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A Novel In Situ Assay for the Identification and Characterization of Soluble Nuclear Mobility Factors
Cem Elbi,1 Dawn A. Walker,1 Marcia Lewis,2 Guillermo Romero,2 William P. Sullivan,3 David O. Toft,3 Gordon L. Hager,1* and Donald B. DeFranco2*
(Published 22 June 2004) (Revised 6 July 2004)

INTRODUCTION MATERIALS
Cell Culture Reagents and Supplies Cell Lines Purified Chaperone and Cochaperone Proteins Chemicals Fluorescent Protein Tags and Expression Vectors

EQUIPMENT
Cell Culture Temperature Control Microscope Quantitative Image Analysis

RECIPES INSTRUCTIONS
Biochemical Permeabilization and Extraction In Situ Nuclear Mobility Factor Assay FRAP Parameters Quantitative FRAP Analysis

NOTES AND REMARKS REFERENCES AND NOTES

of Receptor Biology and Gene Expression, Building 41, Room B602, National Cancer Institute, Bethesda, MD 208925055, USA. of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. 3Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, MN 55905, USA.
2Department

1Laboratory

*Corresponding authors: E-mail, hagerg@exchange.nih.gov (G.L.H.); dod1@pitt.edu (D.B.D.)

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Abstract
The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.

Introduction
The visualization and precise quantitation of protein mobility in live cells through the use of photobleaching techniques has led to an increased understanding of the mechanisms responsible for intracellular protein trafficking (1, 2). Although protein mobility within the nucleus can approach the limits predicted for the diffusion of free solute, low- or high-affinity interactions with specific soluble or solid-state targets can reduce the kinetics of nuclear protein trafficking (3). However, even nuclear proteins that participate in high-affinity interactions with large macromolecular complexes are highly mobile and possess diffusion coefficients that can approach 0.5 m2 s1, as measured by photobleaching techniques in live cells (3). In recent years, many green fluorescent protein (GFP) chimeras of nuclear proteins have been assessed for their localization within specific subnuclear compartments and their intranuclear mobility. One family of nuclear proteins that has been the subject of particular scrutiny in this regard is the nuclear receptor (NR) superfamily. NR proteins participate in various physiological responses to small ligands such as hormones, vitamins, and the metabolites of steroids, bile acids, and fatty acids (4, 5). NRs regulate the transcription of unique sets of target genes, usually in response to ligand binding (4, 5). NRs associate with their target genes through direct interactions with specific DNA sequences or tethering to other DNA-bound factors, resulting in the recruitment of large co-regulator complexes (6, 7). These co-regulators include coactivator and co-repressor complexes that modulate RNA polymerase II activity, in part through direct modification of histone proteins within core nucleosomes (810). In some cases, the mobility of NR co-regulator proteins has been examined in live cells. For example, nuclear mobility of the glucocorticoid receptor-interacting protein-1 (GRIP-1) coactivator is reduced when it interacts with the ligand-bound glucocorticoid receptor (GR) on target genes in live cell nuclei, implying that the assembly of functional complexes of NRs and coactivators on active promoters provides one type of scaffold that may limit nuclear protein mobility (11). Despite the increasing number of nuclear proteins assessed for mobility in live cells, our knowledge of the factors that regulate protein trafficking and mobility within the nucleus is limited. Both biochemical and live-cell imaging experiments reveal that the assembly and disassembly of large nuclear receptor-coactivator complexes on active genes is very rapid (12, 13), yet we know little about the mechanisms that ensure the efficient turnover of such supramolecular assemblies. In many paradigms of protein trafficking, model in situ systems have been instrumental in the identification of both specific targeting signals and soluble transport factors that function in interorganelle protein trafficking. Here, we describe an in situ assay for nuclear protein mobility in which the effects of specific subnuclear trafficking factors can be assessed. Importantly, cells analyzed with this assay maintain their transcriptional competence. Digitonin has been used extensively as a permeabilization agent that maintains both transcriptional competence and the energy- and transport factor-dependent trafficking of proteins between the cytoplasm and nucleus (14, 15). However, most proteins added to digitonin-permeabilized cells will not enter the nucleus unless specific nuclear transport factors are also included to permit their transit through the intact nuclear pore complex. This feature of the digitonin-permeabilized cells limits their usefulness as an assay system to characterize the effects of exogenously added factors on any nuclear function (for example, nuclear mobility). It was therefore necessary to develop additional protocols for permeabilization or extraction or both that would partially disrupt the nuclear pore complex to allow exogenously added proteins to gain access to the nucleus yet maintain nuclear integrity. Previously, we identified a hypotonic buffer treatment of digitonin-permeabilized cells that was efficient at extracting unliganded GR from nuclei, yet incapable of significantly depleting the nucleus of ligand-bound GR (16). We therefore reasoned that this combination of digitonin-permeabilization and hypotonic buffer extraction might allow for selective depletion of some nuclear proteins and not lead to the irreversible disruption of essential nuclear functions. We applied a digitonin-permeabilization plus hypotonic buffer extraction protocol in mouse 3617.4 mammary adenocarcinoma cell line (17) to investigate the roles of putative subnuclear trafficking factors in influencing the localization and mobility of GFP chimeras of GR and PR-B. Mouse 3617.4 and 5953 cells contain an integrated copy of a GFP-GR and GFP-PR-B chimera under the control of a tetracycline (tet)-regulated promoter (18,19). As will be shown below, in 3617.4 cells subjected to our permeabilization and extraction protocol, nuclear GFP-GR chimeras are rendered immobile as assessed by fluorescence recovery after photobleaching (FRAP) analysis. Therefore, the permeabilization and extraction of 3617.4 and 5953 cells provided a useful system to identify and characterize putative subnuclear trafficking factors that could be revealed by their ability to recover GFP-GR and GFP-PR-B mobility. We have found that this procedure can easily be adapted to other cell types for the analysis of nuclear mobility of other proteins.
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Materials

Cell Culture Reagents and Supplies

Charcoal-stripped fetal bovine serum (Hyclone, #SH30068.03) Dulbeccos modified eagle medium (DMEM; Gibco, #11960) Fetal bovine serum (FBS; Atlanta Biologicals, #S11150) Hanks balanced salt solution (HBSS; Gibco, #14025) L-Glutamine, 100 (Invitrogen, #25030) Nonessential amino acids, 100 (Invitrogen, #11140) Penicillin-streptomycin, 100 (Invitrogen, #15140) Phosphate-buffered saline (PBS; Quality Biologicals, #114-057-100) Rabbit reticulocyte lysate (Promega, #L4960) Sodium pyruvate, 100 (Invitrogen, #11360) Tissue culture dishes, 35 mm and 100 mm (Falcon, #353001 and 353003) Trypan blue dye, 0.4% (Invitrogen, #15250) Trypsin in 1 mM ethylenediamine tetraacetic acid (EDTA) (Invitrogen, #25200) Two-chambered Labtek II coverglass (Nalgene, #155360)

Cell Lines
Note: The assay described in this Protocol can be performed with any cell line expressing GFP fusion protein stably. C127 mouse fibroblast derivatives Mouse mammary adenocarcinoma cell line (3617.4) stably expressing GFP-rat GR (18) Mouse mammary adenocarcinoma cell line (5953) stably expressing enhanced GFP-human progesterone receptor (PR)-B

Purified Chaperone and Cochaperone Proteins

Note: See (20) for details on purification unless otherwise noted. Human Hsp70 (recombinant form prepared in Sf9 cells) Human Hop (p60) (recombinant form prepared in bacteria) Human CHIP [as described in (17), recombinant His-tagged CHIP expressed in bacteria was purified using a Stratagene Talon column] Human FKBP51 (recombinant form prepared in bacteria) Human Hsp90 (recombinant form prepared in Sf9 cells) Human p23 (recombinant form prepared in bacteria) Yeast Ydj-1 (recombinant form prepared in bacteria)

Chemicals
Adenosine triphosphate, disodium salt (ATP) Bovine serum albumen (BSA)

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Creatine phosphate Creatine phosphokinase Dexamethasone (Sigma, #D-1756) Digitonin Note: Some vendors do not supply digitonin at 100% purity. The purity of digitonin is indicated in the chemical specification sheet supplied by the vendor. Thus, always check purity of digitonin and vary the amount added if necessary. Dimethyl sulfoxide (DMSO) Dithiothreitol (DTT) Ethanol, 200-proof Ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N,N-tetraacetic acid (EGTA) G418 (Gibco, #11811) Hepes, 1 M (pH 7.3) (Quality Biologicals, #118-089-060) Magnesium acetate Magnesium chloride, 1 M (Quality Biologicals, #351-033-060) Potassium acetate (Quality Biologicals, #351-035-060) Potassium chloride, 1 M (Quality Biologicals, #351-044-100) R5020 (NEN Life Science, #NLP-004) Sodium acetate Sodium acetate, 3 M (pH 5.2) (Quality Biologicals, #351-035-060) Tetracycline (Fisher, #BP912-100) Triton X-100

Fluorescent Protein Tags and Expression Vectors

Green fluorescent protein (GFP) and Enhanced GFP (Clontech) Tet-Off inducible system (Invitrogen): pTet-Splice, pTet-tTAK, and pTKneo

Equipment

Cell Culture
Hemacytometer Tissue culture incubator at 37C, 5% CO2 (Forma Scientific)

Temperature Control
ASI 400 Air Stream incubator (Nevtek)

Microscope
Confocal microscope LSM 510 (Zeiss) equipped with 40-mW argon laser 63 or 100 1.3 numerical aperture oil immersion objective

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Quantitative Image Analysis

LSM 510 image analysis software (Zeiss) Microsoft Excel or any other spread sheet software

Recipes

Recipe 1: Complete Culture Medium

DMEM FBS Nonessential amino acids, 100 Sodium pyruvate, 100 L-Glutamine, 100 G418, 300 mg/ml stock Penicillin-streptomycin, 100 430 ml 50 ml 5 ml 5 ml 5 ml 1.6 ml 5 ml

Dissolve 3.72 g of G418 in 11.4 ml of water and 1 ml of 1 M Hepes, pH 7.3 to make a 300-mg/ml stock solution. Filter sterilize and store at 4C. Dissolve 500 mg of tetracycline in 50 ml of 200-proof ethanol and store in the dark at 20C. Immediately before changing the medium, add 50 l of this 10 mg/ml tetracycline to 500 ml of Complete Culture Medium for a final concentration of 10 g/ml. Complete culture medium containing tetracycline must be used immediately. Note: Cells expressing GFP-GR or GFP-PR-B under the control of a tet-regulated promoter are maintained in this medium to suppress GFP-GR or GFP-PR-B expression.

Recipe 2: Tetracycline-Free Culture Medium

DMEM FBS Nonessential amino acids, 100 Sodium pyruvate, 100
L-Glutamine, 100

430 ml 50 ml 5 ml 5 ml 5 ml 1.6 ml 5 ml

G418, 300 mg/ml stock Penicillin-streptomycin, 100 Store this medium at 4C for 20 days.

Recipe 3: Tetracycline-Free Culture Medium with Charcoal-Stripped FBS

DMEM Charcoal-stripped FBS Nonessential amino acids, 100 Sodium pyruvate, 100
L-Glutamine, 100

430 ml 50 ml 5 ml 5 ml 5 ml 1.6 ml 5 ml

G418, 300 mg/ml stock Penicillin-streptomycin, 100 Store this medium at 4C for 20 days.

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Recipe 4: 100 M Dexamethasone

Dissolve 3.925 g of dexamethasone in 10 ml of ethanol to make a 1 M stock solution. Dilute this stock in ethanol to make a 100-M stock solution, and store at 20C. Chill dexamethasone on ice before use. Note: Dexamethasone is used at a working concentration of 100 nM to stimulate GR.

Recipe 5: 30 M R5020
Dissolve 5 mg of R5020 in 1.53 ml of ethanol to make a 10-mM stock. Dilute this stock in ethanol to make a 30-M stock solution, and store at 20C. Chill R5020 on ice before use. Note: R5020 is used at a working concentration of 30 nM to stimulate PR.

Recipe 6: Transport Buffer

Reagent
1 M Hepes-KOH, pH 7.8 1 M Potassium acetate 1 M Sodium acetate 1 M Magnesium acetate 0.5 M EGTA, pH 8.0

Amount
10 ml 55 ml 2.5 ml 1 ml 1 ml

Final concentration
20 mM, pH 7.8 110 mM 5 mM 2 mM 1 mM

Dissolve 238.3 g of Hepes in 1000 ml of deionized water to make a 1 M stock solution. Titrate to pH 7.8 with 1 M KOH (56.1 g of KOH dissolved in 1000 ml of deionized water). Dissolve 214.5 g of magnesium acetate in 1000 ml of deionized water to make a 1 M stock solution. Dissolve 190.2 g of EGTA in 1000 ml of deionized water final volume to make a 0.5 M stock solution. Titrate to pH 8.0 with 1 M KOH. Dissolve 98.2 g of potassium acetate in 1000 ml of deionized water to make a 1 M stock solution. Dissolve 136.1 g of sodium acetate in 1000 ml of deionized water to make a 1 M stock solution. Prepare Transport Buffer in 500 ml of water, filter sterilize, and store at 4C. Chill on ice before use.

Recipe 7: Permeabilization Buffer

Dissolve 40 mg of digitonin in 1 ml of DMSO to make a 40 mg/ml stock solution and store at room temperature shielded from light. Add 5 l of this 40 mg/ml digitonin stock to 10 ml of Transport Buffer (Recipe 6) just before use, for a final concentration of 20 g/ml. Dissolve 154.3 mg of DTT in 1 ml of deionized water to make a 1 M stock solution. Store at 20C. Add 10 l of this 1M DTT stock to 10 ml of Transport Buffer (Recipe 6) just before use, for a final concentration of 1 mM. Chill on ice before use.

Recipe 8: Wash Buffer A

Dissolve 10 mg of BSA in 1 ml of deionized water to make a 10 mg/ml stock solution. Filter-sterilize and store at 4C. Add 10 l of this 10 mg/ml BSA stock to 10 ml of Transport Buffer (Recipe 6) just before use, for a final concentration of 10 g/ml. Chill on ice before use.

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Recipe 9: Hypotonic Extraction Buffer

Reagent
1 M Hepes-KOH, pH 7.8 1 M Potassium chloride 1 M Magnesium chloride Triton X-100 1 M DTT

Amount
5 ml 5 ml 0.75 ml 0.25 ml 10 l

Final concentration
10 mM 10 mM 1.5 mM 0.05% 1 mM

Prepare Hypotonic Extraction Buffer in 500 ml of deionized water without adding DTT; filter-sterilize and store at 4C. Just before use, add 10 l of 1M DTT stock to 10 ml of Hypotonic Extraction Buffer, for a final concentration of 1 mM. Chill on ice before use.

Recipe 10: Recovery Buffer

Reagent
1 M Hepes-KOH, pH 7.8 1 M Potassium acetate 1 M Sodium acetate 1 M Magnesium acetate 0.5 M EGTA BSA 1 M DTT* 0.3 M ATP* 0.5 M Creatine phosphate* 2 U/l Creatine phosphokinase*

Amount
0.2 ml 1.1 ml 50 l 20 l 20 l 200 mg 10 l 167 l 100 l 100 l

Final concentration
20 mM, pH 7.8 110 mM 5 mM 2 mM 1 mM 20 mg/ml 1 mM 5 mM 5 mM 0.02 U/l

Dissolve 1.8 g of ATP in 10 ml of PBS to make a 0.3 M stock solution. Store in 500 l aliquots at 20C. Dissolve 1.6 g of creatine phosphate in 10 ml of deionized water to make a 0.5 M stock solution. Store in 500 l aliquots at 20C. Dissolve 200 U of creatine phosphokinase in 100 l of deionized water to make a 2 U/l stock solution. Store in 10 l aliquots at 20C. Prepare Recovery Buffer in 10 ml of deionized water without adding DTT or the components of ATP regeneration system marked with an *. Filter sterilize and store at 4C. Add components of ATP regenerating system and DTT just before use. Chill on ice before use. Note: When necessary, rabbit reticulocyte lysate or purified chaperones and cochaperones are added to Recovery Buffer just before use.

Instructions

Biochemical Permeabilization and Extraction

The following procedure has been designed for use with derivatives of C127 mouse fibroblasts and mouse mammary adenocarcinoma cell lines stably expressing GFP-GR (3617.4 cell line) or GFP-PR-B (5953 cell line). Addition of tetracycline to the growth medium is not necessary if a tetracycline-regulated system is not used. Moreover, replacement of medium with growth medium containing charcoal-stripped FBS is not necessary if the experiments do not involve steroid receptors or other proteins responsive to hormones found in FBS. The critical parameters that influence the extent of permeabilization and nuclear protein extraction are the concentration of digitonin in the permeabilization buffer, the concentration of Triton X-100 in the extraction buffer, the duration of the incubations with digitonin- and Triton X-100-containing buffers, and the metabolic rate and density of the cells. These parameters will vary depending upon the cell type and must be optimized for successful permeabilization and extraction (see Notes and Remarks). Recommended initial concentration ranges are 5 to 50 g/ml for digitonin and 0.01% to 0.5% for Triton X-100. Recommended ranges of incubation times are 2 to 10 min with digitonin-containing buffer and 0.5 to 5 min with Triton X-100-containing buffer.

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1. Grow cells in Complete Culture Medium (Recipe 1). 2. Plate 40,000 cells in each chamber of a Labtek II two-chambered coverglass along with 1 ml of Tetracycline-Free Culture Medium (Recipe 2). Incubate for 24 hours at 37C, 5% CO2. Note: If cells will be processed for biochemistry protocols (such as a Western blot), rather than for FRAP analysis as described here, plate 70,000 cells in 2 ml of medium per 35 mm plate. If a different size of tissue culture dish is used, scale the cell number and culture medium volumes proportionally. 3. Remove all media and wash the cells three times with 1 ml of HBSS per wash. Note: When changing solutions or washing cells, completely remove old solution by tilting the Labtek II two-chambered coverglass and aspirating the pooled solution with a glass capillary pipette. 4. Add 1 ml of Tetracycline-Free Culture Medium with Charcoal-Stripped FBS (Recipe 3) to each Labtek II chamber. Place cells in incubator at 37C, 5% CO2 for at least 16 hours (or overnight). 5. Add 100 M Dexamethasone (Recipe 4) at 1:1000 for a final concentration of 100 nM, or 30 M R5020 (Recipe 5) at 1:1000 for a final concentration of 30 nM. Incubate for 1 hour. Note: Dexamethasone and R5020 are used to stimulate translocation of GFP-GR and GFP-PR-B to the nucleus, respectively. This step is not necessary if the experiments do not involve investigation of steroid receptors. 6. Place Labtek II chambered coverglass on ice to keep cells at 4C. Note: Keep them on ice for the remaining steps in this procedure (steps 7 through 14). 7. Gently rinse the cells once with 2 ml of ice-cold PBS per Labtek II chamber. 8. Gently rinse the cells once with 2 ml of ice-cold Transport Buffer (Recipe 6) per chamber. Completely aspirate the buffer with a glass capillary pipette. 9. Add 1 ml of ice-cold Permeabilization Buffer (Recipe 7) into each chamber and incubate the cells on ice for 5 min. 10. Remove Permeabilization Buffer and rinse cells once with 2 ml of ice-cold Transport Buffer (Recipe 6) per chamber. 11. Remove Transport Buffer and rinse cells once with 2 ml of ice-cold Wash Buffer A (Recipe 8) per chamber. Note: These permeabilized cells can be maintained for up to 30 min on ice in 2 ml of Wash Buffer A without any obvious loss of nuclear GFP-GR protein. However, for in situ mobility assays, we recommend that users immediately proceed to step 12, because the stability of other nuclear proteins with long-term storage on ice has not been tested. 12. For hypotonic extraction, remove Wash Buffer A and add 2 ml of ice-cold Hypotonic Extraction Buffer (Recipe 9) to each chamber and incubate the cells on ice for 2 min. 13. Rinse the cells twice with 2 ml of ice-cold Transport Buffer (Recipe 6) per chamber. 14. Rinse the cells once with 2 ml of ice-cold Wash Buffer A (Recipe 8) per chamber. Note: Extracted cells can be kept in Wash Buffer A on ice (up to 30 min if necessary), without any obvious loss of nuclear GFP-GR protein, while awaiting further treatments. However, we recommend that users immediately proceed to the in situ nuclear mobility factor assay, because the stability of other nuclear proteins to long-term storage on ice has not been tested.

In Situ Nuclear Mobility Assay

Recovery conditions with either added reticulocyte lysate or purified molecular chaperones were also optimized for derivatives of C127 mouse fibroblasts and mouse mammary adenocarcinoma cell lines stably expressing GFP-GR (3617.4) or GFP-PR-B (5953). Recovery conditions were adapted from parameters established in experiments analyzing the recovery of hormone-binding activity of steroid receptor heteromeric complexes (20, 21). Different buffer components, concentrations, and incubation conditions may be optimal for other nuclear mobility factors or cell types. Thus, these parameters will vary depending upon cell type and must be optimized to develop a successful in situ mobility assay (see Notes and Remarks). 1. Remove Wash Buffer A completely (added in step 14 above) by tilting the Labtek II two-chambered coverglass and aspirating the buffer with a glass capillary pipette. 2. Add 0.25 ml of Recovery Buffer (Recipe 10) to each Labtek II chamber. Place the cells in tissue culture incubator set at 37C, 5% CO2 for 10 min.

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Note: To test for ATP-dependent recovery of nuclear mobility in the absence of exogenously added factors, proceed immediately to FRAP Parameters. To test for the recovery of nuclear mobility with reticulocyte lysate or with purified chaperone and cochaperone proteins, follow step 3 or step 6, respectively. 3. Add 50 l of rabbit reticulocyte lysate to 200 l of Recovery Buffer (Recipe 10). 4. Gently add 250 l of the lysate-buffer mix to each chamber. 5. Incubate the cells for 10 min at 37C, 5% CO2. Note: Reticulocyte lysate can be preincubated for 5 min at 37C with compounds that inhibit the activity of specific components (for instance, geldanamycin inhibits Hsp90 function) before incubating the mixture with permeabilized and extracted cells. 6. Add purified chaperone or cochaperone proteins to Recovery Buffer (Recipe 10). Note: Purified chaperone or cochaperone proteins are used in the following amounts per 250 l of Recovery Buffer (Recipe 10) in the recovery assay: 20 g of Hsp70, 2 g of Ydj-1, 5 g of Hop, 20 g of Hsp90, 5 g of p23, 10 g of FKBP51, and 10 g of CHIP. 7. Gently add 250 l of chaperone or cochaperone and buffer mix to each chamber. 8. Incubate the cells for 10 min at 37C, 5% CO2. Note: Cells are maintained at 37C after the recovery period and during FRAP analysis. Cells are processed for FRAP analysis immediately after the 10-min recovery period.

FRAP Parameters
In FRAP experiments, the GFP-GR (or GFP-PR-B) fluorescence signal in a small region of the nucleus is irreversibly photobleached by a short and intense laser pulse. Immediately after the bleach, the recovery of the GFP-GR (or GFP-PR-B) fluorescence signal in the bleached area is monitored by fast sequential imaging. The signal in the bleached area is measured and the fluorescence recovery curve (normalized fluorescence intensity as a function of time) is generated by image analysis software. Images are acquired until the FRAP recovery is complete (until the quantitative FRAP recovery curve shows a plateau). Use the least number of images and the longest time intervals that are acceptable to minimize photobleaching due to imaging. Photobleaching due to monitoring should result in less than 10% decrease in the fluorescence signal intensity as compared to prebleach value. To compare individual data sets obtained under various experimental conditions or on different days, users must employ exactly the same FRAP settings. Thus, magnification and zoom settings can be modified according to the size of the cell and the region of interest, but the same settings must be used in different experiments to compare individual data sets without correcting for the size of the bleached area. Initially, test the efficiency and the depth of bleach on a fixed sample [samples can be fixed in 2% paraformaldehyde for 20 min at room temperature as in (22)]. In the fixed sample, the average fluorescence intensity in the bleached region should be less than 30% of that in the unbleached region. If necessary, increase the number of short bleach pulses. The size of a nuclear bleach region influences the quantitative FRAP analysis. Depending on the imaging area of interest, the size of a bleach region may vary. We have successfully used bleach regions in the size of a small circular spot or a 2- to 4-m strip across the width of a cell. The following FRAP procedure has been designed for use on the Zeiss LSM 510 confocal microscope. In-depth discussions of various photobleaching techniques are available (23, 24). 1. Place the Labtek II two-chambered coverglass containing the permeabilized and extracted cells onto the stage of a confocal microscope. Note: Maintain cell temperature at 37C with an air stream incubator during image acquisition. 2. Use an oil immersion lens with 63 or 100 magnification. 3. Adjust laser output from a 40-mW argon laser and choose a small pinhole diameter of 1 Airy unit. 4. Find a cell of interest, set the zoom, and capture image with maximum image acquisition speed without averaging. During imaging, set the GFP fluorescence signal intensity to below the saturation level by adjusting the detector gain. 5. Adjust the number of prebleach images to five.

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6. Determine the number of images to collect after photobleaching and the time interval between them. 7. Set the laser power to 100% for photobleaching and define the size of a nuclear region to bleach. 8. Check the focal plane and the position of the cell. Begin the FRAP experiment.

Quantitative FRAP Analysis

The following steps involve quantitation and analysis of time-lapse data collected on a Zeiss LSM 510 confocal microscope. LSM 510 image analysis software is used to perform quantitative measurements, and Microsoft Excel is used to calculate and generate FRAP recovery curves. During this process, background fluorescence intensity values are subtracted from all measured values, the decrease in the fluorescence intensity due to imaging is normalized, and the corrected prebleach intensity is normalized to one. As a result, a FRAP recovery curve-normalized fluorescence intensity as a function of time is generated. 1. Measure the average fluorescence intensity of the bleached region at each time point (IB). 2. Measure the average fluorescence intensity of the entire nucleus at each time point (IN). 3. Measure the average fluorescence intensity of the region outside of cell (background fluorescence intensity) at each time point (BB). 4. Measure the average fluorescence intensity of the bleached region during prebleach (IP). 5. Measure the average fluorescence intensity of the entire nucleus during prebleach (INP). 6. Measure the average fluorescence intensity of the region outside of cell (background fluorescence intensity) at each time point during prebleach (BP). 7. Calculate the relative fluorescence intensity (IRFI) for each time point using the following formula.

IRFI = (INP BP) (IB BB) / (IN BB) (IP BP)

8. Plot the relative fluorescence intensity for each time point as a function of time. Note: Data collected at different experiments are combined. Thus, the standard deviation for each time point is calculated and included in the final FRAP recovery curve.

Notes and Remarks

The current protocol has been extensively used for adherent cell lines. However, it can also be used for cell lines in suspension. Cell density and metabolic rate are important parameters contributing to the consistency of permeabilization and extraction of cells. It is imperative to process cells that are growing exponentially (in mid-log phase) and are not confluent. Because enhanced GFP and its variants diffuse relatively freely in the nucleus without substantial binding, they can be used as markers for effective permeabilization and extraction. For example, we have found that a GFP chimera containing a single minimal nuclear localization signal sequence (NLS) is highly sensitive to our permeabilization and extraction conditions and is not retained in the nuclei of the extracted cells. We recommend using cell lines stably expressing GFP-fusion proteins, because the same GFP fusion proteins expressed transiently are more susceptible to extraction. The efficacy of extraction should be assessed by qualitative FRAP analysis in every experiment before proceeding further. This can be done quickly by observing the mobility of the nuclear protein of interest in live cells (Fig. 1, A and C) and comparing it to the mobility observed in permeabilized and extracted cells. If permeabilization and extraction results in the depletion of nuclear mobility factors for the nuclear protein of interest, no fluorescence recovery should be observed (Fig. 1, B and D). Once this is established, purified proteins (for instance, molecular chaperones) or cell extracts can be included in the Recovery Buffer to identify the activity of nuclear mobility factors. Using this method, we have determined that molecular chaperones function as selective steroid receptor mobility factors within the nucleus (Fig. 2). A number of parameters can be examined to assess nuclear integrity after permeabilization and extraction. For example, permeabilized cells can be subjected to indirect immunofluorescence analysis to visualize nuclear lamina proteins (such as lamin B or lamin C) and heterochromatin-specific proteins (such as HP1). In this way, the overall integrity of the nuclear envelope and heterochro-

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Fig. 1. FRAP analysis of GFP-GR in nuclei of (A) live or (B) permeabilized and extracted mouse mammary adenocarcinoma cells. Cells were treated with 100 nM dexamethasone for 1 hour. Single optical z-sections from the mid-planes of cells were acquired before photobleaching and during recovery at 154 s. A bleached nucleoplasmic region is indicated by a rectangle and is shown as an enlarged pseudocolor image in the lower panels. Quantitative FRAP analysis of GFP-GR in (C) live or in (D) permeabilized and extracted cells. Although highly mobile in live cells (A and C), GFP-GR that remains within nuclei of cells after digitonin permeabilization and hypotonic extraction is immobile (B and D). All quantitative data values in FRAP recovery kinetics represent averages s.d. from at least 10 cells imaged in three independent experiments. Scale bars, 3 m.

Fig. 2. Recovery of GR and PR mobility within nuclei of permeabilized and extracted mouse mammary adenocarcinoma cells upon treatment with purified molecular chaperones and cochaperones. FRAP analysis of GFP-GR in permeabilized and extracted cells. Cells were treated with 100 nM dexamethasone for 1 hour and incubated with combinations of purified molecular chaperone or cochaperone proteins in the presence of ATP. (A) Single optical z-sections from the mid-planes of cells were acquired before photobleaching and during recovery at 154 s. The bleached nucleoplasmic region is indicated by a rectangle and shown as an enlarged pseudocolor image in the lower panel. (B) Quantitative FRAP analysis of GFP-GR and GFP-PR-B in permeabilized and extracted 3617.4 and 5953 cells, respectively. 5953 cells were treated with 30 nM R5020 for 1 hour. The chaperone mixture in the GFP-GR experiment (A and B) contained Hsp90, Hsp70, p23, p60/Hop, Ydj-1, FKBP51, and CHIP, whereas the chaperone mixture in the GFP-PR-B experiment (B) contained Hsp90, Hsp70, p23, p60/Hop, and Ydj-1. All experiments with chaperone mixtures were performed in the presence of ATP. All quantitative data values in FRAP recovery kinetics represent means SD from at least 10 cells imaged in three independent experiments. Scale bar, 3 m.

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matin can be assessed. As a more precise measure of the differential extraction of nuclear proteins by the Hypotonic Extraction Buffer, we have examined the relative retention of unliganded versus liganded GR after extraction. GFP-GR in hormone treated cells is quite resistant to the combined digitonin-permeabilization and Hypotonic Extraction Buffer extractions. In contrast, unliganded GFPGR, which is generated by a brief hormone withdrawal, is highly sensitive to the permeabilization and extraction regimen, in agreement with the low affinity of unliganded nuclear GR detected in hormone-withdrawn cells by conventional indirect immunofluorescence analysis (16). Finally, it is important to assess the functional status of cells after permeabilization and extraction, especially if the cell line of interest has never been used before or substantial modifications are introduced to the current protocol. By labeling the sites of active transcription in situ by 5bromo-UTP incorporation, one can assess the transcriptional status of the permeabilized and extracted cells. A protocol for the visualization of transcription sites can be found in (24).
Fig. 3. Chaperone and cochaperone proteins remaining in permeabilized and extracted mouse mammary adenocarcinoma cells stably expressing GFP-GR. Cells were treated with 100 nM dexamethasone for 1 hour. Total protein was extracted from intact (Total) or permeabilized and extracted cells (Perm). Expression of endogenous Hsp90, Hsp70, p60/Hop, Hsp40, p23, lamin B, and GR was detected by Western blot analysis using specific antibodies.

If this method is used to identify the activity of nuclear mobility factors, conditions must be established that dramatically reduce or even eliminate putative nuclear mobility factors. As shown in Figure 3, the combined digitonin permeabilization and Hypotonic Extraction Buffer extraction leads to very efficient loss of chaperone and cochaperone proteins. Thus, if novel nuclear mobility factors are identified in this assay, the endogenous levels of such mobility factors remaining must be assessed in permeabilized and extracted cells. It is unclear whether the digitonin permeabilization step is absolutely necessary for efficient extraction of nuclear mobility factors by the Hypotonic Extraction Buffer (16). Importantly, as mentioned in the Introduction, the Hypotonic Extraction Buffer extraction seems necessary to eliminate the requirement for active nuclear transport to restore nuclear mobility factors to the nucleus of permeabilized cells. In a recently published manuscript (25) from the Nickerson group that described an in vitro FRAP assay, digitonin permeabilization alone was used to show the ATP-dependent mobility of some RNA splicing factors. It seems likely that protein mobility factors were not removed from the nucleus in these in vitro assays, highlighting the need for additional extractions, as we have described, to identify macromolecular components of the nuclear protein mobility machinery.
References and Notes
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. G. L. Hager, Studying nuclear receptors with GFP fusions. Methods Enzymol. 302, 7384 (1999). R. Y. Tsien, The green fluorescent protein. Annu. Rev. Biochem. 67, 509544 (1998). R. D. Phair, T. Misteli, High mobility of proteins in the mammalian cell nucleus. Nature 404, 604609 (2000). N. J. McKenna, B. W. OMalley, Nuclear receptor coactivatorsan update. Endocrinology 143, 24612465 (2002). A. Aranda, A. Pascual, Nuclear hormone receptors and gene expression. Physiol. Rev. 81, 12691304 (2001). C. K. Glass, M. G. Rosenfeld, The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121141 (2000). L. P. Freedman, Increasing the complexity of coactivation in nuclear receptor signaling. Cell 97, 58 (1999). V. V. Ogryzko, R. L. Schiltz, V. Russanova, B. H. Howard, Y. Nakatani, The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953959 (1996). T. E. Spencer, G. Jenster, M. M. Burcin, C. D. Allis, J. Zhou, C. A. Mizzen, N. J. McKenna, S. A. Onate, S. Y. Tsai, M. J. Tsai, B. W. OMalley, Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389, 194198 (1997). H. Chen, R. J. Lin, W. Xie, D. Wilpitz, R. M. Evans, Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase. Cell 98, 675686 (1999). M. Becker, C. T. Baumann, S. John, D. Walker, M. Vigneron, J. G. McNally, G. L. Hager, Dynamic behavior of transcription factors on a natural promoter in living cells. EMBO Rep. 3, 11881194 (2002). G. L. Hager, C. Elbi, M. Becker, Protein dynamics in the nuclear compartment. Curr. Opin. Genet. Dev. 12, 137141 (2002). J. G. McNally, W. G. Muller, D. Walker, R. Wolford, G. L. Hager, The glucocorticoid receptor: Rapid exchange with regulatory sites in living cells. Science 287, 12621265 (2000). S. A. Adam, R. S. Marr, L. Gerace, Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807816 (1990). M. S. Moore, G. Blobel, The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell 69, 939950 (1992). J. Yang, J. Liu, D. B. DeFranco, Subnuclear trafficking of glucocorticoid receptors in vitro: Chromatin recycling and nuclear export. J. Cell Biol. 137, 523538 (1997). C. Elbi, D. A. Walker, G. Romero, W. P. Sullivan, D. O. Toft, G. L. Hager, D. B. DeFranco, Molecular chaperones function as steroid receptor nuclear mobility factors. Proc. Natl. Acad. Sci. U.S.A. 101, 28762881 (2004). D. Walker, H. Htun, G. L. Hager, Using inducible vectors to study intracellular trafficking of GFP-tagged steroid/nuclear receptors in living cells. Methods 19, 386393 (1999). P. K. Chakraborti, M. J. Garabedian, K. R. Yamamoto, S. S. Simons Jr., Creation of super glucocorticoid receptors by point mutations in the steroid binding domain. J. Biol. Chem. 266, 2207522078 (1991).

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20. H. Kosano, B. Stensgard, M. C. Charlesworth, N. McMahon, D. Toft, The assembly of progesterone receptor-hsp90 complexes using purified proteins. J. Biol. Chem. 273, 3297332979 (1998). 21. K. D. Dittmar, W. B. Pratt, Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70dependent step is sufficient for creating the steroid binding conformation. J. Biol. Chem. 272, 1304713054 (1997). 22. C. Elbi, T. Misteli, G. L. Hager, Recruitment of dioxin receptor to active transcription sites. Mol. Cell. Biol. 13, 20012015 (2002). 23. M. Dundr, T. Misteli, in Current Protocols in Cell Biology, Chapter 13: Organelle Motility, Unit 13.5, Measuring Dynamics of Nuclear Proteins by Photobleaching. John Davey, Mike Lord, Eds. (Wiley and Sons, New York, 2004). 24. J. Ellenberg, J. Lippincott-Schwartz, in Cells, A Laboratory Manual, Volume 2: Microscopy and Cell Structure, Chapter 79. Fluorescence Photobleaching Techniques. David L. Spector, Robert D. Goldman, Leslie A. Leinwand, Eds. (Cold Spring Harbor Laboratory Press, Plainview, NY, 1998). 25. S. Wagner, S. Chiosea, M. Ivshina, J. A. Nickerson, In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160. J. Cell Biol. 164, 843850 (2004). 26. We thank P. Badger for technical assistance on FRAP analysis and B. Stesgard for help with protein purification. Imaging was carried out in the Fluorescence Imaging Facility, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute.

Citation: C. Elbi, D. A. Walker, M. Lewis, G. Romero, W. P. Sullivan, D. O. Toft, G. L. Hager, D. B. DeFranco, A novel in situ assay for the identification and characterization of soluble nuclear mobility factors. Sci. STKE 2004, pl10 (2004).

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