Fluorescent labeling of proteins in living cells using the FKBP12 (F36V) tag

Original Article Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag Matt Robers,1 Patrick Pinson,2 Louis Leong,2 Robert H. Batchelor,2 Kyle R. Gee,2 Thomas Machleidt1* Invitrogen Discovery Sciences, 501 Charmany Dr, Madison, Wisconsin 53719 1 2 Molecular Probes, 29851 Willow Creek Road, Eugene, Oregon 97402 Received 9 April 2008; Accepted 19 August 2008 *Correspondence to: Thomas Machleidt; Invitrogen Discovery Sciences, 501 Charmany Dr, Madison, WI 53719 Email: thomas.machleidt@invitrogen.com Published online 3 October 2008 in Wiley InterScience (www.interscience. wiley.com) DOI: 10.1002/cyto.a.20649 © 2008 International Society for Advancement of Cytometry
Abstract Over the past decade live cell imaging has become a key technology to monitor and understand the dynamic behavior of proteins in the physiological context of living cells. The visualization of a protein of interest is most commonly achieved by genetically fusing it to green fluorescent protein (GFP) or one of it variants. Considerable effort has been made to develop alternative methods of protein labeling to overcome the intrinsic limitations of fluorescent proteins. In this report we show the optimization of a live cell labeling technology based on the use of a mutant form of FKBP12 (FKBP12(F36V)) in combination with a synthetic high affinity ligand (SLF’) that specifically binds to this mutant. It had been previously shown that the use of a fluorescein-conjugated form of SLF’ (50 -fluorescein-SLF’) allowed the labeling of proteins genetically fused to FKBPF36V in living cells. Here we describe the identification of novel fluorescent SLF’dye conjugates that allow specific labeling of FKBP12(F36V) fusion proteins in living cells. To further increase the versatility of this technology we developed a number of technical improvements. We implemented the use of pluronics during the labeling process to facilitate the uptake of the SLF’-dye conjugates and the use suppression dyes to reduce background signal. Furthermore, the time and dose dependency of labeling was investigated in order to determine optimal labeling conditions. Finally, the specificity of the FKBP12(F36V) labeling technology was extensively validated by morphological analysis using a diverse set of FKBP12(F36V) fusions proteins. In addition we show a number of different application examples, such as translocation assays, the generation of biosensors, and multiplex labeling in combination with different labeling technologies, such as FlAsH or GFP. In summary we show that the FKBP12(F36V)/SLF’ labeling technology has a broad range of applications and should prove useful for the study of protein function in living cells. ' 2008 International Society for Advancement of Cytometry
Key terms fluorescence microscopy; FKBP12; protein labeling; biosensors; protein translocation; live cell imaging; green fluorescent protein; FRET; FALI; high content analysis IMAGING of proteins in living cells has proven to be invaluable for the study of protein function and dynamics within the cellular environment. The most prominent way of labeling proteins in living cell is the genetic fusion of a protein of interest to green fluorescent protein (GFP) or one of its color variants (1), which allows the real time analysis by imaging. Despite its unquestionable value as research tool, GFP has a number of limitations that are dictated by the fact that its structure and fluorescent properties are interdependent. A major issue associated with the use of GFP is its relatively large size (27 kDA), which can potentially interfere with the proper localization or function of the protein of interest (2–4). Despite the generation of numerous GFP color variants there appear to be certain inherent limitations for the modification of the spectral and biochemical properties attainable for GFP. The development of red fluorescent versions of GFP has proven difficult and appears to be accompanied by substantial shortcomings including poor fluorescent performance and a Cytometry Part A
75A: 207224, 2009 ORIGINAL ARTICLE requirement for oligomerization (5). Other limitations include the broad excitation and emission spectra, sensitivity to photobleaching and long maturation times. It should be also noted that the permanent nature of fluorescent proteins (FPs) is a limiting factor for certain applications that require time-dependent labeling strategies such as pulse labeling experiments. To address some of the limitations of GFP, a number of alternative methods for the selective labeling of proteins with small molecules have been developed. The majority of these technologies take advantage of the specific interaction between a receptor protein and its small molecule ligand. For labeling purposes the ligand is typically modified by chemical conjugation to organic fluorophores (6). Analogous to GFP, the receptor protein can be genetically fused to the protein of interest and expressed in cells. Specific labeling is achieved by adding the fluorophore-conjugated ligand to the cells. This receptor– ligand approach has yielded a number of chemical protein labeling technologies, including the tetra-cysteine (TC) tag (7), O6-alkyl guanine DNA transferase (hAGT-tag or SNAPtag) (8), prokaryotic dehalogenase (Halo tag) (9) and dihydrofolate reductase (10,11), and several specific dye-binding peptide tags (12). However, with the exception of the FlAsH tag, the protein receptors used for chemical labeling are mostly comparable in size to GFP, and hence facing the same size-related problems as GFP. Another source of concern is the presence of endogenous equivalents of the protein receptor, such as for hAGT, which might lead to high levels of background signal and might require the use of specifically engineered cell lines (8). The small peptide-based FlAsH tag ,although ideal in size, requires the use of thiol-based reagents to suppress extremely high levels of unspecific labeling and because of its requirement for reducing environment it is mostly limited to intracellular applications (13,14). The use of the receptor–ligand principle for the manipulation of proteins in living cells was pioneered using FKBP12 and its natural small molecule ligand FK506 (15). The human protein FKBP12, an immunophilin with a size of 12 kDa, binds to FK06 and rapamycin with high affinity (16). Further studies led to the development of dimeric ligands, which were used to control intracellular signaling pathways that relied on regulation by inducible protein association (15,17). This chemically induced dimerization technology was further improved with the creation of a completely synthetic ligand (synthetic ligand for FKBP12, SLF) and the generation of a mutant FKBP12-ligand combination, where the mutant FKBP12 (F36V) recognizes a ‘‘bumped’’ version of the synthetic FKBP12 ligand (SLF’) with extremely high affinity (18,19). In an extension of this technology, Marks et al. recently developed a new chemical labeling technology utilizing the high affinity interaction between the FKBP12 mutant (V36F) and its specific synthetic ligand (SLF’) for the specific labeling of FKBP12(F36V) (20). In this elegant study Marks et al. were able to show that a cell-permeable fluorescein conjugated SLF’ derivative could be used to label FKBP12 (F36V) fusion proteins in living cells for a number of different appli208 cations, such as imaging, FACS analysis, and fluorescenceassisted laser protein inactivation (FALI). On the basis of this work we sought to further improve and expand the utility of this technology for new applications. We conducted an extensive evaluation of a panel of novel SLF’-dye conjugates and identified a number of suitable derivatives with improved fluorescent properties and increased cellular retention time. We also introduced the use of pluronics to improve solubility and cellular uptake of SLF’ conjugates, and the use of suppression dyes to improve signal to noise ratios in live cell labeling applications. In addition we extensively validated the use of FKBP12 (F36V) for labeling purposes in living cells using both morphological as well as functional analysis of more than a dozen different proteins fused to FKBP12 (F36V). Some of these experiments provided also direct evidence that the use of the FKBP12 (F36V) tag might be advantageous over GFP for some applications. Finally we demonstrated the utility of this technology for use in FRET-based biosensors and multiplex applications in combination with established live cell labeling methods, such as GFP or FlAsH labeling. MATERIALS AND METHODS Cell Culture HeLaS3 and CHO K1 cells were purchased from ATCC. The HEK293 GripTite cell line was supplied by Invitrogen. All cell lines were maintained in full growth medium, consisting of 90% high glucose DMEM (Invitrogen, Carlsbad), supplemented with 10% dialyzed FBS, 100 U/mL penicillin, 100 lg/ mL streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 25 mM HEPES pH 7.3. The cell lines were kept in a humidified incubator at 378C in a 5% CO2 atmosphere. For live imaging experiments the cells were temporarily maintained in OPTI-MEM I. Expression Plasmids N-terminal expression constructs were generated via gateway cloning using pcDNA6.2 nYFP-DEST, pcDNA6.2 nTC-DEST (Invitrogen) and an analogous pcDNA6-nFKBP F36V-DEST (custom-made) cloning vectors. Because of the presence of gateway att sites, the following amino acid linkers were sandwiched between flanking coding regions: pcDNA6.2 nYFP-DEST -GSSPSTSLYKKAGT, pcDNA6.2 nTC-DEST— GSSPSTSLYKKAGT, pcDNA6 nFKBP-DEST—SRGPFDQTSL YKKAGT. The following ORFs (ultimate ORF, Invitrogen) were cloned into these vectors using Gateway cloning technology: Map4/IOH5750, rac1/IOH27054, TubA1/IOH13040, RhoA/ IOH7574, PDLIM5/IOH5919 or ioh14002, and Beta-actin/ IOH3654. C-terminal expression constructs were generated via standard restriction site cloning techniques using a custom CMV-driven pcDNA 6.2 cYFP and pcDNA cFKBP F36V. Using standard molecular biology techniques, the following genes were PCR amplified from HeLa cell cDNA: GR (Glucocorticoid Receptor) matching ref seq NM 001018077.1, TIF2 Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE matching ref seq NM 006549.2, Caveolin1 matching ref seq NM 001753.3, and PKCalpha (Protein Kinase C) matching ref seq NM 002723.2. The PCR fragments were then subcloned using standard cloning techniques to generate in-frame C-terminal fusions of YFP or FKBP F36V. The linker region of these vectors consisted of the amino acid sequence SRAAA between coding regions of the fusion partners. The pcDNA3 CFPFKBP12 (F36V) expression construct was generated using standard restriction site cloning techniques and contained a 3x-Gly amino acid linker between CFP and FKBP coding domains. The C2FK and C0FK caspase sensor expression constructs were generated based upon previously made C2Y and C0Y caspase sensors (21). FKBP F36V coding DNA was substituted for the YFP coding DNA using standard molecular biology techniques. The amino acid sequence of the DEVD domain consisted of the following amino acids: RMHERPYACPVESC— DEVD—SRSDELTRHIRIHTGQKELLARL sandwiched between the CFP and FKBP coding regions. All plasmids were amplified and purified using standard molecular biology methods. Generation of Stable Cell Lines The stable CHO-K1 CFP and CHO-K1 CFP-FKBP12 (F36V) cell lines were generated by transfecting the expression constructs pCDNA3.1 CFP and pCDNA3.1 CFP-FKBP12 (F36V) in to CHO-K1 cells using Lipofectamine 2000 according to manufacturers’ recommendations. After antibiotic selection with G418 for 14 days we isolated clonal populations by limiting dilution. We selected clones which exhibited comparable expression of CFP and CFP-FKBP12 (F36V), which was determined by fluorescent intensity measurements. Synthesis of SLF’ Conjugates All reagents, unless otherwise noted, were obtained from Invitrogen or Sigma-AldrichTM. Compounds were characterized by ESI HPLC/MS. Fluorescein-SLF’ compounds. The aniline derivative of SLF’ (Figure 1, structure 1) was synthesized via methods of Keenan et al. (22) and the Clackson group (18). The acid chloride of the desired fluorophore (5-carboxyfluorescein diacetate (Invitrogen, P/N C1361) or 5-carboxy-20 -70 difluorofluorescein diacetate (23) was prepared with excess oxalyl chloride and a catalytic amount of DMF in dichloromethane at 08C. After 15 min the solution was evaporated under vacuum, and the oil was diluted in dichloromethane and added to a solution of one in dichloromethane and N,N-diisopropylethylamine (20 equiv.) at ambient temperature. The volatile components were removed under vacuum. The acetate protecting groups were cleaved by dissolving the oil in 1:1:1:1 dioxane/DMF/triethylamine/water and stirring for 6 h at ambient temperature. The solution was concentrated to dryness and purified on a silica gel column using chloroform/methanol as the mobile phase (Figure 1, structures 4 and 5). Cytometry Part A
75A: 207224, 2009 50 -TAMRA-gly-SLF’. The acid chloride of SLF’-Gly (2) (18), prepared with oxalyl chloride and cat. DMF, as above, was added to a solution of 50 -tetramethylrhodamine aniline (24) in dichloromethane in the presence of excess N,N,-diisopropylethylamine at ambient temperature. The solution was concentrated to dryness and purified on a silica gel column using chloroform/methanol as the mobile phase (Figure 1, structure 6). SLF’ethylenediamine linked conjugates. SLF’-Gly (Figure 1, structure 2) was reacted with N,N,N0 ,N0 -tetramethyl-O-(Nsuccinimidyl)uronium tetrafluoroborate (1.5 equiv.) and N,Ndiisopropylethylamine (2.0 equiv.) in anhydrous DMF to form the succinimidyl ester. This solution was added in a dropwise fashion to ethylenediamine (10 equiv.) in DMF and the solution was concentrated to dryness and purified on a silica gel column using chloroform/methanol as mobile phase giving SLF’-Gly-EDA (Figure 1, structures 3, 7–9). To a solution of the desired fluorophore activated ester (1.2 equiv: 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (Invitrogen P/N C1171 for structure 7); 4,4difluoro-1,3-dimethyl-5-1(4-methoxyphenyl)-3a,4a-diaza-sindacene-3-propionic acid, 4-sulfo-2,3,5,6-tetrafluorophenyl ester, sodium salt (B10002) for 8; 4,4-difluoro-5-(2-pyrrolyl)4-bora-3a,4a-diaza-s-indacene-3-propionic acid, succinimidyl ester (D2225) for 9) in DMF, was added 3 (1.0 equiv.) followed by N,N,-diisopropylethylamine (2.0 equiv.). After 2 h, all volatile components were removed under vacuum and the remaining crude was extracted with chloroform/water, dried over sodium sulfate, and purified on a silica gel column using chloroform/methanol as the mobile phase. General Protocol for FKBP12(F36V) Fusion Protein Labeling with SLF’-Dye Conjugates This protocol was developed as a result of our optimization experiments (see results) and used for most validation experiments. The protocol should provide a good starting point but might require further optimization depending on the choice of cell line, target protein, and SLF’ conjugate. Any deviations from this protocol are indicated and described in the results section. Labeling experiments were in general carried out in either 96-well plate or 8-well chambered coverslips. On the day prior to the experiment, cells were plated in growth medium at 30 or 95% confluence for microscopy or dye uptake experiments, respectively. The dye-SLF’ was premixed at the indicated concentration with growth medium containing 0.1% F127 pluronic acid. On the day of the experiment, the original medium was exchanged against the dye-SLF’/growth medium/pluronic solution and incubated for the indicated period of time (4 h unless otherwise indicated). The concentrations of dye-SLF’ varied depending on the specific linker/fluorophore combination used. For high-resolution imaging, the following concentrations were used: 4 lM 50 -fluorescein-SLF’, 4 lM 50 -Oregon Green-SLF’, 1 lM 50 (60 )-TAMRA-EDA-gly-SLF’, 200 nM 50 TAMRA-gly-SLF’, The dye-SLF’ labeling solution was removed by aspiration and replaced with phenol-red free Opti-MEM1 (Invitrogen) containing suppression dye. We used either 209 ORIGINAL ARTICLE Figure 1. Chemical structure of all SLF’ derivatives that exhibited specific labeling of FKBP12 (F36V) fusion proteins in living cells. GeneBlazer solution C (Invitrogen, Carlsbad) at a final dilution of one to six or 1 mM patent blue V as suppression dye. Following the addition of the medium/suppression dye solution the samples were analyzed by microscopy or fluorescence spectroscopy. Dose Response/Time Course Labeling Experiments Dose response and time course labeling experiments were conducted in black clear bottom 96-well plates (Corning). Cells were seeded at 50,000 cells/well in 100 lL full growth medium and incubated overnight to allow cell adherence. The cells were labeled by an addition of 100 lL 23 labeling mix including the indicated concentration of dye-SLF’ in the presence of 0.1% F127 pluronic (final concentration) in a fullgrowth medium. After incubation for the indicated period of time the labeling solution was removed and replaced by phenol-red free OPTI-MEM I. Images were acquired using a Zeiss Axiovert 25 inverted microscope equipped with a 103 Fluar 210 objective and Penguing Pixera CL600 CCD camera. Identical exposure settings were used for all images in comparative experiments. For quantitative analysis of dye uptake the samples the fluorescence intensity of each sample was determined using a monochromator-based Tecan Safire (2) plate reader. Excitation and emission parameters were set according to the fluorophore properties (see Table 1 for fluorophore emission and excitation peaks) using a 10-nm bandwidth setting. Cytotoxicity Assay Cell viabitlity was determined using the tetrazolium saltbased WST-1 cell proliferation reagent (Roche), according to the manufacturers instructions. Briefly, 10,000 cells were seeded per well in a 96-well plate in full growth medium and incubated for 24 h. The cells were treated as described for additional 24 h, followed by addition of 20 mL WST-1 reagent per well to determine mitochondrial activity. The conversion of WST-1 into formazan was determined after 2 h of incuba- Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE Table 1. SLF’dye conjugates PARENT DYE Marina blue Bimane 5-Fluorescein 5-Fluorescein Oregon green Oregon green Oregon green Rhodamine110 Rhodamine110 Rhodamine110 BODIPY1-FL 5’ TAMRA 5’ TAMRA BODIPY1-TMR BODIPY 576/589 5(6)-ROX Texas red BODIPY-TR DDAO LINKER EXCITATION/EMISSION LIVE CELL LABELING EDA-gly EDA-gly Zero EDA-gly zero Cadaverine-gly Gly EDA-gly EDA-PEG4-3OH- propionamide-gly MePiperidine-gly EDA-gly EDA-gly Gly EDA-gly EDA-gly EDA-gly EDA-gly Cadaverine-gly EDA-gly 362/459 380/460 494/518 494/518 495/521 495/521 495/521 502/527 504/528 507/529 502/510 546/576 544/572 544/570 576/588 567/601 591/612 588/616 646/659 1/2 2 111 1 1 1/2 1/2 1/2 2 2 2 111 11 (washout) 1/2 1/2 2 2 2 2 tion under cell culture conditions by measuring absorbance at 450 nm in a Tecan Ultra384 plate reader. Western Blotting Expression of CFP and CFP-FKBP-F36V was carried out following standard electrophoresis and western blotting protocols. Briefly, cells were seeded at 106 cells per well (in 5 mL of complete growth medium) into a 6-well plate and incubated overnight. On the second day, cells were collected by scraping, washed once with PBS, and lysed in 100 lL lysis buffer (1% NP-40 substitute150 mM NaCl, 20 mM Tris, 5 mM EDTA, 5 mM NaF, 13 SIGMA protease inhibitor mix). After 30-min incubation on ice the samples were centrifuged at 12,000 3 g to pellet cell debris. Next, supernatants were transferred to fresh polypropylene tubes and mixed with 25 lL of SDS-PAGE sample buffer (53 buffer consisting of 10% SDS, 50 mM Tris pH 6.8, 6 M Urea, 1 mg/mL bromophenol blue, 50 mM DTT). Samples were boiled at 1008C for 2 min and 10 lL of the sample was subjected to SDS-PAGE using 4–20% Tris-Glycine gradient gels (Invitrogen) and transferred to nitrocellulose by standard Western blotting methods. To detect GFP and FKBP, blots were probed with either anti-GFP or anti-FKBP mouse monoclonal antibody as primary antibody (1:2,000 final dilution, Zymed) followed by a goat anti-mouse Alkaline Phosphatase (AP) conjugated secondary antibody (Invitrogen). The blot was developed using a chromogenic AP substrate (WesternBreeze1 Kit, Invitrogen). Morphological and Functional Validation Assays For functional analysis of FKBP12 (F36V) fusion proteins Hela S3 cells were seeded in 6-well plates and transiently transfected with the indicated expression constructs using LipofecCytometry Part A
75A: 207224, 2009 tamine 2000 according to the manufacturer’s protocol. Briefly, 500,000 cells were seeded into six well plates in normal growth medium at a density of 60–80%. Before addition of the transfection mix, growth medium was exchanged for growth medium without antibiotic supplements. The transfection mix was prepared in OPTI-MEM I according to manufacturer’s recommendations and added to the cells followed by incubation for 24 h under standard cell culture conditions. The cells were then trypsinized and plated in chambered coverslips at 50% confluency and incubated for 16 h. The cells were then labeled with the indicated dye-SLF’ conjugate for 4 h, followed by removal of the labeling solution and addition of phenol-red free growth medium containing suppression dye (patent blue V or solution C, see standard labeling protocol). High magnification/resolution images were acquired on an Applied Precisision Deltavision RT System. This imaging platform is equipped with an environmental chamber to allow cell handling and manipulation at 378C for extended periods of time. The images were acquired with a CoolSnap HQ camera using a PlanApo 603 1.4 objective. All images were processed using Applied Precision’s image processing software softWorx. Image processing was limited to linear adjustments (gain/black level) and deconvolution using the iterative constrained algorithm described by Agard (25). Immunofluorescence For immunofluorescence analysis of beta-tubulin distribution HeLa cells were transiently transfected with pCDNA3.1-beta-tubulin-V5 and plated onto 12 mm #1 coverslips in 24-well plates at a density of 12,5000 cells/well. The cells were fixed for 30 min with 4% paraformaldehyde in PBS 211 ORIGINAL ARTICLE at room temperature and permeabilized with 0.1% Triton X100 for 5 min on ice. The sample was blocked with PBS 1 0.3% BSA for 30 min followed by incubations with anti-V5 mAb (1:200, Invitrogen) and goat anti mouse AlexaFluor488 secondary antibody (1:1,000, Invitrogen) for 60 min each at room temperature. The samples were then mounted in Aquamount (Polysciences) and analyzed by microscopy. ReAsH Staining For experiments involving ReAsH/TC imaging, cells were transiently transfected with the indicated expression construct and plated on the day prior to the experiment at 90% confluency in a chambered coverslip. On the day of the experiment, growth medium was aspirated and replaced with OPTIMEM I containing 2 lM ReAsH and incubated 1 h. After incubation, cells were washed 33 with OPTI-MEM I containing 0.25 mM ethane dithiol (Sigma) with a 5 min room temperature incubation per wash. Caspase Biosensor Assay HEK 293 Griptite cells were transfected in 6-well plates with pcDNA C2FK, C0FK, C2Y, and C0Y using Lipofectamine 2000 (Invitrogen). The caspase biosensors COY and C2Y were described previously (21). After overnight incubation, cells were trypsinized and transferred to poly-lysine coated 96-well clear bottom plates (Corning) at approximately 90% confluency and FKBP F36V fusions were treated with 4 lM 50 -fluorescein-SLF’, in the presence of 0.1% F127 pluronic, and allowed to incubate overnight. After overnight incubation, cells were treated with 100 lg/mL cycloheximide, and left treated/untreated with 40 ng/mL TRAIL. Following 8-h incubation at 378C, background suppression dye (GeneBlazer solution C, Invitrogen) was added as described previously and plates were read on a Tecan Saffire2 fluorescence plate reader. C2Y/C0Y controls were treated in the same manner, but were not incubated in the presence of FKBP reagents. RESULTS Development of Novel Fluorescent SLF’ Conjugates for Chemical Labeling of FKBP12(F36V) in Living Cells To provide greater versatility for the FKBP12(F36V) labeling technology we synthesized and tested a large panel of SLF’dye conjugates (see Table 1), using a variety of different organic fluorophore and linker combinations. All SLF’dye conjugates were analyzed in live cell labeling experiments (Figs. 2a–2h). For this purpose we generated two stable CHOK1 cell lines expressing either CFP or CFP-FKBP12 (F36V) fusion protein. We selected the combination of cyan fluorescent protein (CFP) and CFP-FKBP12 (F36V) as our experimental system since it would allow us to determine specific labeling in multiple ways. First, specific labeling by the SLF’dye conjugate can be analyzed by imaging and by fluorescence intensity measurements (Figs. 2a–2g). In addition specific binding to CFP-FKBP12 (F36V) was further validated by measuring Foerster resonance energy transfer (FRET) between CFP 212 (donor) and the SLF’ conjugated fluorophore (acceptor) (data not shown). Finally the use of CFP also allows the labeling intensity to be normalized against protein expression levels in comparative experiments (data not shown). The cells express equivalent levels of CFP and CFP-FKBP12 (F36V) as shown by western blot analysis using an anti GFP antibody which permits direct comparison. Furthermore, the expression level of CFP-FKBP12 (F36V) fusion protein is about 10-fold higher than endogenous FKBP12 as shown Figure 2h. All SLF’dye conjugates were evaluated in 7-point dose response experiments starting with a maximum initial concentration of 10 lM (see Material and Methods) followed by a twofold serial dilution. The SLF’dye conjugate solution was incubated for a period of 16 h to permit the detection of specific labeling for SLF’dye conjugates with slow cellular uptake rates. Figures 2a and 2b shows a typical dose response experiment using the original 50 -Fluorescein-SLF’ as label. Also shown are images taken with a CFP specific filter (Ex:460 nm, Em: 485 nm) to provide additional information about the relative expression levels of CFP and CFP-FKBP12 (F36V). In the course of testing all newly synthesized SLF’-conjugates, we were able to identify a number of new SLF’ conjugates that exhibited highly specific labeling of CFP-FKBP12 (F36V) fusion protein in living cells. Most notably we identified the red fluorescent 50 (60 )-TAMRA-EDA-gly-SLF’ (Figs. 2c and 2d) which showed in dose response experiments similar staining properties as 50 -FluoresceinSLF’ in respect to labeling intensity. However, labeling with 50 (60 )-TAMRA-EDA-gly-SLF’ resulted in an improved signal to noise ratio (Figs. 2c and 2d). Fluorescence intensity measurements showed that specific dye uptake is directly proportional to the concentration of 50 -Fluorescein-SLF’ (Fig. 2b) or 50 (60 )-TAMRA-EDA-gly-SLF’ (Fig. 2d) conjugate in the labeling solution up to a concentration of 5 lM. Both SLF’-conjugates show clearly detectable labeling by microscopy and in fluorescence intensity measurements at concentrations above 156 nM. At concentrations above 5–10 lM, background staining becomes more pronounced most likely due to the binding of SLF’ to endogenous FKBP12, which is not entirely unexpected since SLF’ has a Kd of 67 nM for wildtype FKBP12 (as opposed to 0.094 nM for FKBP12 (F36V)) (18). We identified a second red-fluorescent SLF’-dye conjugate, 50 -TAMRA-gly-SLF’, which exhibited somewhat unusual labeling properties in living cells. 50 -TAMRA-gly-SLF’ shows efficient labeling of CHO CFP-FKBP12 (F36V) cells at very low concentrations (Figs. 2e and 2f), but also showed considerable amount of unspecific labeling in CHO CFP cells, especially at concentrations above 250 nM, which resulted in an overall poor signal to noise ratio. However, removal of the staining solution followed by 16-h incubation in growth medium resulted in a dramatic loss of background signal with little loss of the CFP-FKBP12 (F36V) specific signal, therefore dramatically improving signal to noise ratio. In addition 50 TAMRA-gly-SLF’ is effectively retained for a considerably longer period of time than any other SLF’-dye conjugate tested (Fig. 2d). Specific labeling could be detected for as long as 96 h after removal of the labeling solution, whereas other SLF’dye Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE Figure 2. Evaluation of new SLF’-dye conjugates for live cell labeling of FKBP-F36V fusion proteins (a–g) CHO CFP or CHO CFP-FKBP12 (F36V) cells were plated in 96-well plates and labeled for 16 h with the indicated concentrations of, (a, b) 50 -Fluorescein-SLF’, (c, d) 50 (60 )TAMRA-EDA-gly-SLF’, (e, f) 50 ;-TAMRA-gly-SLF’ (g) Bodipy576-EDA-gly-SLF’, (g) BodipyTMR-EDA-gly-SLF’, and (g) 50 -Orgeon Green-SLF’ in the presence of 0.1% L127 pluronic in full growth medium. The labeling solution was then replaced with OPTIMEM containing 16.6% solution C (a,b) or 1 mM patent blue V (c–f) as suppression dye. Images were taken for all SLF’ conjugates immediately after addition of the suppression dye solution using identical exposure settings for each SLF’dye conjugate (a, c, e, g). For 50 -TAMRA-gly-SLF’ additional images were taken 24 h and 96 h after replacement of the labeling solution using the same exposure settings. The fluorescence intensity values for the labeling of CHO-CFP (squares) and CHO CFP-FKBP12 (F36V) (triangles) with 50 -Fluorescein-SLF’ (b), 50 (60 )-TAMRA-EDA-glySLF’ (d) and 50 ;-TAMRA-gly-SLF’ (f) are shown in the diagrams below the respective image panel. (h) Expression of CFP and CFP-FKBP12 (F36V) was determined by western blotting using an anti-GFP or anti-FKBP12 primary antibody as indicated. conjugates showed almost complete loss of signal within 18– 24 h after dye removal (data not shown). These rather unique properties should make 50 -TAMRA-gly-SLF’ useful for labeling experiments that require extended periods of observation. Cytometry Part A
75A: 207224, 2009 In addition to the TAMRA-SLF’ conjugates, we identified a number of additional SLF’dye conjugates that exhibit specific labeling of CFP-FKBP12 (F36V) in living cells (Fig. 2g), including 50 -Oregon green-SLF’, BODIPY-SP-TMR-EDA-gly213 ORIGINAL ARTICLE Figure 2. (Continued). SLF’, and BODIPY-SP-576-EDA-gly-SLF’. However, these SLF’conjugates showed inferior labeling properties (in regard to fluorescence intensity and signal-to-noise-ratio) in comparison to 50 Fluorescein-SLF’, 50 (60 )-TAMRA-EDA-gly-SLF’ and 50 -TAMRAgly-SLF’ and were therefore not further investigated. All other SLF’dye conjugates we tested (Table 1) showed no FKBP12(F36V)-specific labeling in this cell model as determined by imaging and fluorescence intensity measurements and were therefore not further investigated (data not shown). In most cases staining patterns suggested unspecific incorporation into membrane compartments, which is not surprising considering the relatively hydrophobic nature of many dyes. However, some SLF’dye conjugates showed a high degree of specificity in FRET analysis (data not shown), which might be useful for FRET-based applications such as biosensors. Optimization of FKBP12 (F36V)/SLF’ Labeling Technology The report published by Marks et al. required the incubation of 1 lM 50 -Fluorescein-SLF’ for a period of 16 h to achieve effective labeling. Initial experiments conducted in our labs confirmed these results (data not shown). To optimize 214 labeling conditions and streamline the experimental workflow we introduced two principal modifications to the staining protocol. First, the SLF’dye conjugate was premixed with 0.1% of the pluronic L127 in order to increase solubility and cellular uptake of the SLF’-dye conjugate. Pluronics are nonionic surfactant polyols that have been found to facilitate the solubilization of water-insoluble dyes in physiological media. The second modification of the labeling protocol was the use of a suppression dye in the assay medium after the labeling procedure. Typically labeling experiments require multiple wash steps to remove residual label to avoid unwanted background signal. The function of the suppression dye is the quenching of all fluorescent signals originating from extracellular sources (such as residual label). For our initial experiments with 50 -Fluorescein-SLF’ we used a proprietary dye mixture, which is also used as suppression dye in Invitrogens GeneBlazer assay (www.invitrogen.com). However this suppression dye shows considerable intrinsic fluorescence at wavelengths above 550 nm and was therefore not suitable as suppression dye in combination with red fluorescent SLF’dye conjugates. Instead we tested a number of dyes as potential suppression dyes and identified patent blue V as most suitable for this purpose. We Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag Figure 3. Optimization of SLF’ labeling protocol: (a) CHO-K1 CFP and CHO-K1 CFP-FKBP12 (F36V) were plated in a 96-well plate and labeled for 16 h with 2.5 mM 50 (60 )-TAMRA-EDA-gly-SLF’ in the presence or absence of 0.1% pluronic L127. The labeling solution was then replaced with OPTI-MEM I with or with out 1 mM patent blue V as suppression dye. Images were taken immediately after addition of OPTI-MEM I using identical exposure setting for all samples. (b) Fluorescence intensity was determined for the samples shown in (a) using a plate reader. The fluorescence intensity values for the different labeling protocols are shown for CHO-CFP (white bars) and CHO CFP-FKBP12 (F36V) (black bars). The signal to noise ratio (squares) was derived by dividing the intensity value of CHO-K1-CFP-FKBP12 (F36V) by the intensity value for CHO-K1 CFP. (c) CHO-K1 CFP and CHO-K1 CFP-FKBP12 (F36V) cells were plated in 96-well plate and labeled for the indicated period of time with 2.5 lM 50 (60 )-TAMRA-EDA-gly-SLF’ in the presence of 0.1% pluronic L127. The labeling solution was then replaced with OPTI-MEM I containing 1 mM patent blueV as suppression dye. Images were taken immediately after addition of OPTI-MEM I using identical exposure setting for all samples. (d) The fluorescence intensity values for 50 (60 )-TAMRA-EDA-gly-SLF’ labeling of CHO CFP (square) and CHO CFP-FKBP12 (F36V) (triangle) are plotted against incubation time. (e) Cell viability/proliferation was determined by measuring overall metabolic activity of the sample. CHO-CFP cells (hatched bars) or CHO CFP-FKBP12 (F36V) (solid bars) cells were left untreated or treated for 18 h with 10 lM of the indicated SLF’dye conjugate in the presence of 0.1% pluronic L127. The results are normalized against the untreated control sample (equals 100% viability). ORIGINAL ARTICLE Figure 4. Labeling of FKBP12 (F36V)-beta actin and FKBP12 (F36V)-a/b-tubulin expressed in HeLa cells: (a) HeLa cells were transiently transfected with either GFP-beta-tubulin, FKBP12 (F36V)-beta tubulin, beta-tubulin-V5, YFP-beta-actin, or FKBP12 (F36V)-beta actin. The cells were plated on chambered coverslips and labeled with the indicated SLF’dye conjugate in the presence of 0.1% L127 for 4 h at 378C. The labeling solution was then replaced with OPTI-MEM I containing 1 mM patent blueV as suppression dye. Images were taken immediately after the addition of OPTI-MEM I. The cells were maintained at 378C throughout the image acquisition. (b) HeLa cells were transiently transfected with GFP-alpha-tubulin, alpha-tubulin-GFP, FKBP12 (F36V)-alpha tubulin, or alpha-tubulin-FKBP12 (F36V). The cells were plated on chambered coverslips and labeled with the indicated SLF’dye conjugate in the presence of 0.1% L127 for 4 h at 378C. The labeling solution was then replaced with OPTI-MEM I containing 1 mM patent blueV as suppression dye. Images were taken immediately after addition of OPTI-MEM I. The cells were maintained at 378C throughout the image acquisition. Size bar equals 10 lm. continued to use patent blue V as standard suppression dye at a concentration of 1 mM. The experiment in Figure 3 shows a representative example for the application of pluronic and suppression dye and its 216 effect on dye uptake as well as signal to noise ratio. For this experiment we plated CHO-K1 CFP or CFP-FKBP12 (F36V) in a 96 well plate in growth medium followed by 24 h incubation. After aspiration of the growth medium the cells were Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE Figure 4. (Continued). incubated with the labeling mix (full medium 1 2.5 lM 50 (60 )-TAMRA-EDA-gly-SLF’) in absence or presence of 0.1% L127 for a period of 16 h. The labeling mix was removed by aspiration and replaced with phenol-red free OPTI-MEM I without or with 1 mM patent blue V. The labeling of CFP or CFP-FKBP12(F36V) was analyzed by microscopy and fluorescence intensity measurement. Surprisingly, no increase of specific 50 (60 )-TAMRA-EDA-gly-SLF’ uptake in the presence of pluronic was noticed in this model (Figs. 3a and 3b). However, a substantial reduction of unspecific staining was observed in the presence of L127, resulting in an improved signal to noise ratio. It should be also noted that the effect of pluronic on specific dye uptake into living cells is greatly dependent on the SLF’dye conjugate and cell line used in the experiment (data not shown). In general, the use of pluronic lead to substantial improvements of signal to noise for every cell model we tested without any notable signs of toxicity or morphological change. The application of patent blue V resulted in a dramatic reduction of unspecific background signal compared to untreated samples (Figs. 3a and 3b). We tested numerous concentrations of patent blue V and determined that a final concentration of 1 mM yielded excellent results for all applications. Although not strictly required for successful labeling, the use of suppression dye nevertheless provides several advantages: First, it circumvents the need for extensive washing steps. Furthermore excessive washing can cause loss of weakly adherent cells and is challenging to implement for nonadherent cells. Finally, it will also suppress most of background signal originating from dye that adheres nonspecifically to plastic surfaces, which can be difficult to remove by Cytometry Part A
75A: 207224, 2009 washing. In this experiment the combination of pluronic and patent blue V lead to an almost sixfold improved signal to noise ratio, without any requirement for multiple wash steps after the labeling procedure. Similar improvements of signal to noise ratio were achieved in experiments using different SLF’dye conjugates and cellular models. On the basis of these results we incorporated the use of pluronic L127 and patent blue V in our standard labeling procedure. We conducted time course labeling experiments in order to determine the optimal labeling time required for dye-SLF’ conjugates. CHO-K1 CFP and CHO-K1 CFP-FKBP12 (F36V) cells were plated in 96-well plates and incubated them with 2.5 lM of 50 (60 )-TAMRA-EDA-gly-SLF’ in growth medium in the presence of 0.1% L127 for different periods of time. Following the replacement of the labeling solution with OPTI-MEM I and 1 mM patent blue, each sample was analyzed by imaging and by fluorescence intensity measurements and observed specific labeling after as little as 15 min of incubation with 50 (60 )TAMRA-EDA-gly-SLF’ (Figs. 3c and 3d). Labeling with 50 (60 )TAMRA-EDA-gly-SLF’ resulted in a time-dependent linear increase in fluorescence intensity, which approached saturation after 4 h under the conditions used in this experiment (Fig. 3d). Time course experiments conducted for 50 -Fluorescein-SLF’ and 50 -TAMRA-gly-SLF’ revealed similar labeling kinetics (data not shown). In addition we evaluated the potentially cytotxic effect of 50 -Fluorescein-SLF’, 50 (60 )-TAMRA-EDA-gly-SLF’ and 50 TAMRA-gly-SLF’, which could be a concern especially after prolonged periods of incubation. A standard metabolic cell proliferation assay based on the conversion of tetrazolium salts to a colored formazan product by mitochondrial dehydrogenases was used to determine dye-mediated changes in cell viability or proliferation. As shown in Figure 3e, CHO-CFP or CHO-CFP-FKBP12 (F36V) treated with up to 10 lM SLF’-dye conjugate for 18 h exhibited no significant change in metabolic activity for any SLF’dye conjugate, compared to untreated cells. This result was further supported by the absence of any visible morphological or functional changes during or following the staining procedure. Following these experiments a standardized labeling protocol (outlined in the Material and Methods section) was adopted, which yielded excellent results for most applications. However, we found that even under identical labeling conditions, fluorescence intensity as well as signal to noise ratios can vary substantially depending on the cell line and type of SLF’-dye conjugate used (data not shown). Using our ‘‘standard’’ labeling protocol (4 h labeling, 0.1% L127, 1–4 lM dye-SLF’ conjugate, replacement of labeling solution with phenol-red free OPTI-MEM I containing suppression dye) signal to noise ratios between 10:1 and 50:1 were typically achieved, as determined by fluorescence intensity measurements in a plate reader. However, it should be mentioned that single cell analysis by flow cytometry or image analysis, produces typically better signal to noise ratios compared to fluorescence intensity determinations in a microplate format due to the complete absence of cell-independent background. 217 ORIGINAL ARTICLE Figure 5. Labeling of transiently expressed FKBP12 (F36V)-fusion proteins in HeLa cells: HeLa cels were transiently transfected with the indicated FKBP12 (F36V) fusion protein. The cells were plated on chambered coverslips and labeled with 1 mM 50 (60 )-TAMRA-EDA-gly-SLF’ in the presence of 0.1% L127 for 4 h at 378C. The labeling solution was then replaced with OPTI-MEM I containing 1 mM patent blueV as suppression dye. Images were taken immediately after the addition of OPTI-MEM I. The cells were maintained at 378C throughout the image acquisition. Size bar equals 10 lm. FKBP12 (F36V) Fusion Proteins Show the Anticipated Subcellular Distribution Patterns and Translocation Behavior in Living Cells The genetic fusion of protein or peptide tags bears the inherent risk that the tag might interfere with the proper function and localization of the target protein. One of the main 218 concerns for the use of GFP is its relatively large size which might result in unwanted interference with protein function. FKBP12 (F36V) is considerably smaller than GFP (11 kDa vs. 27 kDa) and might therefore be more suitable as a molecular tag for target proteins that form oligo/polymers (e.g., cytoskeleton proteins such as actin and tubulin) or participate in large Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE multi protein complexes such as the signalosome. We investigated the potential effect of FKBP12 (F36V) on protein localization by generating N-terminal fusions of FKBP12 (F36V) with b-tubulin and b-actin. These cytoskeleton proteins exhibit a distinct and well-defined subcellular distribution patterns, which requires their proper assembly into polymers. We also compared the FKBP12 (F36V) fusion proteins against their respective GFP and V5 epitope tagged versions. Hela cells were transiently transfected with expression constructs for FKBP12 (F36V) beta tubulin and beta actin (Fig. 4a). After 24 h the cells were trypsinized and plated in chambered coverslips, followed by 16 h of incubation under cell culture conditions. The samples were stained with different SLF’dye conjugates as indicated in Figure 4a using our standardized labeling protocol. Both FKBP12 (F36V) b-tubulin and FKBP12 (F36V) b-actin exhibited the anticipated distribution patterns regardless of the SLF’dye conjugates used for labeling (Fig. 4a). FKBP12 (F36V) b-tubulin/b-actin fusion proteins also matched the subcellular distribution pattern found for GFP-beta-tubulin and GFP-beta-actin as well as the immuno-staining pattern of the V5-peptide tagged betatubulin. We also discovered an example that provides some direct evidence for the assumption that GFP has the potential to interfere with the proper localization and function of his fusion partner. We noticed that both, N or C-terminal fusion of YFP to a-tubulin would lead to a diffuse cytosolic distribution pattern indicating that the YFP a-tubulin fusion protein is not effectively integrated into microtubules (Fig. 4b). In contrast both FKBP12 (F36V) a-tubulin fusion proteins (Nand C-terminal) exhibited the expected phenotype associated with microtubules suggesting that FKBP12 (F36V) doesn’t impede the polymerization of a-tubulin into microtubules (Fig. 4b). This example underscores the value of alternative labeling technologies that complement FPs for protein labeling applications in living cells. To further validate the use of FKBP12 (F36V) for protein labeling applications we analyzed the subcellular distribution of numerous FKBP12 (F36V) fusions with different proteins that represent a wide range of functions, sizes, and subcellular localizations. The use of multiple examples in this context is important considering the variability in expression levels, subcellular localization, and protein–protein interactions required for proper target protein localization. The proteins we investigated included cytoskeletal proteins (vimentin (26), MAP4 (27)), membrane proteins (caveolin-1 (28)) signal transduction proteins (rac, rho (29)), ion channels (hERG), cofactors (tif2 (30)), and focal adhesion proteins (PDLIM5, (31)) and organelle specific proteins (golgin97 (32))(Fig. 4a). All FKBP12 (F36V) fusion proteins were transiently expressed in HeLa cells and labeled with 1 lM 50 (60 )-TAMRA-EDA-glySLF’ according to our standard labeling protocol. We found that the subcellular distribution of all FKBP12 (F36V) fusion proteins correspond well to immunostaining results reported in the literature (Fig. 5). In addition, the FKBP12 (F36V) fusion proteins showed similar distribution patterns compared to their respective GFP versions (data not shown). Cytometry Part A
75A: 207224, 2009 Finally we tested the functional integrity of FKBP12 (F36V) fusion proteins in protein translocation models. Protein translocation often depends on a ligand or modificationinduced conformational change which leads to selective redistribution of the target protein between different cellular compartments. The translocation of key proteins is frequently used as surrogate readouts to measure activation of signaling pathways. We selected for our experiments two well-defined translocation models, the PMA-induced membrane translocation of PKC alpha (33) and the dexamethasone-induced nuclear translocation of the glucocorticoid receptor (GR) (34). We generated expression constructs of FKBP12 (F36V) fusions for both proteins, which were transiently expressed in HeLa cells. The cells were then plated on chambered coverslips and labeled with 4 lM 50 -Fluorescein-SLF’ (PKC-alpha) or 1 lM 50 (60 )-TAMRA-EDA-gly-SLF’ for 4 h. Stimulation of FKBP12 (F36V)-PKC-alpha cells with 1 lM PMA resulted in the expected redistribution of PKC from the cytosol to the plasmamembrane within 8 min. Similarly, treatment with 1 mM dexamethasone induced the nuclear translocation of FKBP12(F36V)-GR fusion protein within 10 min (Fig. 6). The images represent the start and endpoint of each experiment. These data show that FKBP12 (F36V) can be used for monitoring protein dynamics in living cells. Use of FKBP12-(F36V) in a Caspase Biosensor An increasingly important area for live cell labeling is the use of biosensors to monitor signal transduction events, such as calcium release or enzyme activation in real time in living cells. A number of examples have been recently described for the generation of FRET-based biosensors, which utilize a pair of FPs, typically CFP yellow fluorescent protein and (YFP), connected by a sensor domain (35–37). A stimulus-induced change of the sensor domain causes a change in distance between the FPs which results in changes in the amount of FRET occurring between donor and acceptor fluorophores, which can be detected by changes in acceptor and donor emission intensity. We were interested if FKBP12 (F36V) could be potentially used as FRET partner in combination with a FP. To determine the use of FKBP12 (F36V) in a biosensor we selected a well-defined caspase biosensor (21) for comparative analysis. The caspase censor (C2Y) consists of CFP and YFP connected by a caspase3 (DEVD) cleavage site. In untreated cells CFP and YFP are kept in close proximity resulting in FRET occurring between CFP and YFP (Fig. 7, upper right panel). Treatment of C2Y transfected HEK293 cells with proapoptotic stimuli such as TRAIL leads to the activation of caspases and proteolysis of the DEVD motif. The separation of CFP and YFP can be determined by the loss of FRET which is indicated by increased donor emission and decreased acceptor emission (Fig. 7, upper right panel). In contrast cells transfected with a control construct without the DEVD linker exhibited no change in FRET following TRAIL treatment (Fig. 7, upper left panel). To test the utility of FKBP12 (F36V) for use in FRETbased biosensors we exchanged YFP against FKBP12 (F36V) in both constructs (renamed C2FK and COFK). 219 ORIGINAL ARTICLE Figure 6. FKBP-F36V-fusion protein can be used for translocation assays:Hela cells were transiently transfected with pCDNA3.1-PKC-aFKBP12 (F36V) or pCDNA3.1-GR-FKBP12 (F36V) and plated on chambered coverslips. The cells were labeled with either 4 lM 50 -Fluorescein-SLF’ or 1 lM 50 (60 )-TAMRA-EDA-gly-SLF’. The labeling solution was then replaced with OPTI-MEM I containing 1 mM patent blueV as suppression dye. Images were taken before and 10 min after addition of either 1 lM PMA (PKC-a-FKBP12 (F36V)) or 1 lM Dexamethasone (GR-FKBP12 (F36V)). Size bar equals 10 lm. We transiently expressed either C2FK or C0FK in HEK293 cells. The cells were then left untreated or treated with 40 ng/mL TRAIL for 8 h in the presence of 50 -Fluorescein-SLF’. We were able to detect a modest but reproducible TRAIL-inducible FRET change in C2FK-transfected cells (Fig. 7, lower right panel). In contrast, COFK-transfected cells showed no detectable change in FRET independent of treatment (Fig. 7, lower left panel). The observed change in FRET is considerably smaller for C2FK compared to the original C2Y. One reason might be the use of 50 -Fluorescein-SLF’as acceptor, which shows considerable overlap for its emission spectrum with CFP. The change in emission signal for fluorescein might therefore be masked by ‘bleed-through’ signal from CFP. Furthermore, the spectral overlap and conformational dipole orientation of the C2Y sensor has been previously optimized using genetic permutations (21), but the C2FK was not optimized for use in this study. Despite the modest dynamic range of the C2FK FRET sensor, the example demonstrates the potential utility of FKBP12-F36V for the design of biosensors for the analysis of signaling in living cells. The availability of red fluorescent dye-SLF’ conjugates might be used to create highly efficient FRET pairs for biosensors. In addition, the smaller size of FKBP12 (F36V) might be also pro220 vide better accessibility for ligands or modifying enzymes to a small biosensor effector domains (such as the DEVD caspase site) if sandwiched between the acceptor and donor domains. Use of FKBP12(F36V) in Combination with Other Labeling Technologies for Multiplexed Analysis of Protein Dynamics The development of novel labeling technologies such as FKBP12 (F36V) also enable multiplexed applications by pairing it with existing labeling technologies. The combination of multiple technologies allows the observation and measurements of a greater number of individual parameters simultaneously, which might be valuable for certain applications, such as high content analysis. Since FPs have been established as the predominant way for the visualization of proteins in living cells we wanted to demonstrate that GFP and FKBP12 (F36V) fusions could be used in combination for live cell imaging applications. We selected MAP4 (a microtubule associated protein) and vimentin as an example for combined imaging of GFP and FKBP12 (F36V) fusion proteins. MAP4 and vimentin are representative markers for microtubules and intermediate filaments, respec- Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE Figure 7. Use of FKBP12 (F36V) in a caspase biosensor: In Griptite 293 cells transiently transfected with Caspase III biosensors, CFP-DEVDYFP is inducibly cleaved by treatment with TRAIL (right panel), resulting in a change in FRET. The corresponding control lacking the DEVD recognition site is not cleaved by TRAIL treatment. (b) In the same format as described in (a), CFP-DEVD-FKBP F36V:Fluorescein SLF’ is inducibly cleaved by TRAIL treatment, resulting in a change in FRET. The corresponding control lacking the DEVD recognition site is not cleaved by TRAIL treatment. tively. To illustrate in a better way the use of these technologies for the analysis of protein dynamics we also included imaging of drug-induced phenotypical changes for both proteins. HeLa cells were transiently transfected with expression constructs for FKBP12 (F36V)-vimentin and GFP-MAP4 and plated on chambered coverslips. The cells were then treated for 6 h with 10 lM of each, paclitaxel, nocodazole, or vinblastine. All three compounds are well-characterized antineoplastic drugs which interfere with microtubule polymerization (38). Following drug treatment the cells were labeled with 1 lM 50 (60 )-TAMRA-SLF’ and counterstained with 1 lg/mL of the cell permeable DNA stain Hoechst 33342 for a period of 2 Cytometry Part A
75A: 207224, 2009 h. The samples were then analyzed by microscopy. In untreated cells GFP-MAP4 and FKBP12 (F36V)-vimentin localize mostly to two distinct filamentous networks (Fig. 8, upper left image) as described in the literature. Treatment with all three antimicrotubule drugs induced dramatic changes in the subcellular distribution of GFP-MAP4 and FKBP12 (F36V)-vimentin (Fig. 8). Both nocodazole and vinblastine inhibit the incorporation of tubulin into microtubules and eventually cause microtubule depolymerization as shown by the partial or complete dissolution of GFP-MAP4 associated microtubule structures. Vinblastine is also known to induce the formation of so-called tubulin paracrystals. These 221 ORIGINAL ARTICLE with 50 -fluorescein-SLF’ and the biarsenical dye ReAsH. The labeling protocols for tetra-cysteine/ReAsH and FKBP/SLF FL proved to be compatible despite the relatively short labeling time and wash requirements for ReAsH. We observed distinct localization patterns for hTC-ReAsH-beta actin (red), CFPbeta-tubulin (green), and FKBP12(F36V)-rac (magenta) (Fig. 9). The subcellular distribution for all three fusion proteins are in accordance with results reported in literature. Together, these examples illustrate how different protein labeling technologies can be combined to simultaneously monitor the dynamic behavior of multiple proteins in living cells. This combinatorial approach offers the opportunity to match the most suitable labeling technology with the protein of interest, which might be determined by a number of factors, such as tag size limitations, labeling requirements, and experimental needs. DISCUSSION Figure 8. Use of FKBP12 (F36V) in combination with other labeling technologies to monitor drug-induced morphological changes: (a) HeLa cells were transiently transfected with the expression constructs for GFP-MAP4 and FKBP12 (F36V)-vimentin. The cells were plated on chambered coverslips and left untreated or treated for 6 h with 10 lM of vinblastine, nocodazole, or paclitaxel as indicated. The cells were than labeled with 1 lM 50 (60 )-TAMRA-EDA-gly-SLF’ and 1 lg/mL Hoechst 33342 for 2 h at 378C. The labeling solution was then replaced with OPTIMEM containing 1 mM patent blueV as suppression dye. Images were taken immediately after addition of OPTI-MEM I. The cells were maintained at 378C throughout the image acquisition. Size bar equals 10 lm. structures are clearly visible in the form of concentrated GFPMAP4 aggregates. Similarly nocodazole and vinblastin treatment causes the collapse of vimentin fibers, which are indicated by the dissolution of intermediate fibers (nocodazole) or by the appearance of solid vimentin bundles (vinblastine). Contrary to nocodazole and vinblastine, paclitaxel inhibits the depolymerization of microtubules that leads to the appearance of massive and dysfunctional GFP-MAP4 (microtubule) bundles. Paclitaxel treatment also causes the collapse of vimentin filaments into an unstructured perinuclear complex. These results are in agreement with previous studies that used conventional immunodetection methods to show the effects of antimicrotubule drug on microtubules and intermediate filaments (39–41). In an extension of this dual-labeling approach we also successfully tested the simultaneous use of three different protein labeling technologies, FKBP12 (F36), FPs, and TCFlAsH, for the simultaneous imaging of rac1, b-tubulin, and b-actin in Hela cells (Fig. 9). We transiently transfected HeLa cells with CFP-b-tubulin, TC-beta-actin, and FKBP12 (F36V)rac1, plated them on chambered coverslips and stained them 222 Over the past decade imaging technologies, especially fluorescence microscopy, have become invaluable research tools to elucidate the spatial and temporal complexity of protein dynamics in physiological context. Rapid improvements in instrumentation and detection technologies have driven the development of numerous tools Figure 9. Use of FKBP12 (F36V) in combination with GFP and TCReAsH labeling technologies: HeLa cells were transiently transfected with CFP-beta-tubulin, FKBP12 (F36V)-rac1 and TC-actin. The cells were plated on chambered coverslips and labeled for 60 min with 4 lM 50 -fluorescein and 2 lM ReAsH. The labeling solution was then replaced with OPTI-MEM I. Images were taken immediately after addition of OPTI-MEM I. The cells were maintained at 378C throughout the image acquisition. Individual channels are shown by false coloring. Size bar equals 10 lm. Fluorescent Labeling of Proteins in Living Cells Using the FKBP12(F36V) Tag ORIGINAL ARTICLE for the specific labeling of proteins in cells (42). However, as discussed previously, each labeling technique comes with advantages and disadvantages and has to be evaluated carefully for a particular application. It is therefore imperative to develop novel and improved protein-labeling methods to complement existing technologies. In addition the increasing use of imaging in large scale imaging applications such as genome wide RNAi screens (43), the generation of protein localization maps (44), and drug screening (45) also increases demand for novel and robust approaches to protein labeling in living cells. In an effort to address these needs, we report the development of novel labels and applications of the FKBP12 (F36V)/ SLF’ protein-labeling method. We evaluated a number of novel SLF’ fluorophore conjugates and have successfully demonstrated the versatility and modularity of the SLF’ scaffold. Specifically, the development of novel red fluorescent SLF’dye conjugates for the fast and efficient labeling of FKBP12 (F36V) fusion proteins in living cells expands the overall versatility of this labeling technology, and makes it an ideal complement for existing labeling technologies. The availability of different live cell compatible protein labeling technologies is particularly valuable for the analysis of the dynamic behavior of multiple proteins. In this report we demonstrated the successful use of FKBP12 (F36V) in combination with two different labeling methods, FPs and FlAsH tag, for the simultaneous labeling and functional analysis of multiple cytoskeleton proteins in living cells as an example. The use of multiple protein labeling methods in combination with spectrally resolved imaging might offer an even greater potential for simultaneous analysis of an unprecedented number of proteins in living cells (46). In addition, the identification of the stable labeling properties of 50 -TAMRA-gly-SLF’, could open completely new applications for the use of the FKBP12 (F36V) tag, such as pulse chase labeling (47) or the analysis of protein trafficking over extended periods of time. The availability of different labeling methods (with multiple fluorophores to choose from) also allows matching labeling methods with the needs and constraints dictated by experimental design and target protein. The importance of being able to choose from multiple labeling strategies is underscored by our finding that the fusion of GFP to certain isoforms of btubulin results in mis-localization and likely loss of function, which corroborates earlier publications that reported the occurrence of functional interference in fusion proteins caused by GFP (2–4). In contrast, the same beta-tubulin isoform was found to exhibit the anticipated morphology when fused to FKBP12 (F36V). The differences in localization might be explained by the fact that FKBP12 (F36V) is substantially smaller than GFP (12 kDa for FKBPF12 (F36V) vs. 28 dDa for GFP) and most other genetically encoded tags used for chemical protein labeling (8,9,11). FKBP12 (F36V) might be therefore less prone to perturb protein function due to steric interference. It might be interesting to carry out a systematic analysis of multiple protein labeling methods for a limited set of proteins in order to directly compare the influence of different protein-tags on localization as well as signal-induced translocation. Cytometry Part A
75A: 207224, 2009 Another field of applications where FKBP12 (F36V) might provide a critical advantage due its small size and the flexible choice of fluorophores, are proximity dependent assays such as FRET or FALI. FRET describes the radiation less transfer of energy between a donor and acceptor fluorophore, which occurs only at distances less than 10 nm. FRET efficiency varies to the sixth power of the distance between donor and acceptor, which makes it an ideal technology to study intramolecular rearrangements of proteins as well as intermolecular protein– protein interactions. The use of FP pairs has become popular for intracellular FRET application due to the ease of targeting. However, the use FPs for FRET applications has some disadvantages directly related to the intrinsic properties of FP’s. First, compared to most organic fluorescent dyes, the excitation and emission spectra of most FPs are relatively broad with extended tails (48), which leads to substantial bleedthrough signal in FRET applications between commonly used FP pairs, such as CFP/YFP. The combination of FPs with FKBP12 (F36V) and its fluorescent dye conjugated ligand (e.g., GFP and 50 (60 )-TAMRA-EDA-gly-SLF’) might provide a significant advantage over FP pairs since organic fluorophores have better-resolved spectral properties which would result in the reduction of bleed-through signal. Interestingly, a large number of the SLF’dye conjugates evaluated in this study exhibited high background staining in microscopy applications but produced a strong and specific signal in a FRETbased readout (data not shown). In addition the relatively large size of FP’s uses most of the useful FRET distance, which results in limited maximal FRET efficiencies (48). The use of FKBP12 (F36V) in combination with a FP might provide therefore some improvement in respect to usable FRET distance. Similarly to FRET, small tag size benefits better efficiency in FALI applications as shown by Marks et al. (20). In addition, the newly introduced SLF’dye conjugates might provide greater flexibility in the application of FALI. This might be especially interesting for multiplex applications in combination with other labeling techniques (49), were multiple, differentially labeled proteins (e.g., FlAsH in combination with FKBP12 (F36V)) could be simultaneously or sequentially inactivated using label-specific illumination for FALI. This approach might be particularly valuable for the functional investigation of multisubunit protein complexes. In conclusion, the development of novel, red fluorescent SLF’dye conjugates and their use in a wide variety of experimental procedures greatly expands the versatility of the FKBP12 (F36V)-tag as a tool for the analysis of proteins dynamics in living cells. We intend this report to serve as a template to drive further studies expanding the number of useful SLF’ dyes and applications for this promising labeling technology. ACKNOWLEDGMENTS The authors thank Kurt Vogel, Brian Pollok, Joe Beechem, Mike O’Grady, and Larry Greenfield for helpful discussions and Josh Thompson for the invaluable help with the preparation of compounds. 223 ORIGINAL ARTICLE LITERATURE CITED 1. Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods 2005;2:905–909. 2. Zacharias DA, Violin JD, Newton AC, Tsien RY. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 2002;296:913–916. 3. Andresen M, Schmitz-Salue R, Jakobs S. 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