfarb, D. S. translocation process, interaction of receptors with compo- (1994) J. Cell Biol. 127, 1527-1536. nents of the eukaryotic interphase nuclei, and mechanisms of 28. Greber, U. F. & Gerace, L. (1995) J. Cell Biol. 128, 5-14. 29. Ning, Y. M. & Sanchez, E. R. (1993) J. Biol. Chem. 268, transcriptional activation by steriod receptors. In addition, 6073-6076. GFP-GR will surely be invaluable in helping shape our 30. DeFranco, D. B., Qi, M., Borror, K. C., Garabedian, M. J. & understanding of the architecture and dynamics of the nucleus. Brautigan, D. L. (1991) Mol. Endocrinol. 5, 1215-1228. 31. Kaufmann, S. H., Okret, S., Wikstrom, A. C., Gustafsson, J. A. Note Added in Proof. While this paper was.in press, Ogawa et al. (40) & Shaper, J. H. (1986) J. Biol. Chem. 261, 11962-11967. described a fusion between a fragment of the human GR and wild-type 32. Nelson, W. G., Pienta, K. J., Barrack, E. R. & Coffey, D. S. (1986) GFP. These authors reported a requirement for lower temperature Annu. Rev. Biophys. Biophys. Chem. 15, 457-475. preincubation (30°C versus 37°C) to obtain both GFP as well as GR 33. van Steensel, B., Brink, M., van der Meulen, K., van Binnendijk, activity. In addition, they did not observe subnuclear localization of GR. E. P., Wansink, D. G., de Jong, L., de Kloet, E. R. & van Driel, R. (1995) J. Cell Sci. 108, 3003-3011. Stoney S. Simons, Jr. (National Institute of Diabetes and Digestive 34. Ktistaki, E., Ktistakis, N. T., Papadogeorgaki, E. & Talianidis, I. and Kidney Diseases) generously provided the C656G mutant GR (1995) Proc. Natl. Acad. Sci. USA 92, 9876-9880. receptor for this work and also supplied RU486 ligand for use in these 35. Manuelidis, L. (1990) Science 250, 1533-1540. experiments. We thank Ravi Dhar (National Cancer Institute) and 36. Yokota, H., van den Engh, G., Hearst, J. E., Sachs, R. K. & Trask, Mike Moser (University of Washington, Seattle) for providing the B. J. (1995) J. Cell Biol. 130, 1239-1249. plasmid pZA69, and Barbour Warren and Cathy Smith for helpful 37. Cook, P. R. (1995) J. Cell Sci. 108, 2927-2935. comments on the manuscript. The variant GFP allele was used with the 38. Brasch, K. & Ochs, R. L. (1995) Int. Rev. Cytol. 159, 161-194. kind permission of Roger Y. Tsien (Howard Hughes Medical Insti- 39. Carter, K. C., Bowman, D., Carrington, W., Fogarty, K., McNeil, tute/University of California, San Diego). Carl A. Miller (National J. A., Fay, F. S. & Lawrence, J. B. (1993) Science 259, 1330-1335. Instiute of Diabetes and Digestive and Kidney Diseases) helped with 40. Ogawa, H., Inouye, S., Tsuji, F. I., Yasuda, K. & Umesono, K. three-dimensional image reconstruction and display. (1995) Proc. Natl. Acad. Sci. USA 92, 11899-11903. 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