- Email: [email protected]
mKOκ fluorescent protein pairs
Biosensors and Bioelectronics 46 (2013) 97–101
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOk fluorescent protein pairs Ting Su a,b, Shaotao Pan a,b, Qingming Luo a,b, Zhihong Zhang a,b,n a Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics–Huazhong University of Science and Technology, Wuhan 430074, China b MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e i n f o
abstract
Article history: Received 13 December 2012 Received in revised form 6 February 2013 Accepted 11 February 2013 Available online 27 February 2013
¨ Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) biosensors are powerful tools for dynamically measuring cellular molecular events because they offer high spatial and temporal resolution in living cells. Despite the broad use of FP-based FRET biosensors in cell biology, imaging of multiple molecular events (multi-parameter molecular imaging) in single cells using current FRET pairs remains difficult because it usually requires a control group for additional data calibration. Hence, spectrally compatible FRET pairs that do not require complex image calibration are the key to widespread applications of FP-based FRET biosensors in multi-parameter molecular imaging. Here, we report a new combination of spectrally distinguishable FRET pairs for dual-parameter molecular imaging: mTagBFP/sfGFP (blue and green FP, B/G) and mVenus/mKOk (yellow and orange FP, Y/O). We demonstrate that additional image correction is not necessary for these dual FRET pairs. Using these dual FRET pairs, we achieve simultaneous imaging of Src and Ca2 þ signaling in single living cells stimulated with epithelial growth factor (EGF). By converting traditional FRET biosensors into B/G and Y/O-based biosensors, additional applications are available to elucidate the dynamic relationships of multiple molecular events within a single living cell. & 2013 Elsevier B.V. All rights reserved.
Keywords: Fluorescent protein FRET biosensor Multi-parameter imaging mTagBFP sfGFP mVenus mKOk
1. Introduction ¨ Forster resonance energy transfer (FRET) is a physical phenomenon in which an excited chromophore (donor) non-radiatively transfers its energy to another chromophore (acceptor) when the distance between donor and acceptor is between 1 and 10 nm. FRET, when applied to optical microscopy, enables researchers to visualize molecular interactions or conformational changes in single living cells, bypassing the intrinsic diffraction limit of optical microscopy. The advent of green fluorescent protein (GFP) and its combination with FRET greatly enhance the application of this technology within the field of cell biology (DiPilato and Zhang, 2010; Yu and Xiao, 2012; Zhang et al., 2002, 2008). The use of fluorescent protein (FP)-based FRET has been particularly advantageous in the field of biosensors (Campbell, 2009). Because FRET technology is inherently quantitative, FRET-based biosensor measurements are easily read in the ratiometric manner, greatly
n Corresponding author at: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. Tel.: þ86 27 87792033; fax: þ 86 27 87792034. E-mail address: [email protected] (Z. Zhang).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.02.024
simplifying experimental procedures. Amongst FP pairs, the CFP/ YFP pair has been shown to be superior in providing sensitive FRET signals, making it the preferred candidate for construction of FP-based FRET biosensors (Carlson and Campbell, 2009). Although it is possible to monitor signals in spatially segregated subcellular locations by targeting CFP/YFP-based biosensors to these locations, spectral overlap between biosensors hinders the imaging of multiple signals within the same intracellular location. Molecules in living cells form complex, interconnected networks, and there is a strong demand from cell biologists to synchronously study interacting molecules in single cells (Welch et al., 2011). The discovery and optimization of red-shifted FPs provide numerous candidate FRET pairs and make imaging of multiple pairs of FP biosensors (multi-parameter imaging) spectrally possible (Chudakov et al., 2010). Because most of the current FP-based FRET biosensors are engineered on the basis of CFP and YFP pair, the simplest FP combination is to adopt red-shift OFP/RFP (O/R) FRET pair such as mOrange/mCherry (Ouyang et al., 2010) or mKOk/mLumin (Su et al., 2012). Unfortunately, the relatively low quantum yield of RFP prevents O/R pairs from providing satisfactory sensitivity in FRET biosensors (Carlson and Campbell, 2009). Alternative solutions involve the use of fluorescent proteins with unique
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T. Su et al. / Biosensors and Bioelectronics 46 (2013) 97–101
spectral properties (Ai et al., 2008; Nagai et al., 2009). For example, Wataru et al. introduced the ultramarine FP Sirius and made a new combination of dual FRET pairs, including SiriusmseCFP and Sapphire-DsRed (Nagai et al., 2009). Ai et al. (2008) generated the large stoke shift violet-excitable yellow-fluorescing FP mAmetrine and developed mAmetrine-tdTomato and mCitrine-mTFP1 dual FRET pairs. Although both dual FRET pairs can visualize dual-molecular events in single living cells, extra correction, involving either linear un-mixing (Nagai et al., 2009) or calibration coefficients (Ai et al., 2008), is needed to acquire non-interfered ratiometric images. Recently, we reported a mVenus/mKOk (Y/O) pair that performs better than the mKOk/mLumin (O/R) pair in FRET biosensors (Su et al., 2012). In the present study, we introduce a new FRET pair that is composed of the blue fluorescent protein mTagBFP (Subach et al., 2008) and the green fluorescent protein sfGFP (Pedelacq et al., 2006). We show that the mTagBFP/sfGFP (B/G)based FRET biosensor can be simultaneously imaged in the presence of Y/O FRET-based biosensor without the need for further correction. Monitoring of two signals demonstrates the power of this combination in acquiring kinetic information that is unattainable previously.
100 U/ml penicillin and 100 mg/ml streptomycin. The day before transfection, the cells were plated on 35-mm-diameter coverglassbottom dishes (MatTek Corporation, Ashland, MA) at approximately 50% confluence. Plasmids encoding the fluorescent biosensors were transfected into the cells using the Lipofectamine 2000 reagent according to the manufacturer’s instructions. For transfection of a single biosensor, 0.2 mg of DNA was used. For dual transfections, equal amounts of DNA were used for each sensor (0.1 mg each). For the EGF stimulation experiments, cells were allowed to rest for 36 h after transfection and then serumstarved for 6–12 h in DMEM before EGF stimulation. 2.3. Ratiometric imaging
Plasmids encoding each biosensor were genetically engineered. For details, please see the methods section of the Supplemental data.
Ratiometric fluorescent imaging was performed with a confocal laser-scanning microscope FV1000 (Olympus, Japan) and FluoView software (Version 1.5) (Olympus, Japan). Immediately before imaging cells, DMEM was replaced with CO2-independent media to maintain cells at physiological pH (7.3). For the BG-Src biosensors, mTagBFP was excited at 405 nm, and mTagBFP and sfGFP emissions were collected at 440–480 nm and 500–560 nm, respectively. For the YO-Src and YO-TnC biosensors, mVenus was excited at 514 nm and mVenus and mKOk emissions were collected at 524–540 nm and 560–620 nm, respectively. In the dual ratiometric imaging experiment with the BG-Src and Y/O-based biosensors, the ‘‘time-control’’ function in the FluoView software was used to quickly switch between imaging of each biosensor. Under our imaging conditions, there was an approximate 3-s delay between imaging of the two biosensors.
2.2. Cell culture and transfection
2.4. Image processing
HeLa cells were cultured at 37 1C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS,
‘‘Image J’’ software (http://rsbweb.nih.gov/ij/) was used to process confocal images and to generate emission ratio images
2. Materials and methods 2.1. Plasmid construction
Fig. 1. Characterization of the BG-Src biosensor in HeLa cells. (A). Pseudo-color BFP/GFP ratio images of BG-Src upon EGF (100 ng/ml) stimulation of HeLa cells. (B). Graphical representation of (A) demonstrating the BFP/GFP ratio time course of the BG-Src biosensor. (C). BFP/GFP ratio time course of the BG-Src biosensor in HeLa cells upon sequential addition of EGF and the Src inhibitor PP1. (D). Photo-bleaching experiment demonstrating stable intensities of BFP, GFP and the BFP/GFP ratio upon illumination with a Violet blue 405 nm laser. Imaging parameters are identical with those used in the subsequent dual-FRET experiments. A total of 120 image frames were scanned. Scale bar: 10 mm.
T. Su et al. / Biosensors and Bioelectronics 46 (2013) 97–101
for the BG-Src (or YO-Src and YO-TnC) biosensor. For the BG-Src biosensor, the original mTagBFP and sfGFP images were first background subtracted at each time point. The updated images were then subjected to Gaussian blur (sigma value 1) for image smoothing. Because the sfGFP images had the largest signal-tonoise ratio, the sfGFP image at each time point was used to generate a binary mask image with pixel values equal to 1 or 0. The mTagBFP and sfGFP images were then multiplied by this mask image. Finally, the updated mTagBFP and sfGFP images were used to generate the mTagBFP/sfGFP ratio images. For a better presentation of ratio images, the brightness/contrast was adjusted and a linear pseudo-color lookup table was applied. For quantification, the ratio values were normalized to the average value of the ratio before EGF stimulation. The aforementioned procedures were also used to generate the mVenus/mKOk (or mKOk/mVenus) ratio images for the YO-Src (or YO-TnC) biosensor. Kinetic curves for the biosensors were plotted using OriginPro 8 software.
3. Results and discussions 3.1. Characterization of the sfGFP/mTagBFP-based Src biosensor (BG-Src) in living cells The key to implementing dual-parameter imaging experiments is that the two biosensors must be spectrally compatible. When using dual-FRET biosensors, one FRET biosensor must be ratiometrically imaged without interference from the other. By analyzing the spectra of current FPs, we determined that the B/G FRET pair might be combined with the Y/O FRET pair with minimal interference in dual-FRET imaging. From all blue FPs and green FPs, mTagBFP (Subach et al., 2008) and sfGFP (Pedelacq et al., 2006) were chosen based on their high brightness, fast maturation and high photostability (Crivat and Taraska, 2012; Day and Davidson, 2009).
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The substantial overlap between the emission spectra of mTagBFP and the absorbance spectra of sfGFP, together with their desired optical properties (quantum yield of mTagBFP and sfGFP is 0.63 and 0.65, respectively; extinction coefficients of mTagBFP and sfGFP are 52,000 and 83,000 M–1 cm–1, respectively) result in highly ¨ sensitized emissions (Forster radius: 5.5 nm), and facilitate quantitative measurements via direct ratio imaging. To demonstrate the usefulness of the mTagBFP/sfGFP pair, we constructed a FRET-based biosensor for Src kinase (denoted as BG-Src). The specific response element of BG-Src is a Src substrate peptide from p130cas and a SH2 domain from Src kinase and is the same as the YC-based Src biosensor previously reported (Wang et al., 2005). During ratiometric imaging, the ratio change was used to characterize the response of the BG-Src biosensor to Src kinase. When expressed in HeLa cells, the BG-Src biosensor exhibited marked FRET response upon EGF stimulation, with a 65% ratio change (Fig. 1A and B). The ratio change of the BG-Src biosensor was substantially larger than that of the initial Src biosensor based on ECFP/mCitrine (43%) (Wang et al., 2005) and was comparable to the updated Src biosensor, which was based on ECFP and circular permutated mVenus (77%) (Ouyang et al., 2008). Treatment with the Src-specific inhibitor PP1 readily reversed the FRET response, indicating that BG-Src is specific to Src kinase (Fig. 1C). The high photo-stability of mTagBFP and sfGFP ensures that the ratio of BG-Src biosensor is not distorted by UV illumination, as represented by the 405 nm laser in our confocal system (Fig. 1D). Together, our results demonstrate that the mTagBFP/sfGFP pair can be used as a FRET biosensor. 3.2. mTagBFP/sfGFP and mVenus/mKOk FRET pairs are spectrally compatible for dual-FRET imaging in living cells To image two pairs of FRET biosensors in the same cellular location, it is imperative that imaging of one biosensor does not
Fig. 2. mTagBFP/sfGFP (B/G) and mVenus/mKOk (Y/O) FRET pairs are spectrally compatible in vivo. (A). Spectra of the B/G and Y/O FRET pairs. A 405 nm laser was used to excite the B/G-based FRET biosensor and a 514 nm laser was used to excite the Y/O-based biosensor. (B). Spectral bleed-though of the two FRET biosensors after transfection into HeLa cells. (C, D) Control experiments confirm that the ratio change of the BG-Src biosensor does not affect the Y/O-based caspase-3 (YO-C3) biosensor, and the ratio change of the Y/O-based calcium (YO-TnC) biosensor does not affect the BG-Src biosensor. Stimulation of HeLa cells with EGF (100 ng/ml) changed the emission ratio of BG-Src without affecting the emission ratio of YO-C3 (C), and stimulation with histamine (100 mM) changed the emission ratio YO-TnC without affecting the emission ratio of BG-Src (D). BG-Src is targeted to cytosol by adding nuclear export sequence (NES) to the 30 -end of the biosensor.
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Fig. 3. Simultaneous monitoring of Src and Ca2 þ signal in single living HeLa cells using dual biosensors. (A). Pseudo-color ratio images of HeLa cells expressing dual biosensors (BG-Src and YO-TnC biosensors). EGF (100 ng/ml) was added at time 0. (B) Emission ratio (black curve) time course of the BG-Src biosensor and emission ratio (red curve) time course of YO-TnC in (A). Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
affect imaging of the other. In the case of mTagBFP/sfGFP- and mVenus/mKOk-based FRET biosensors, spectral contamination is attributed to crosstalk between sfGFP and mVenus (Fig. 2A). To gain quantitative insights, we transfected one biosensor in HeLa cells and measured bleed-through emissions into channels used by the other biosensor. In the YO-Src biosensor, only 1% of mVenus fluorescence bled into the sfGFP channel. However, in BG-Src biosensor, 7% of sfGFP fluorescence bled into the mVenus channel (Fig. 2B). To determine the effect of this contamination on the ratio of each biosensor, both B/G- and Y/O-based biosensors were co-transfected into HeLa cells. Addition of EGF stimulated a dramatic response from the BG-Src biosensor but the Y/O-based caspase-3 biosensor (YO-C3) remained stable (Fig. 2C), indicating that fluorescence contamination from BG-Src does not distort the ratio of YO-C3. Similarly, a ratio change in the Y/O-based calcium biosensor (YO-TnC) did not influence the ratio of the BG-Src biosensor (Fig. 2D). In summary, our results demonstrate that the B/G- and Y/O-based biosensors can work together to monitor dual molecular events in the same cellular location. 3.3. Simultaneous imaging of Src and calcium signal in single living cells using B/G-Src and Y/O-TnC biosensor, respectively Upon binding to exogenous ligands, many plasma membrane receptors initiate multiple signaling pathways. In many cases, a complicated signaling network is activated, causing diverse effects ranging from cytoskeletal changes to gene expression alterations (Schlessinger, 2000). One example is EGF-induced signal transduction; it is well known that EGF can trigger Src signaling in cytosol and Ca2 þ release from the ER into the cytosol (Oda et al., 2005). To gain insight into the dynamics of these two processes, BG-Src and YO-TnC were co-transfected into HeLa cells (Supplemental Fig. S1). Upon EGF administration, cytosolic levels of Ca2 þ were robustly increased, as indicated by YO-TnC, peaking at approximately 2 min and then decreasing back to baseline levels (Fig. 3). In contrast, Src signaling, as indicated by BG-Src, was sustained throughout the imaging period, consistent with its role in regulating long-term events such as STAT-mediated gene expression and changes in cell morphology (Yeatman, 2004). In the other example, simultaneous imaging of cytoplasmic and plasma membrane Src signaling was achieved in single living cells using BG-Src and YO-Src biosensors, respectively (Supplemental Fig. S2). 4. Discussion Here, we demonstrate that a combination of FRET pairs, which is based on mTagBFP/sfGFP and mVenus/mKOk FPs, allows for
simultaneous kinetic measurements of two molecular events within the same subcellular location without the need for further image correction. The traditional YC-based biosensors can be easily converted to mTagBFP/sfGFP or mVenus/mKOk-based biosensors. We thus expect that more dual signaling events will be simultaneously studied, providing previously unattainable dynamic molecular information.
Acknowledgments We thank Dr. Atsushi Miyawaki for providing mVenus and mKO. The authors also thank the Analytical and Testing Center (Huazhong University of Science and Technology) for spectral measurements. This work was supported by the National Basic Research Program of China (Grant no. 2011CB910401), Science Fund for Creative Research Group of China (Grant no. 61121004), National Natural Science Foundation of China (Grant no. 81172153), and National Science and Technology Support Program of China (Grant no. 2012BAI23B02).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.02.024.
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Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Monitoring of dual bio-molecular events using FRET biosensors based on mTagBFP/sfGFP and mVenus/mKOk fluorescent protein pairs Ting Su a,b, Shaotao Pan a,b, Qingming Luo a,b, Zhihong Zhang a,b,n a Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics–Huazhong University of Science and Technology, Wuhan 430074, China b MoE Key Laboratory for Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e i n f o
abstract
Article history: Received 13 December 2012 Received in revised form 6 February 2013 Accepted 11 February 2013 Available online 27 February 2013
¨ Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) biosensors are powerful tools for dynamically measuring cellular molecular events because they offer high spatial and temporal resolution in living cells. Despite the broad use of FP-based FRET biosensors in cell biology, imaging of multiple molecular events (multi-parameter molecular imaging) in single cells using current FRET pairs remains difficult because it usually requires a control group for additional data calibration. Hence, spectrally compatible FRET pairs that do not require complex image calibration are the key to widespread applications of FP-based FRET biosensors in multi-parameter molecular imaging. Here, we report a new combination of spectrally distinguishable FRET pairs for dual-parameter molecular imaging: mTagBFP/sfGFP (blue and green FP, B/G) and mVenus/mKOk (yellow and orange FP, Y/O). We demonstrate that additional image correction is not necessary for these dual FRET pairs. Using these dual FRET pairs, we achieve simultaneous imaging of Src and Ca2 þ signaling in single living cells stimulated with epithelial growth factor (EGF). By converting traditional FRET biosensors into B/G and Y/O-based biosensors, additional applications are available to elucidate the dynamic relationships of multiple molecular events within a single living cell. & 2013 Elsevier B.V. All rights reserved.
Keywords: Fluorescent protein FRET biosensor Multi-parameter imaging mTagBFP sfGFP mVenus mKOk
1. Introduction ¨ Forster resonance energy transfer (FRET) is a physical phenomenon in which an excited chromophore (donor) non-radiatively transfers its energy to another chromophore (acceptor) when the distance between donor and acceptor is between 1 and 10 nm. FRET, when applied to optical microscopy, enables researchers to visualize molecular interactions or conformational changes in single living cells, bypassing the intrinsic diffraction limit of optical microscopy. The advent of green fluorescent protein (GFP) and its combination with FRET greatly enhance the application of this technology within the field of cell biology (DiPilato and Zhang, 2010; Yu and Xiao, 2012; Zhang et al., 2002, 2008). The use of fluorescent protein (FP)-based FRET has been particularly advantageous in the field of biosensors (Campbell, 2009). Because FRET technology is inherently quantitative, FRET-based biosensor measurements are easily read in the ratiometric manner, greatly
n Corresponding author at: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. Tel.: þ86 27 87792033; fax: þ 86 27 87792034. E-mail address: [email protected] (Z. Zhang).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.02.024
simplifying experimental procedures. Amongst FP pairs, the CFP/ YFP pair has been shown to be superior in providing sensitive FRET signals, making it the preferred candidate for construction of FP-based FRET biosensors (Carlson and Campbell, 2009). Although it is possible to monitor signals in spatially segregated subcellular locations by targeting CFP/YFP-based biosensors to these locations, spectral overlap between biosensors hinders the imaging of multiple signals within the same intracellular location. Molecules in living cells form complex, interconnected networks, and there is a strong demand from cell biologists to synchronously study interacting molecules in single cells (Welch et al., 2011). The discovery and optimization of red-shifted FPs provide numerous candidate FRET pairs and make imaging of multiple pairs of FP biosensors (multi-parameter imaging) spectrally possible (Chudakov et al., 2010). Because most of the current FP-based FRET biosensors are engineered on the basis of CFP and YFP pair, the simplest FP combination is to adopt red-shift OFP/RFP (O/R) FRET pair such as mOrange/mCherry (Ouyang et al., 2010) or mKOk/mLumin (Su et al., 2012). Unfortunately, the relatively low quantum yield of RFP prevents O/R pairs from providing satisfactory sensitivity in FRET biosensors (Carlson and Campbell, 2009). Alternative solutions involve the use of fluorescent proteins with unique
98
T. Su et al. / Biosensors and Bioelectronics 46 (2013) 97–101
spectral properties (Ai et al., 2008; Nagai et al., 2009). For example, Wataru et al. introduced the ultramarine FP Sirius and made a new combination of dual FRET pairs, including SiriusmseCFP and Sapphire-DsRed (Nagai et al., 2009). Ai et al. (2008) generated the large stoke shift violet-excitable yellow-fluorescing FP mAmetrine and developed mAmetrine-tdTomato and mCitrine-mTFP1 dual FRET pairs. Although both dual FRET pairs can visualize dual-molecular events in single living cells, extra correction, involving either linear un-mixing (Nagai et al., 2009) or calibration coefficients (Ai et al., 2008), is needed to acquire non-interfered ratiometric images. Recently, we reported a mVenus/mKOk (Y/O) pair that performs better than the mKOk/mLumin (O/R) pair in FRET biosensors (Su et al., 2012). In the present study, we introduce a new FRET pair that is composed of the blue fluorescent protein mTagBFP (Subach et al., 2008) and the green fluorescent protein sfGFP (Pedelacq et al., 2006). We show that the mTagBFP/sfGFP (B/G)based FRET biosensor can be simultaneously imaged in the presence of Y/O FRET-based biosensor without the need for further correction. Monitoring of two signals demonstrates the power of this combination in acquiring kinetic information that is unattainable previously.
100 U/ml penicillin and 100 mg/ml streptomycin. The day before transfection, the cells were plated on 35-mm-diameter coverglassbottom dishes (MatTek Corporation, Ashland, MA) at approximately 50% confluence. Plasmids encoding the fluorescent biosensors were transfected into the cells using the Lipofectamine 2000 reagent according to the manufacturer’s instructions. For transfection of a single biosensor, 0.2 mg of DNA was used. For dual transfections, equal amounts of DNA were used for each sensor (0.1 mg each). For the EGF stimulation experiments, cells were allowed to rest for 36 h after transfection and then serumstarved for 6–12 h in DMEM before EGF stimulation. 2.3. Ratiometric imaging
Plasmids encoding each biosensor were genetically engineered. For details, please see the methods section of the Supplemental data.
Ratiometric fluorescent imaging was performed with a confocal laser-scanning microscope FV1000 (Olympus, Japan) and FluoView software (Version 1.5) (Olympus, Japan). Immediately before imaging cells, DMEM was replaced with CO2-independent media to maintain cells at physiological pH (7.3). For the BG-Src biosensors, mTagBFP was excited at 405 nm, and mTagBFP and sfGFP emissions were collected at 440–480 nm and 500–560 nm, respectively. For the YO-Src and YO-TnC biosensors, mVenus was excited at 514 nm and mVenus and mKOk emissions were collected at 524–540 nm and 560–620 nm, respectively. In the dual ratiometric imaging experiment with the BG-Src and Y/O-based biosensors, the ‘‘time-control’’ function in the FluoView software was used to quickly switch between imaging of each biosensor. Under our imaging conditions, there was an approximate 3-s delay between imaging of the two biosensors.
2.2. Cell culture and transfection
2.4. Image processing
HeLa cells were cultured at 37 1C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS,
‘‘Image J’’ software (http://rsbweb.nih.gov/ij/) was used to process confocal images and to generate emission ratio images
2. Materials and methods 2.1. Plasmid construction
Fig. 1. Characterization of the BG-Src biosensor in HeLa cells. (A). Pseudo-color BFP/GFP ratio images of BG-Src upon EGF (100 ng/ml) stimulation of HeLa cells. (B). Graphical representation of (A) demonstrating the BFP/GFP ratio time course of the BG-Src biosensor. (C). BFP/GFP ratio time course of the BG-Src biosensor in HeLa cells upon sequential addition of EGF and the Src inhibitor PP1. (D). Photo-bleaching experiment demonstrating stable intensities of BFP, GFP and the BFP/GFP ratio upon illumination with a Violet blue 405 nm laser. Imaging parameters are identical with those used in the subsequent dual-FRET experiments. A total of 120 image frames were scanned. Scale bar: 10 mm.
T. Su et al. / Biosensors and Bioelectronics 46 (2013) 97–101
for the BG-Src (or YO-Src and YO-TnC) biosensor. For the BG-Src biosensor, the original mTagBFP and sfGFP images were first background subtracted at each time point. The updated images were then subjected to Gaussian blur (sigma value 1) for image smoothing. Because the sfGFP images had the largest signal-tonoise ratio, the sfGFP image at each time point was used to generate a binary mask image with pixel values equal to 1 or 0. The mTagBFP and sfGFP images were then multiplied by this mask image. Finally, the updated mTagBFP and sfGFP images were used to generate the mTagBFP/sfGFP ratio images. For a better presentation of ratio images, the brightness/contrast was adjusted and a linear pseudo-color lookup table was applied. For quantification, the ratio values were normalized to the average value of the ratio before EGF stimulation. The aforementioned procedures were also used to generate the mVenus/mKOk (or mKOk/mVenus) ratio images for the YO-Src (or YO-TnC) biosensor. Kinetic curves for the biosensors were plotted using OriginPro 8 software.
3. Results and discussions 3.1. Characterization of the sfGFP/mTagBFP-based Src biosensor (BG-Src) in living cells The key to implementing dual-parameter imaging experiments is that the two biosensors must be spectrally compatible. When using dual-FRET biosensors, one FRET biosensor must be ratiometrically imaged without interference from the other. By analyzing the spectra of current FPs, we determined that the B/G FRET pair might be combined with the Y/O FRET pair with minimal interference in dual-FRET imaging. From all blue FPs and green FPs, mTagBFP (Subach et al., 2008) and sfGFP (Pedelacq et al., 2006) were chosen based on their high brightness, fast maturation and high photostability (Crivat and Taraska, 2012; Day and Davidson, 2009).
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The substantial overlap between the emission spectra of mTagBFP and the absorbance spectra of sfGFP, together with their desired optical properties (quantum yield of mTagBFP and sfGFP is 0.63 and 0.65, respectively; extinction coefficients of mTagBFP and sfGFP are 52,000 and 83,000 M–1 cm–1, respectively) result in highly ¨ sensitized emissions (Forster radius: 5.5 nm), and facilitate quantitative measurements via direct ratio imaging. To demonstrate the usefulness of the mTagBFP/sfGFP pair, we constructed a FRET-based biosensor for Src kinase (denoted as BG-Src). The specific response element of BG-Src is a Src substrate peptide from p130cas and a SH2 domain from Src kinase and is the same as the YC-based Src biosensor previously reported (Wang et al., 2005). During ratiometric imaging, the ratio change was used to characterize the response of the BG-Src biosensor to Src kinase. When expressed in HeLa cells, the BG-Src biosensor exhibited marked FRET response upon EGF stimulation, with a 65% ratio change (Fig. 1A and B). The ratio change of the BG-Src biosensor was substantially larger than that of the initial Src biosensor based on ECFP/mCitrine (43%) (Wang et al., 2005) and was comparable to the updated Src biosensor, which was based on ECFP and circular permutated mVenus (77%) (Ouyang et al., 2008). Treatment with the Src-specific inhibitor PP1 readily reversed the FRET response, indicating that BG-Src is specific to Src kinase (Fig. 1C). The high photo-stability of mTagBFP and sfGFP ensures that the ratio of BG-Src biosensor is not distorted by UV illumination, as represented by the 405 nm laser in our confocal system (Fig. 1D). Together, our results demonstrate that the mTagBFP/sfGFP pair can be used as a FRET biosensor. 3.2. mTagBFP/sfGFP and mVenus/mKOk FRET pairs are spectrally compatible for dual-FRET imaging in living cells To image two pairs of FRET biosensors in the same cellular location, it is imperative that imaging of one biosensor does not
Fig. 2. mTagBFP/sfGFP (B/G) and mVenus/mKOk (Y/O) FRET pairs are spectrally compatible in vivo. (A). Spectra of the B/G and Y/O FRET pairs. A 405 nm laser was used to excite the B/G-based FRET biosensor and a 514 nm laser was used to excite the Y/O-based biosensor. (B). Spectral bleed-though of the two FRET biosensors after transfection into HeLa cells. (C, D) Control experiments confirm that the ratio change of the BG-Src biosensor does not affect the Y/O-based caspase-3 (YO-C3) biosensor, and the ratio change of the Y/O-based calcium (YO-TnC) biosensor does not affect the BG-Src biosensor. Stimulation of HeLa cells with EGF (100 ng/ml) changed the emission ratio of BG-Src without affecting the emission ratio of YO-C3 (C), and stimulation with histamine (100 mM) changed the emission ratio YO-TnC without affecting the emission ratio of BG-Src (D). BG-Src is targeted to cytosol by adding nuclear export sequence (NES) to the 30 -end of the biosensor.
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Fig. 3. Simultaneous monitoring of Src and Ca2 þ signal in single living HeLa cells using dual biosensors. (A). Pseudo-color ratio images of HeLa cells expressing dual biosensors (BG-Src and YO-TnC biosensors). EGF (100 ng/ml) was added at time 0. (B) Emission ratio (black curve) time course of the BG-Src biosensor and emission ratio (red curve) time course of YO-TnC in (A). Scale bar: 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
affect imaging of the other. In the case of mTagBFP/sfGFP- and mVenus/mKOk-based FRET biosensors, spectral contamination is attributed to crosstalk between sfGFP and mVenus (Fig. 2A). To gain quantitative insights, we transfected one biosensor in HeLa cells and measured bleed-through emissions into channels used by the other biosensor. In the YO-Src biosensor, only 1% of mVenus fluorescence bled into the sfGFP channel. However, in BG-Src biosensor, 7% of sfGFP fluorescence bled into the mVenus channel (Fig. 2B). To determine the effect of this contamination on the ratio of each biosensor, both B/G- and Y/O-based biosensors were co-transfected into HeLa cells. Addition of EGF stimulated a dramatic response from the BG-Src biosensor but the Y/O-based caspase-3 biosensor (YO-C3) remained stable (Fig. 2C), indicating that fluorescence contamination from BG-Src does not distort the ratio of YO-C3. Similarly, a ratio change in the Y/O-based calcium biosensor (YO-TnC) did not influence the ratio of the BG-Src biosensor (Fig. 2D). In summary, our results demonstrate that the B/G- and Y/O-based biosensors can work together to monitor dual molecular events in the same cellular location. 3.3. Simultaneous imaging of Src and calcium signal in single living cells using B/G-Src and Y/O-TnC biosensor, respectively Upon binding to exogenous ligands, many plasma membrane receptors initiate multiple signaling pathways. In many cases, a complicated signaling network is activated, causing diverse effects ranging from cytoskeletal changes to gene expression alterations (Schlessinger, 2000). One example is EGF-induced signal transduction; it is well known that EGF can trigger Src signaling in cytosol and Ca2 þ release from the ER into the cytosol (Oda et al., 2005). To gain insight into the dynamics of these two processes, BG-Src and YO-TnC were co-transfected into HeLa cells (Supplemental Fig. S1). Upon EGF administration, cytosolic levels of Ca2 þ were robustly increased, as indicated by YO-TnC, peaking at approximately 2 min and then decreasing back to baseline levels (Fig. 3). In contrast, Src signaling, as indicated by BG-Src, was sustained throughout the imaging period, consistent with its role in regulating long-term events such as STAT-mediated gene expression and changes in cell morphology (Yeatman, 2004). In the other example, simultaneous imaging of cytoplasmic and plasma membrane Src signaling was achieved in single living cells using BG-Src and YO-Src biosensors, respectively (Supplemental Fig. S2). 4. Discussion Here, we demonstrate that a combination of FRET pairs, which is based on mTagBFP/sfGFP and mVenus/mKOk FPs, allows for
simultaneous kinetic measurements of two molecular events within the same subcellular location without the need for further image correction. The traditional YC-based biosensors can be easily converted to mTagBFP/sfGFP or mVenus/mKOk-based biosensors. We thus expect that more dual signaling events will be simultaneously studied, providing previously unattainable dynamic molecular information.
Acknowledgments We thank Dr. Atsushi Miyawaki for providing mVenus and mKO. The authors also thank the Analytical and Testing Center (Huazhong University of Science and Technology) for spectral measurements. This work was supported by the National Basic Research Program of China (Grant no. 2011CB910401), Science Fund for Creative Research Group of China (Grant no. 61121004), National Natural Science Foundation of China (Grant no. 81172153), and National Science and Technology Support Program of China (Grant no. 2012BAI23B02).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.02.024.
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