Using Co-Cultures Expressing Fluorescence Resonance Energy Transfer Based Protein Biosensors to Simultaneously Image Caspase3 and Ca 2+ Signaling

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 Springer 2005

Biotechnology Letters (2005) 27: 1219–1227 DOI 10.1007/s10529-005-0021-0

Using co-cultures expressing fluorescence resonance energy transfer based protein biosensors to simultaneously image caspase-3 and Ca2+ signaling Jason Jui-Hsuan Chiang1,2 & Kevin Truong1,2,* 1

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, M5S 3G9, Toronto, Ontario, Canada 2 Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, M5S 3G4, Toronto, Ontario, Canada *Author for correspondence (Fax: 416-978-4317; E-mail: [email protected]) Received 18 April 2005; Revisions requested 9 May 2005; Revisions received: 8 June 2005; Accepted 11 June 2005

Key words: biosensor, fluorescence imaging, fluorescence resonance energy transfer, protein engineering

Abstract Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca2+ signaling events involved in cell death of COS-7 cells induced by 10 mM H2O2. Interestingly, this H2O2 stimulus induced synchronous caspase-3 activation and Ca2+ signaling. In parallel to caspase-3 activation, cytosolic Ca2+ concentration, [Ca2+]c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca2+]c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H2O2. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.

Introduction Fluorescence resonance energy transfer (FRET)based protein biosensors are emerging as important tools to study the kinetics and location of signaling events in living cells such as Ca2+ signaling, phosphorylation or caspase proteolytic cleavage (Zhang et al. 2002). FRET occurs when a donor and acceptor chromophores with overlapping emission and absorption spectra, such as a Cyan fluorescent protein (CFP) and Yellow fluorescent protein (YFP) are found within the 10–80 Angstrom range, with a suitable orientation (Truong & Ikura 2001). For example, cameleons (Miyawaki et al. 1999, Mizuno et al.

2001, Truong et al. 2001) are a class of FRETbased protein biosensors that consist of a tandem fusion of CFP, calmodulin, calmodulin-binding domain, and YFP. Upon increase in the free Ca2+ concentration, [Ca2+], calmodulin binds Ca2+ and acquires affinity for the calmodulinbinding domain. This results in a conformational change that decreases the distance between the FRET partners and, therefore, an increase in FRET (Miyawaki et al. 1999, Mizuno et al. 2001, Truong et al. 2001). Similarly, to study caspase-3 cleavage kinetics involved in apoptosis, biosensors have been created, where CFP and YFP are separated by a peptide linker containing a caspase-3 cleavage site (Tyas et al. 2000, Luo

1220 et al. 2001, Rehm et al. 2002, Takemoto et al. 2003). Upon caspase-3 proteolysis, the CFP and YFP are separated and, therefore, FRET decreases. The next step involves the simultaneous imaging of multiple events using FRET-based protein biosensors to reveal the spatial and temporal relationships between signaling events of a pathway. Previous experiments using cells that were co-transfected with two different biosensors have been ineffective due to the strong spectral overlap between fluorescent proteins. Here, we showed a method to simultaneous image multiple events using, instead, a co-culture of cells expressing two different FRET-based biosensors. This method is effective given two conditions. First, cells expressing one biosensor must be easily distinguishable from cells expressing another. For this reason, biosensors with a red fluorescent protein acceptor from Discosoma sp. reef coral (DsRed) (Baird et al. 2000, Campbell et al. 2002) were used as they are easily distinguishable from biosensors with a YFP acceptor. Second, to correlate events from two different cells, they must respond to the stimulus at the same time or in other words, synchronously. As a demonstration, we simultaneously imaged both caspase-3 proteolytic cleavage and cytosolic [Ca2+] changes induced by 10 mM H2O2.

Materials and methods Construction of the plasmids pTriEx-3 (Novagen) was chosen as the base plasmid, because it allows expression in both prokaryotic and eukaryotic cells. The plasmid for expressing y3c, pY3ctx3, was created by combining 4 different plasmids: pCfptx3, pVentx3, pDMQDtx3 and pHistx3. To create pCfptx3 and pVentx3, the pECFP-1 (Clontech) and pVenus plasmid (Nagai et al. 2002), respectively, were amplified with the following primers: forward, 5¢CATGCCATGGGCCTGACTAGTGTGAGCAAGGGCGAGGAGCTG-3¢; reverse, 5¢-CCGCT CGAGTTAGCCGCTAGCGGCGGCGGTCAC GAACTCCA-3¢. The forward primer contained NcoI and SpeI, sites, while reverse primer contained XhoI and NheI sites. The resulting PCR

fragments were digested with NcoI–XhoI and ligated into the NcoI–XhoI site of pTriEx-3 to create pCfptx3 and pVentx3. To construct pDMQDtx3 and pHistx3, oligonucleotides were flanked by NcoI–SpeI on the 5¢ end and NheI– XhoI on the 3¢ end that contained the peptide sequences GGASGGASDMQDASGGASGG and GSSHHHHHHSSG in human codon preference, respectively. The oligonucleotides were digested with NcoI–XhoI and ligated into the NcoI–XhoI site of pTriEx-3 to create pDMQDtx3 and pHistx3. To create the intermediate pDM QDctx3, pCfptx3 was digested with SpeI–XhoI and ligated into the NheI–XhoI site of pDMQDtx3. Similarly, to create the intermediate pY3ctx3, pDMQDctx3 was digested with SpeI– XhoI and ligated into the NheI–XhoI site of pVentx3. Finally, to create the pHY3ctx3, pY3ctx3 was digested with SpeI–XhoI and ligated into the NheI–XhoI site of pHistx3. Purification of the y3c biosensor E. coli strain Rosetta (Novagen) were transformed with the pHY3ctx3 plasmid and plated. A single colony was selected and grown overnight in Luria broth at 37 C. Cells were then centrifuged and resuspended in the buffer (50 mM Tris/HCl, pH 7.9, 250 mM NaCl, 1% Nonidet P40 and 0.1 mM PMSF). The cells were lysed by sonication and the biosensor was purified using Ni-NTA resin (Qiagen) and eluted from the column in the buffer (10 mM Tris/HCl, pH 7.9, 250 mM NaCl, and 500 mM imidazole). Finally, the sample was dialyzed and concentrated in the buffer (20 mM Hepes, pH 7.5, and 50 mM NaCl). In vitro caspase-3 cleavage assay and spectroscopy All emission spectra were recorded at 22 C before and after protease treatment with excitation 437 nm using a fluorescence spectrophotometer. The y3c biosensor (1 lM) was diluted in the caspase reaction buffer (20 mM Hepes, pH 7.5, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, and 10 mM DTT). Two units of various recombinant active caspases (Calbiochem) were incubated with the y3c biosensor at 37 C for 2 h. To measure the spectra of the completely cleaved the biosensor, the biosensor (1 lM) was

1221 diluted in the proteinase K reaction buffer (50 mM Tris/HCl, pH 7.8, and 10 mM EDTA) and incubated with 0.1 mg ml)1 proteinase K at 37 C for 1 h. Cell culture and transfection COS-7 cells were grown at 37 C with 5% CO2 in DMEM (Gibco) supplemented with 10% (v/v) FBS (Gibco). Transfection was performed using GeneJuice (Novagen). Annexin V-FITC and MitoSensor staining (BD Biosciences) COS-7 cells grown in glass-bottom culture dishes (Maltek) and treated with 10 mM H2O2 in PBS (pH 7.4) for 30 min. Then, the cells were washed in PBS (pH 7.4). Thereafter, staining was performed as described in the manufacturer’s protocol. Imaging Two glass-bottom 35-mm culture dishes (Maltek) with COS-7 cells at 30--70% confluency were separately transfected with the crc2 plasmid (1 lg) and pY3ctx3 plasmid (1 lg), respectively. After 24 h, the transfection efficiency was estimated by the percentage of fluorescent cells on the culture dishes. Cells were detached from each dish by 200 ll Trypsin-EDTA (Gibco) and the trypsinized cells were mixed to maximize the probability of finding two adjacent cells in the co-culture where one is expressing the y3c biosensor and the other, crc2. To determine this optimal mixing ratio, the higher transfection efficiency was divided by the lower, which yielded a value x. Every 1 ll of the trypsinized cells with higher transfection efficiency was diluted by x ll of the other. This mixture was then re-plated on a new dish. Between 1 and 3 days after co-culturing, cells were imaged at 22 C on an Olympus IX70 microscope with a CCD camera (MicroMax 1300YHS) controlled by MetaMorph 4.5r2 software (Universal Imaging). Two adjacent cells expressing the crc2 and y3c biosensor were found on the eyepiece by the Multiband – Triple XF91 filter set (Omega Optical). The relative expression levels of y3c and crc2 biosensors can be approximated by the intensity of Enhanced cyan

fluorescent protein (ECFP) emission. Best results were obtained when these expression levels were approximately the same. Triple-emission ratio imaging of co-cultures of the biosensors used a 440DF20 excitation for ECFP, a 455DRLP dichotic mirror, and three emission filters (480DF30 for ECFP, 535DF25 for Venus, 600ALP or DsRed mutants) alternated by a Lambda 10–2 filter changer (Sutter Instruments). Interference filters were purchased from Omega Optical.

Results and discussion A FRET-based protein biosensor, named y3c, was constructed to measure caspase-3 activation, which displayed a specific caspase-3 cleavage in vitro (Figure 1). The y3c biosensor was created by the protein fusion of Venus (Nagai et al. 2002) and Enhanced cyan fluorescent protein (ECFP) that sandwiched a 20 amino acid linker containing a caspase-3 specific recognition sequence, DMQD (Hirata et al. 1998) (Figure 1a). Venus (Nagai et al. 2002) was chosen as an

Fig. 1. Design of the y3c biosensor and in vitro spectroscopy. (a) Schematic diagram of the y3c biosensor. The linker sequences between Venus and ECFP are shown with the caspase-3 recognition sequence (DMQD) underlined. (b) Emission spectra of the y3c biosensor was acquired at an excitation of 437 nm before (solid line) and after (dotted line) treatment with purified recombinant caspase-3. The spectra of the fully cleaved biosensor was obtained after treatment with proteinase K. (c) Specificity of the y3c biosensor was determined by estimating the cleavage percentage based on the change in emission ratio after treatment with various recombinant caspases (-: 0–5%; + : 5–25%; ++ : 25– 75%;+++ : 75–100%).


Fig. 2. Characterization of the y3c biosensor in COS-7 cells. (a) Synchronous cleavage of the y3c biosensor in transfected COS-7 cells stimulated with 10 mM H2O2. Images are displayed in intensity modulated display (IMD) mode, which reduces noise from weak fluorescent signals. IMD mode uses the emission ratio to determine the colour hue but uses the intensity from the wavelength images to determine the intensity of the colour hue. The IMD scale is shown where green corresponds to a high ratio; blue, midrange; black, low. (b) Cleavage of the y3c biosensor in transfected COS-7 cells stimulated using different death stimulus: 1 lM staurosporine (short-dashed line); UV irradiation (long-dashed line); 20 lM etoposide (solid line). The emission ratio is normalized such that 0 is the minimum emission ratio and 1 is the maximum.

acceptor instead of enhanced yellow fluorescent protein because it maturates quickly and its molar extinction coefficient is less sensitive to pH changes. The latter property is particularly

important as some forms of cell death, such as apoptosis, are linked with a drop in cytosolic pH (Matsuyama et al. 2000). In the emission spectra of purified y3c biosensor prior to protease treat-


Fig. 3. Annexin V-FITC staining. Annexin V-FITC binds to phospholipid phosphatidylserine (PS) plasma membrane from cells undergoing apoptosis. FITC emission fluorescence (485 ± 10 nm emission) from COS-7 cells treated with (a) no stimulus; (b) 1 lM STS; (c) 10 mM H2O2. Brightfield images of COS-7 cells treated with (d) no stimulus; (e) 1 lM STS; (f) 10 mM H2O2.

ment, there was a strong peak emission of Venus (535 nm) due to FRET from ECFP to Venus (Figure 1b). After treatment with active recombinant caspase-3, the peak emission of Venus decreased and ECFP (480 nm) increased due to an expected loss of FRET. After additional treatment with proteinase K (a non-specific protease), the emission spectra did not change showing that proteinase K does not further cleave the y3c biosensor. Thus, the initial dose of caspase-3 had efficiently cleaved the y3c biosensor. To evaluate the specificity, the same experiment was performed using various caspases, where the y3c biosensor was not cleaved by most other caspases and only moderately cleaved by caspase-7 (Figure 1c). Lastly, the in vitro effect of H2O2 on the y3c biosensor was tested by incubating the biosensor with increasing concentrations of H2O2. The spectra did not change until the concentration of H2O2 was in the range of 1 M, but at such concentrations, the measurement is unreliable as H2O2 dramatically lowers the pH causing Venus to lose fluorescence. The y3c biosensor was cleaved synchronously and quickly in transfected COS-7 cells when stimulated by 10 mM H2O2 (Figure 2a). An ex-

cess H2O2 stimulus upsets the prooxidant–antioxidant balance, resulting in cell injury or death pathways such as apoptosis (Chandra et al. 2000). Apoptotic cell death is distinctly characterized morphologically by cell shrinkage, rounding and blebbing, and molecularly by such events as mitochrondrial dysfunction, the translocation of phospholipid phosphatidylserine (PS) to the extracellular plasma membrane and the activation of the effector caspase-3 (Kanduc et al. 2002). After the H2O2 stimulus, the cleavage of the y3c biosensor started within 2 min and was mostly completed within 5 ± 2 min, represented by a decrease in emission ratio (division of 535 nm by 480 nm intensity) (n=21 experiments). In contrast, when transfected COS-7 cells were stimulated with staurosporine, UV irradiation or etoposide, the onset of biosensor cleavage was asynchronous between cells within a time window of 2–4 h, which is consistent with the observation of other groups (Tyas et al. 2000, Luo et al. 2001, Rehm et al. 2002, Takemoto et al. 2003) (Figure 2b). Furthermore, Annexin V and MitoSensor staining confirmed PS translocation and mitochondrial dysfunction, respectively (Figures 3 and 4). Therefore, the cell death


Fig. 4. MitoSensor staining. MitoSensor localizes to the mitochrondria in normal cells and the cytoplasm in cells undergoing apoptosis as the mitochrondria dysfunctions. MitoSensor fluorescence (485 ± 10 nm emission) from COS-7 cells treated with (a) no stimulus; (b) 1 lM STS; (c) 10 mm H2O2.

induced by this H2O2 stimulus has hallmark features of apoptosis. Using a FRET-based protein Ca2+ biosensor with a DsRed acceptor, named crc2 (Mizuno et al. 2001), a synchronous rise in Ca2+ in COS7 cells was detected following the H2O2 stimulus (Figure 5). A H2O2 stimulus can induce intracellular Ca2+ changes in many cultured cells including osteocytes, HeLa and mouse pancreatic acinar cells (Pariente et al. 2001, Kikuyama et al. 2002, Castro et al. 2004). To evaluate the effect on COS-7 cells, they were transfected with the crc2 biosensor, which consisted of a tandem fusion of ECFP, calmodulin, a Myosin light chain kinase peptide (MLCKp) and DsRed (Mizuno et al. 2001). When Ca2+ increases, calmodulin binds MLCKp and causes an FRET increase between ECFP and DsRed. After the H2O2 stimu-

lus to the transfected COS-7 cells, the emission ratio (division of 600 nm by 480 nm intensity) increased synchronously in all cells in the field of view signifying an increase in cytosolic Ca2+ concentration, [Ca2+]c. This [Ca2+]c increased to a peak of 600 ± 100 nM (n=3 experiments). The same experiment was performed in the absence of extracellular Ca2+ in the media and the same initial [Ca2+]c changes occurred, signifying that the Ca2+ originated from the intracellular Ca2+ stores. As the caspase-3 cleavage and Ca2+ signaling occurred synchronously after the H2O2 stimulus, the two events were simultaneously imaged using a co-culture of crc2 and y3c transfected COS-7 cells (Figure 6). The yellow fluorescence from the cells expressing the y3c biosensors was distributed in the both cytoplasm and the nucleus,

Fig. 5. Synchronous rise in [Ca2+]c cleavage of the crc2 biosensor in transfected COS-7 cells stimulated with 10 mM H2O2 in IMD mode.


Fig. 6. Imaging of simultaneous caspase-3 and Ca2+ signaling using a co-culture of y3c and crc2 in transfected COS-7 cells stimulated with 10 mM H2O2. (a) At various time points, the overlay of the fluorescence image (440 ± 10 nm excitation, 535 ± 12.5 nm emission, coloured green) and the fluorescence image (440 ± 10 nm excitation, >600 nm emission, coloured red) for the co-culture of y3c and crc2. Correspondingly, y3c and crc2 emission ratio images were displayed in IMD mode. (b) The emission ratios for y3c (535 ± 12.5 nm divided by 480 ± 12.5 nm emission intensity) and crc2 (>600 nm divided by 480 ± 12.5 nm emission intensity) along with the emission intensities (480 ± 12.5 nm, 535 ± 12.5 nm and >600 nm) were measured every 10 s. The data corresponding to y3c and crc2 were coloured green and red, respectively.

1226 while cells expressing the crc2 biosensor were easily distinguished by its red fluorescence distributed in the cytoplasm but excluded from the nucleus. The time course for the spatially averaged fluorescence intensities and emission ratios was measured from two adjacent cells. Prior to stimulus both y3c and crc2 emission ratios were constant at 2.80 ± 0.01 and 0.49 ± 0.005, respectively; however, approximately 50 s after the the H2O2 stimulus, the crc2 emission ratio linearly rose to its peak of 0.608 over 50 ± 10 s (n=3 experiments) and simultaneously, the y3c emission ratio exponentially dropped and 95% cleavage was completed in approximately 5 min (Figure 6b). The increase in DsRed and decrease of ECFP emission intensities indicated an increase in FRET resulting from a conformational change of crc2, while the decrease in Venus and increase in ECFP emission intensities indicated a decrease in FRET resulting from the cleavage of the y3c biosensor. Morphology change such as cell shrinkage began approximately 10 ± 1 min after the initial y3c cleavage (n=3 experiments). Furthermore, after reaching its peak, Ca2+ levels gradually dropped to its resting state and then gradually rose again as the cell began acquiring a distinctly round morphology. The second delayed [Ca2+]c rise is shared by many apoptotic inducing agents, associated with the apoptotic morphological changes (i.e. cell rounding and blebbing) and transcriptionally reprogramming of death associated proteins (such as Gadd153) (Tombal et al. 2002).

Conclusions We have demonstrated an approach using FRET-based protein biosensors and co-cultures to simultaneously image multiple events in a signaling pathway within living cells. The problem of spectral overlap between fluorescent proteins was avoided by using a co-culture of cells expressing different FRET-based protein biosensors, one with Venus acceptor and another, DsRed. This is a useful approach to studying signaling pathways that progress synchronously in a population of cells as in the case of caspase-3 and Ca2+ signaling events from cell death induced by a 10 mM stimulus of H2O2. Such

experiments applied to other similar pathways would yield a better knowledge of their exact timing, kinetics and location that are important to understanding signaling output.

Acknowledgements We thank Mitsuhiko Ikura for the use of fluorescence imaging facilities. This work was supported by grants from the National Science and Engineering Research Council (NSERC) and from the Banting Foundation.

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