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Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes
Biochemical and Biophysical Research Communications xxx (2017) 1e6
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes Sonia Colombo a, c, Serena Broggi a, d, Maddalena Collini b, Laura D'Alfonso b, Giuseppe Chirico b, Enzo Martegani a, c, * a
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Department of Physics, University of Milano-Bicocca, Milan, Italy SysBio Centre of Systems Biology, Piazza della Scienza 2, I-20126 Milan, Italy d S.C. di Ematologia e Trapianto di Midollo Osseo, Ospedale Santa Maria della Misericordia, S. Andrea delle Fratte Perugia, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2017 Accepted 18 April 2017 Available online xxx
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour). © 2017 Elsevier Inc. All rights reserved.
Keywords: Cyclic AMP Protein kinase activity Fluorescence resonance energy transfer Live cell imaging Yeast
1. Introduction In the yeast Saccharomyces cerevisiae, the cAMP/PKA pathway plays an important role in the control of metabolism, stress resistance, proliferation and it also affects morphogenesis and development, including pseudohyphal, invasive growth and sporulation [1e5]. The central component of this pathway is adenylate cyclase whose activity is controlled by two G-protein systems, the Ras proteins and the Ga protein Gpa2 [6,7]. Two triggers are known to activate the cAMP/PKA pathway: the addition of glucose to derepressed cells and intracellular acidification. Cyclic AMP is synthesized by adenylate cyclase, encoded by
* Corresponding author. Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. E-mail address: [email protected] (E. Martegani).
CYR1 gene, and induces the activation of the cAMP-dependent protein kinase A (PKA). In turn, PKA phosphorylates a variety of proteins involved in key cellular processes. The whole signaling cascade is tightly regulated and experimental evidences indicate that multiple feedback mechanisms operate within the pathway by the generation of a complex interplay between the cascade components [8e10]. Ras proteins are positively controlled by the activity of Cdc25, that stimulates the GDP-GTP exchange, and negatively regulated by Ira1 and Ira2, that stimulate the GTPase activity of Ras. The inactivation of cAMP is governed by phosphodiesterases that constitute the major feedback mechanism in the pathway [8], although Colombo et al. [9] demonstrated that the feedback inhibition mechanism acts also by changing the Ras2 proteins activation state. A basal level of cAMP is required for growth, while a transient increase of cAMP induced by addition of glucose is required for transition from respiratory to fermentative metabolism. Although the cAMP/PKA pathway has been extensively studied in yeast and both upstream and downstream
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Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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elements are known, the changes in cAMP and in the activity of this pathway were measured in cell populations and usually for a very short time and data on the spatiotemporal variation of cAMP and PKA activity in single cells are till now lacking. Some years ago Nikolaev et al. developed an Epac-based FRET probes for monitoring cAMP levels in vivo in single mammalian cells [11]. These sensor consist of part of the cAMP-binding protein Epac1 or Epac2 sandwiched between cyan and yellow fluorescent proteins(CFP and YFP). The construct unfolds upon binding of the second messenger cAMP to the Epac moiety and cAMP increases are thus easily followed as a drop in FRET. A couple of years later, Allen and Zhang developed a FRET-based A-kinase activity reporter (AKAR), AKAR3, for monitoring PKA activity in vivo in single mammalian cells [12]. AKAR is a recombinant protein composed of a phosphoamino acid binding domain and a PKA-specific substrate sandwiched between CFP and cp-Venus fluorescent protein. When phosphorylated by PKA, intramolecular binding of the substrate by the phosphoamino acid binding domain drives a conformational reorganization, leading to an increase in FRET between CFP and cpVenus. Since key cellular processes, including components and mechanisms used for signal transduction, are conserved between human and yeast, methodologies setups and successfully used in mammalian cells might work in yeast as well. In this regard, we recently studied the localization of Ras-GTP in vivo in single S. cerevisiae cells by expressing a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3) [13], which was proven to be a useful live-cell biosensor for Ras-GTP in mammalian cells [14]. Aim of this work was to develop and test FRET probes for monitoring cAMP levels and PKA activity in vivo in single S. cerevisiae cells, starting from methodologies setups and successfully used in mammalian cells. 2. Materials and methods 2.1. Yeast strains and media Strains used in this study: W303-1A (MATa ade2-1 can1-100 his3-11,15 leu2-3112 trp1-1 ura3-1) [15]; X4004-3A (MATa lys5 met2 ura3 trp1); SP1 (MATa his3 leu2 ura3 trp1 ade8 can1) [16]; GG104 (MATa W303-1A cdc35::KanMX pde2::TRP1 msn2::HIS3 msn4::TRP1) [17]; ASY62 (MATa SP1 tpkl::ADE8 tpk2::HIS3 tpk3::TRPl msn2::HIS3 msn4::LEU2) [18]. Synthetic complete media (SD) contained 2% glucose, 6.7 g/l YNB w/o aminoacids (supplied by ForMedium™, United Kingdom) and the proper selective drop-out CSM (Complete Synthetic Medium, supplied by ForMedium™, United Kingdom). Culture density was measured with a Coulter Counter (Coulter mod. Z2) on mildly sonicated samples. 2.2. Plasmids To obtain the pYX212-YFP-EPAC2-CFP and the pYX242-YFPEPAC2-CFP vectors we used the following strategy. The YFP-EPAC2CFP fragment, obtained digesting the pcDNA3- YFP-EPAC2-CFP construct (kindly provided by Dr. V.O. Nikolaev, University of Wuerzburg, Germany) with XhoI and HindIII, was ligated into the expression vectors, pYX212 and pYX242, digested with the same enzymes. To obtain the pYX212-AKAR3 vector we used the following strategy. The CFP-AKAR3-cpVenus fragment, obtained digesting the pcDNA3- AKAR3 construct (kindly provided by Dr. Jin Zhang, The Johns Hopkins University, Baltimore, USA) with BamHI and XbaI, was ligated into the expression vector pYX212 digested with BamHI and NheI.
2.3. Fluorescence microscopy and FRET determination Cells were grown in medium containing 2% glucose at 30 C till exponential phase, collected by centrifugation, resuspended in 25 mM MES buffer, pH 6 (about 5 107 cells/ml) and incubated at 30 C for at least 1 h. Subsequently, 40 ml of glucose-starved cells were seeded on concanavalin A (Sigma-Aldrich, Milano, Italy)coated cover glass for 10 min [13]. The cover glass was washed four times using 1 ml of 25 mM MES buffer (pH 6), mounted on a custom chamber and covered by 500 ml of the same buffer. Time stacks of images (512 512 pixels, typical field of view 150 mm 150 mm, 400 Hz scanning frequency) were acquired before and after addition of either glucose, cAMP or H2O by means of a Leica SP5 confocal microscope (Leica BLA Germany) through a 40X oil objective (HCX PL APO CS 1.30). The pinhole was set at 150e170 mm in order to detect a higher signal from each cell and to avoid losing the focal plane in long time acquisitions. CFP was excited at 458 nm, its emission detected in the range 465e495 nm and either YFP or cpVenus emission were detected in the range 514e600 nm. Glucose was added by pipetting, directly into the chamber, 25 ml of 40% glucose dissolved in 25 mM MES buffer, pH 6. cAMP was added by pipetting, directly into the chamber, 75 ml of 20 mM cAMP dissolved in 25 mM MES buffer, pH 6. The acquisition started with non-stimulated cells and continued after the glucose or cAMP addition without interruption for 30e60 min, typically. For each sample, image time series have been acquired selecting field of view populated with more than 50 cells. After acquisition, data have been analyzed by means of the Leica Application Suite Software (Leica Microsystem, Germany). A ROI has been selected including each cell and the CFP and YFP or cp Venus fluorescence signals in the two acquisition channels have been saved together with their ratio versus time. In this way, both single cell behavior and average values have been calculated for each sample. The raw data were then further elaborated with Excel ™. Preliminary experiments were performed on a two photon scanning microscope (BX51 equipped with FV300, Olympus, Japan) modified for direct (non descanned) detection of the signal and coupled to a femtosecond Ti:sapphire laser (Mai Tai, Spectra Physics, CA) [19]. The microscope was equipped with a highly efficient objective (N.A. ¼ 0.95, 20X, water immersion, Olympus, Japan). CFP was excited at l ¼ 820 nm and the fluorescence was detected through a short-pass 670 nm filter (Chroma Inc. Brattelboro, VT) and selected by a band-pass filter at 485/30 nm (Chroma Inc. Brattelboro, VT) for the CFP channel at 560/50 (Chroma Inc. Brattelboro, VT) for the YFP channel. Time stacks of images 512pixel x 512 pixel were acquired in fast scan mode with a field of view of 250 mm 250 mm. 3. Results and discussion 3.1. Monitoring cAMP changes in a single Saccharomyces cerevisiae cell We used a FRET (Fluorescence Resonance Energy Transfer) sensor to monitor the changes in cAMP level in single S. cerevisiae cell. To avoid any interference with the cAMP/PKA signaling, we used a sensor based on the mammalian protein EPAC2 (Epac2camps), originally developed by Nikolaev et al. to monitor cAMP changes in mammalian cells. This sensor exhibits a decrease of FRET in response to cAMP concentration with a SD50 of about 1 mM and is therefore suitable for measuring the changes in cAMP in yeast [11]. The sequence coding for the sensor, a fusion between Cyan-fluorescent protein-Epac2 (aa 285e443)-Yellow fluorescent protein (CFP-EPAC2-YFP), was cloned in a yeast expression vector
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
S. Colombo et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
and expressed in different wild type strains. We used a confocal microscope system to measure in a single cell the CFP and YFP fluorescence and the relative FRET efficiency was determined from the YFP/CFP fluorescence ratio. Since in the presence of cAMP the FRET decreases, we measured the reverse ratio (i.e.CFP/YFP fluorescence ratio) as an indication of intracellular cAMP levels. Preliminary experiments were done using a two-photon microscope (see Materials and Methods for details). Unfortunately, with this systems we observed a relevant bleaching of the fluorescence and we were able to follow the behavior for a limited time (5e10 min). In addition, an high level of background noise was present that strongly limited the quality of the signals (data not shown). A significant improvement was obtained using a Leica Confocal inverted microscope, using a low magnification in order to reduce the bleaching and to capture images of many cells in the same frame (up to 50 cell); an example is shown in Supplementary Fig. S1. Using this systems we could measure the CFP and YFP fluorescence for a long time in many single yeast cells without any apparent decrease of the fluorescence signal, moreover the CFP/YFP fluorescence ratio was almost unchanged in living SP1 yeast cells left in buffer (Supplementary Fig. S2). Our results show that in all the strains analyzed the CFP/YFP fluorescence ratio increases immediately after addition of glucose to derepressed yeast cells, although with a slightly different kinetics. In Fig. 1A we report an example of the changes in CFP/YFP fluorescence ratio in single yeast cells of wild type SP1 strain. Each cell responds in a slightly different way, although a first peak of CFP/YFP fluorescence ratio is clearly detectable after glucose addition, followed by an increase of ratio that last for long times, suggesting that cAMP levels remain high. A similar fast increase of CFP/YFP fluorescence ratio was detected in other wild type strains (W303-1A and X4004) (Fig. 2 B,C). A relevant noise is still present, likely related to the low amount of fluorescence presents in yeast cells, in comparison with that obtained with the same probe in mammalian cells [11]. However, the noise can be reduced by filtering the raw data as shown in Fig. 1C,D, where the results for different SP1 single cells are reported. After the first peak, that lasts about 100e120 s in some cells, damped noisy oscillations are evident especially after a smoothing (using a moving average) of the raw data. Interestingly, the noise can be reduced also by averaging the CFP/YFP fluorescence ratio of many cells (up to 15), as shown in Fig. 1 B. However, in this case after the first peak of cAMP, no oscillations were detectable, since the different cells respond in a rather asynchronous way. To test the specificity of the signal, we used the GG104 strain (cyr1D pde2D msn2D msn4D). This mutant strain bears a deletion of the gene coding for adenylate cyclase (CYR1) and therefore is not able to produce cAMP, but the PKA can be activated by the addition of extracellular cAMP [17,20]. As expected, we did not observe relevant changes in the CFP/YFP fluorescence ratio when glucose was added to this strain (Fig. 2A). Taken together, these data indicate that the CFP-EPAC2-YFP probe is a useful live-cell biosensor for monitoring cAMP levels in S. cerevisiae cells. With this method it is possible to monitor changes in cAMP in single cells for a long time (up to 1 h) and to put in evidence individual variability of response to a stimulus. In addition, as predicted by a model of the Ras/cAMP pathway developed in our laboratory [10], we were able to give experimental evidence of the presence of damped oscillations in cAMP levels after stimulation of derepressed cells with glucose. 3.2. Detection of PKA activity in a single yeast cell, using a FRETbased A-kinase activity reporter To monitor changes in PKA activity in single S. cerevisiae cells,
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Fig. 1. CFP/YFP fluorescence ratio in single starved SP1 yeast cells expressing the Epac2camps probe after addition of glucose. The arrows indicate the time of glucose addition; images of the cells were taken every 15 s. The relative fluorescence ratio was normalized to the value of one for cells before the glucose addition. A) The signals of three single yeast cells are reported; B) the mean of the relative fluorescence ratio of 15 single yeast cells is reported; C and D) the raw signals of two yeast cells (blue lines with small squares) are reported. The red thick line represents the moving average values (n ¼ 4) of the raw data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
we used a FRET-based A-kinase activity reporter (AKAR), originally developed by Allen and Zhang to monitor PKA activity in mammalian cells [12]. The sequence coding for the sensor, a fusion between Cyan-fluorescent protein-AKAR3-cpVenus fluorescent protein (CFP-AKAR3-cpVenus), was cloned in a yeast expression vector and expressed in two different wild type strains, SP1 and W303-1A. We used a Leica confocal inverted microscope system to measure in a single cell the CFP and cpVenus fluorescence and the relative FRET efficiency was determined from the cpVenus/CFP fluorescence ratio on low magnification images, as shown before
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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Fig. 2. CFP/YFP fluorescence ratio in single starved yeast cells of different strains expressing the Epac2-camps probe after addition of glucose. The arrows indicate the time of glucose addition; images of the cells were taken every 10 s. The relative fluorescence ratio was normalized to the value of one for cells before the glucose addition. A) Relative fluorescence ratio for a single GG104 cell bearing a deletion of adenylate cyclase (cyr1D pde2D msn2D msn4D); B and C) raw signals (blue lines) for a single W303-1A and X4004 cell. The red line represents a smoothing (moving average n ¼ 8) of the raw data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for cAMP detection. Our results show that in a wild type strain the FRET increases after addition of glucose to derepressed yeast cells (Fig. 3A). Also with this probe, a substantial noise was detected but after smoothing of the raw data the increase is well apparent and sustained in time (Fig. 3A). In this case the presence of damped oscillation is less apparent (if any), but this may be related to the different biochemical mechanism that originates the FRET signal: in the Epac probe the association/dissociation of cAMP are very fast [11], while in the AKAR probe a dephosphorylation is required to reduce the FRET signal, with the intervention of protein phosphatases and this may require some additional time and may not allow to follow fast kinetic changes. The noise can be reduced also by averaging the fluorescence ratio on several cells, as shown in Fig. 3B. To test the specificity of the probe we used again the GG104
strain (cyr1D pde2D msn2D msn4D), previously described. As expected, we did not observe changes in PKA activity when glucose was added to this strain, which is not able to produce cAMP (Fig. 3C), while the cpVenus/CFP fluorescence ratio increased immediately after addition of cAMP directly to the cell culture (Fig. 3D). A comparable result was obtained using a strain with absent PKA activity (tpk1D tpk2D tpk3D) [18]. As expected, we did not observe changes in PKA activity when glucose was added to this strain (Fig. 3E). Taken together, these data indicate that the CFP-AKAR3cpVenus probe is a useful live-cell biosensor for monitoring PKA activity in S. cerevisiae. In our opinion, this is a relevant point, since it is not an easy task to monitor in vivo the PKA activity, also in cell population. At present, the most used way is to measure the activity
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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Acknowledgements We thank Dr. V.O. Nikolaev, University of Wuerzburg, Germany for providing the pcDNA3- YFP-EPAC2-CFP construct and Dr. Jin Zhang, The Johns Hopkins University, Baltimore, USA for providing the pcDNA3- AKAR3 construct. This work was supported by FAR grants to E.M. and S.C., by Program Sys-BioNet, Italian Roadmap Research Infrastructure 2012 grant to S.C. and by fundings from University of Milano-Bicocca to M.C. and G.C. (“large infrastructures 201100 and “unimib competitive grant”). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.097. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.097. References
Fig. 3. cpVenus/CFP fluorescence ratio in single yeast cells expressing the AKAR3 probe. The arrows indicate the addition of either glucose (in A,B,C and E) or cAMP (in D). Images were taken every 4 s. The relative fluorescence ratio was normalized to the value of one for cells before the glucose or cAMP addition. The blue lines report the raw data, while the red lines represent a smoothing (moving average n ¼ 16) of the raw data. A) Signal of a single SP1 cells in response to glucose addition; (B) mean value of 9 single SP1 cells; (C) signal of a single GG104 cell (cyr1D pde2D msn2D msn4D) in response to glucose addition or D) to cAMP addition; E) signal of a single tpk1D tpk2D tpk3D cell in response to glucose addition. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of threalase as an indication of PKA activation [21], that however is an indirect way and may give experimental bias.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes Sonia Colombo a, c, Serena Broggi a, d, Maddalena Collini b, Laura D'Alfonso b, Giuseppe Chirico b, Enzo Martegani a, c, * a
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy Department of Physics, University of Milano-Bicocca, Milan, Italy SysBio Centre of Systems Biology, Piazza della Scienza 2, I-20126 Milan, Italy d S.C. di Ematologia e Trapianto di Midollo Osseo, Ospedale Santa Maria della Misericordia, S. Andrea delle Fratte Perugia, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 April 2017 Accepted 18 April 2017 Available online xxx
In Saccharomyces cerevisiae the second messenger cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) play a central role in metabolism regulation, stress resistance and cell cycle progression. To monitor cAMP levels and PKA activity in vivo in single S. cerevisiae cells, we expressed an Epac-based FRET probe and a FRET-based A-kinase activity reporter, which were proven to be useful live-cell biosensors for cAMP levels and PKA activity in mammalian cells. Regarding detection of cAMP in single yeast cells, we show that in wild type strains the CFP/YFP fluorescence ratio increased immediately after glucose addition to derepressed cells, while no changes were observed when glucose was added to a strain that is not able to produce cAMP. In addition, we had evidence for damped oscillations in cAMP levels at least in SP1 strain. Regarding detection of PKA activity, we show that in wild type strains the FRET increased after glucose addition to derepressed cells, while no changes were observed when glucose was added to either a strain that is not able to produce cAMP or to a strain with absent PKA activity. Taken together these probes are useful to follow activation of the cAMP/PKA pathway in single yeast cells and for long times (up to one hour). © 2017 Elsevier Inc. All rights reserved.
Keywords: Cyclic AMP Protein kinase activity Fluorescence resonance energy transfer Live cell imaging Yeast
1. Introduction In the yeast Saccharomyces cerevisiae, the cAMP/PKA pathway plays an important role in the control of metabolism, stress resistance, proliferation and it also affects morphogenesis and development, including pseudohyphal, invasive growth and sporulation [1e5]. The central component of this pathway is adenylate cyclase whose activity is controlled by two G-protein systems, the Ras proteins and the Ga protein Gpa2 [6,7]. Two triggers are known to activate the cAMP/PKA pathway: the addition of glucose to derepressed cells and intracellular acidification. Cyclic AMP is synthesized by adenylate cyclase, encoded by
* Corresponding author. Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy. E-mail address: [email protected] (E. Martegani).
CYR1 gene, and induces the activation of the cAMP-dependent protein kinase A (PKA). In turn, PKA phosphorylates a variety of proteins involved in key cellular processes. The whole signaling cascade is tightly regulated and experimental evidences indicate that multiple feedback mechanisms operate within the pathway by the generation of a complex interplay between the cascade components [8e10]. Ras proteins are positively controlled by the activity of Cdc25, that stimulates the GDP-GTP exchange, and negatively regulated by Ira1 and Ira2, that stimulate the GTPase activity of Ras. The inactivation of cAMP is governed by phosphodiesterases that constitute the major feedback mechanism in the pathway [8], although Colombo et al. [9] demonstrated that the feedback inhibition mechanism acts also by changing the Ras2 proteins activation state. A basal level of cAMP is required for growth, while a transient increase of cAMP induced by addition of glucose is required for transition from respiratory to fermentative metabolism. Although the cAMP/PKA pathway has been extensively studied in yeast and both upstream and downstream
http://dx.doi.org/10.1016/j.bbrc.2017.04.097 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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elements are known, the changes in cAMP and in the activity of this pathway were measured in cell populations and usually for a very short time and data on the spatiotemporal variation of cAMP and PKA activity in single cells are till now lacking. Some years ago Nikolaev et al. developed an Epac-based FRET probes for monitoring cAMP levels in vivo in single mammalian cells [11]. These sensor consist of part of the cAMP-binding protein Epac1 or Epac2 sandwiched between cyan and yellow fluorescent proteins(CFP and YFP). The construct unfolds upon binding of the second messenger cAMP to the Epac moiety and cAMP increases are thus easily followed as a drop in FRET. A couple of years later, Allen and Zhang developed a FRET-based A-kinase activity reporter (AKAR), AKAR3, for monitoring PKA activity in vivo in single mammalian cells [12]. AKAR is a recombinant protein composed of a phosphoamino acid binding domain and a PKA-specific substrate sandwiched between CFP and cp-Venus fluorescent protein. When phosphorylated by PKA, intramolecular binding of the substrate by the phosphoamino acid binding domain drives a conformational reorganization, leading to an increase in FRET between CFP and cpVenus. Since key cellular processes, including components and mechanisms used for signal transduction, are conserved between human and yeast, methodologies setups and successfully used in mammalian cells might work in yeast as well. In this regard, we recently studied the localization of Ras-GTP in vivo in single S. cerevisiae cells by expressing a probe consisting of a GFP fusion with a trimeric Ras Binding Domain of Raf1 (eGFP-RBD3) [13], which was proven to be a useful live-cell biosensor for Ras-GTP in mammalian cells [14]. Aim of this work was to develop and test FRET probes for monitoring cAMP levels and PKA activity in vivo in single S. cerevisiae cells, starting from methodologies setups and successfully used in mammalian cells. 2. Materials and methods 2.1. Yeast strains and media Strains used in this study: W303-1A (MATa ade2-1 can1-100 his3-11,15 leu2-3112 trp1-1 ura3-1) [15]; X4004-3A (MATa lys5 met2 ura3 trp1); SP1 (MATa his3 leu2 ura3 trp1 ade8 can1) [16]; GG104 (MATa W303-1A cdc35::KanMX pde2::TRP1 msn2::HIS3 msn4::TRP1) [17]; ASY62 (MATa SP1 tpkl::ADE8 tpk2::HIS3 tpk3::TRPl msn2::HIS3 msn4::LEU2) [18]. Synthetic complete media (SD) contained 2% glucose, 6.7 g/l YNB w/o aminoacids (supplied by ForMedium™, United Kingdom) and the proper selective drop-out CSM (Complete Synthetic Medium, supplied by ForMedium™, United Kingdom). Culture density was measured with a Coulter Counter (Coulter mod. Z2) on mildly sonicated samples. 2.2. Plasmids To obtain the pYX212-YFP-EPAC2-CFP and the pYX242-YFPEPAC2-CFP vectors we used the following strategy. The YFP-EPAC2CFP fragment, obtained digesting the pcDNA3- YFP-EPAC2-CFP construct (kindly provided by Dr. V.O. Nikolaev, University of Wuerzburg, Germany) with XhoI and HindIII, was ligated into the expression vectors, pYX212 and pYX242, digested with the same enzymes. To obtain the pYX212-AKAR3 vector we used the following strategy. The CFP-AKAR3-cpVenus fragment, obtained digesting the pcDNA3- AKAR3 construct (kindly provided by Dr. Jin Zhang, The Johns Hopkins University, Baltimore, USA) with BamHI and XbaI, was ligated into the expression vector pYX212 digested with BamHI and NheI.
2.3. Fluorescence microscopy and FRET determination Cells were grown in medium containing 2% glucose at 30 C till exponential phase, collected by centrifugation, resuspended in 25 mM MES buffer, pH 6 (about 5 107 cells/ml) and incubated at 30 C for at least 1 h. Subsequently, 40 ml of glucose-starved cells were seeded on concanavalin A (Sigma-Aldrich, Milano, Italy)coated cover glass for 10 min [13]. The cover glass was washed four times using 1 ml of 25 mM MES buffer (pH 6), mounted on a custom chamber and covered by 500 ml of the same buffer. Time stacks of images (512 512 pixels, typical field of view 150 mm 150 mm, 400 Hz scanning frequency) were acquired before and after addition of either glucose, cAMP or H2O by means of a Leica SP5 confocal microscope (Leica BLA Germany) through a 40X oil objective (HCX PL APO CS 1.30). The pinhole was set at 150e170 mm in order to detect a higher signal from each cell and to avoid losing the focal plane in long time acquisitions. CFP was excited at 458 nm, its emission detected in the range 465e495 nm and either YFP or cpVenus emission were detected in the range 514e600 nm. Glucose was added by pipetting, directly into the chamber, 25 ml of 40% glucose dissolved in 25 mM MES buffer, pH 6. cAMP was added by pipetting, directly into the chamber, 75 ml of 20 mM cAMP dissolved in 25 mM MES buffer, pH 6. The acquisition started with non-stimulated cells and continued after the glucose or cAMP addition without interruption for 30e60 min, typically. For each sample, image time series have been acquired selecting field of view populated with more than 50 cells. After acquisition, data have been analyzed by means of the Leica Application Suite Software (Leica Microsystem, Germany). A ROI has been selected including each cell and the CFP and YFP or cp Venus fluorescence signals in the two acquisition channels have been saved together with their ratio versus time. In this way, both single cell behavior and average values have been calculated for each sample. The raw data were then further elaborated with Excel ™. Preliminary experiments were performed on a two photon scanning microscope (BX51 equipped with FV300, Olympus, Japan) modified for direct (non descanned) detection of the signal and coupled to a femtosecond Ti:sapphire laser (Mai Tai, Spectra Physics, CA) [19]. The microscope was equipped with a highly efficient objective (N.A. ¼ 0.95, 20X, water immersion, Olympus, Japan). CFP was excited at l ¼ 820 nm and the fluorescence was detected through a short-pass 670 nm filter (Chroma Inc. Brattelboro, VT) and selected by a band-pass filter at 485/30 nm (Chroma Inc. Brattelboro, VT) for the CFP channel at 560/50 (Chroma Inc. Brattelboro, VT) for the YFP channel. Time stacks of images 512pixel x 512 pixel were acquired in fast scan mode with a field of view of 250 mm 250 mm. 3. Results and discussion 3.1. Monitoring cAMP changes in a single Saccharomyces cerevisiae cell We used a FRET (Fluorescence Resonance Energy Transfer) sensor to monitor the changes in cAMP level in single S. cerevisiae cell. To avoid any interference with the cAMP/PKA signaling, we used a sensor based on the mammalian protein EPAC2 (Epac2camps), originally developed by Nikolaev et al. to monitor cAMP changes in mammalian cells. This sensor exhibits a decrease of FRET in response to cAMP concentration with a SD50 of about 1 mM and is therefore suitable for measuring the changes in cAMP in yeast [11]. The sequence coding for the sensor, a fusion between Cyan-fluorescent protein-Epac2 (aa 285e443)-Yellow fluorescent protein (CFP-EPAC2-YFP), was cloned in a yeast expression vector
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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and expressed in different wild type strains. We used a confocal microscope system to measure in a single cell the CFP and YFP fluorescence and the relative FRET efficiency was determined from the YFP/CFP fluorescence ratio. Since in the presence of cAMP the FRET decreases, we measured the reverse ratio (i.e.CFP/YFP fluorescence ratio) as an indication of intracellular cAMP levels. Preliminary experiments were done using a two-photon microscope (see Materials and Methods for details). Unfortunately, with this systems we observed a relevant bleaching of the fluorescence and we were able to follow the behavior for a limited time (5e10 min). In addition, an high level of background noise was present that strongly limited the quality of the signals (data not shown). A significant improvement was obtained using a Leica Confocal inverted microscope, using a low magnification in order to reduce the bleaching and to capture images of many cells in the same frame (up to 50 cell); an example is shown in Supplementary Fig. S1. Using this systems we could measure the CFP and YFP fluorescence for a long time in many single yeast cells without any apparent decrease of the fluorescence signal, moreover the CFP/YFP fluorescence ratio was almost unchanged in living SP1 yeast cells left in buffer (Supplementary Fig. S2). Our results show that in all the strains analyzed the CFP/YFP fluorescence ratio increases immediately after addition of glucose to derepressed yeast cells, although with a slightly different kinetics. In Fig. 1A we report an example of the changes in CFP/YFP fluorescence ratio in single yeast cells of wild type SP1 strain. Each cell responds in a slightly different way, although a first peak of CFP/YFP fluorescence ratio is clearly detectable after glucose addition, followed by an increase of ratio that last for long times, suggesting that cAMP levels remain high. A similar fast increase of CFP/YFP fluorescence ratio was detected in other wild type strains (W303-1A and X4004) (Fig. 2 B,C). A relevant noise is still present, likely related to the low amount of fluorescence presents in yeast cells, in comparison with that obtained with the same probe in mammalian cells [11]. However, the noise can be reduced by filtering the raw data as shown in Fig. 1C,D, where the results for different SP1 single cells are reported. After the first peak, that lasts about 100e120 s in some cells, damped noisy oscillations are evident especially after a smoothing (using a moving average) of the raw data. Interestingly, the noise can be reduced also by averaging the CFP/YFP fluorescence ratio of many cells (up to 15), as shown in Fig. 1 B. However, in this case after the first peak of cAMP, no oscillations were detectable, since the different cells respond in a rather asynchronous way. To test the specificity of the signal, we used the GG104 strain (cyr1D pde2D msn2D msn4D). This mutant strain bears a deletion of the gene coding for adenylate cyclase (CYR1) and therefore is not able to produce cAMP, but the PKA can be activated by the addition of extracellular cAMP [17,20]. As expected, we did not observe relevant changes in the CFP/YFP fluorescence ratio when glucose was added to this strain (Fig. 2A). Taken together, these data indicate that the CFP-EPAC2-YFP probe is a useful live-cell biosensor for monitoring cAMP levels in S. cerevisiae cells. With this method it is possible to monitor changes in cAMP in single cells for a long time (up to 1 h) and to put in evidence individual variability of response to a stimulus. In addition, as predicted by a model of the Ras/cAMP pathway developed in our laboratory [10], we were able to give experimental evidence of the presence of damped oscillations in cAMP levels after stimulation of derepressed cells with glucose. 3.2. Detection of PKA activity in a single yeast cell, using a FRETbased A-kinase activity reporter To monitor changes in PKA activity in single S. cerevisiae cells,
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Fig. 1. CFP/YFP fluorescence ratio in single starved SP1 yeast cells expressing the Epac2camps probe after addition of glucose. The arrows indicate the time of glucose addition; images of the cells were taken every 15 s. The relative fluorescence ratio was normalized to the value of one for cells before the glucose addition. A) The signals of three single yeast cells are reported; B) the mean of the relative fluorescence ratio of 15 single yeast cells is reported; C and D) the raw signals of two yeast cells (blue lines with small squares) are reported. The red thick line represents the moving average values (n ¼ 4) of the raw data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
we used a FRET-based A-kinase activity reporter (AKAR), originally developed by Allen and Zhang to monitor PKA activity in mammalian cells [12]. The sequence coding for the sensor, a fusion between Cyan-fluorescent protein-AKAR3-cpVenus fluorescent protein (CFP-AKAR3-cpVenus), was cloned in a yeast expression vector and expressed in two different wild type strains, SP1 and W303-1A. We used a Leica confocal inverted microscope system to measure in a single cell the CFP and cpVenus fluorescence and the relative FRET efficiency was determined from the cpVenus/CFP fluorescence ratio on low magnification images, as shown before
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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Fig. 2. CFP/YFP fluorescence ratio in single starved yeast cells of different strains expressing the Epac2-camps probe after addition of glucose. The arrows indicate the time of glucose addition; images of the cells were taken every 10 s. The relative fluorescence ratio was normalized to the value of one for cells before the glucose addition. A) Relative fluorescence ratio for a single GG104 cell bearing a deletion of adenylate cyclase (cyr1D pde2D msn2D msn4D); B and C) raw signals (blue lines) for a single W303-1A and X4004 cell. The red line represents a smoothing (moving average n ¼ 8) of the raw data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
for cAMP detection. Our results show that in a wild type strain the FRET increases after addition of glucose to derepressed yeast cells (Fig. 3A). Also with this probe, a substantial noise was detected but after smoothing of the raw data the increase is well apparent and sustained in time (Fig. 3A). In this case the presence of damped oscillation is less apparent (if any), but this may be related to the different biochemical mechanism that originates the FRET signal: in the Epac probe the association/dissociation of cAMP are very fast [11], while in the AKAR probe a dephosphorylation is required to reduce the FRET signal, with the intervention of protein phosphatases and this may require some additional time and may not allow to follow fast kinetic changes. The noise can be reduced also by averaging the fluorescence ratio on several cells, as shown in Fig. 3B. To test the specificity of the probe we used again the GG104
strain (cyr1D pde2D msn2D msn4D), previously described. As expected, we did not observe changes in PKA activity when glucose was added to this strain, which is not able to produce cAMP (Fig. 3C), while the cpVenus/CFP fluorescence ratio increased immediately after addition of cAMP directly to the cell culture (Fig. 3D). A comparable result was obtained using a strain with absent PKA activity (tpk1D tpk2D tpk3D) [18]. As expected, we did not observe changes in PKA activity when glucose was added to this strain (Fig. 3E). Taken together, these data indicate that the CFP-AKAR3cpVenus probe is a useful live-cell biosensor for monitoring PKA activity in S. cerevisiae. In our opinion, this is a relevant point, since it is not an easy task to monitor in vivo the PKA activity, also in cell population. At present, the most used way is to measure the activity
Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097
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Acknowledgements We thank Dr. V.O. Nikolaev, University of Wuerzburg, Germany for providing the pcDNA3- YFP-EPAC2-CFP construct and Dr. Jin Zhang, The Johns Hopkins University, Baltimore, USA for providing the pcDNA3- AKAR3 construct. This work was supported by FAR grants to E.M. and S.C., by Program Sys-BioNet, Italian Roadmap Research Infrastructure 2012 grant to S.C. and by fundings from University of Milano-Bicocca to M.C. and G.C. (“large infrastructures 201100 and “unimib competitive grant”). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.097. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.097. References
Fig. 3. cpVenus/CFP fluorescence ratio in single yeast cells expressing the AKAR3 probe. The arrows indicate the addition of either glucose (in A,B,C and E) or cAMP (in D). Images were taken every 4 s. The relative fluorescence ratio was normalized to the value of one for cells before the glucose or cAMP addition. The blue lines report the raw data, while the red lines represent a smoothing (moving average n ¼ 16) of the raw data. A) Signal of a single SP1 cells in response to glucose addition; (B) mean value of 9 single SP1 cells; (C) signal of a single GG104 cell (cyr1D pde2D msn2D msn4D) in response to glucose addition or D) to cAMP addition; E) signal of a single tpk1D tpk2D tpk3D cell in response to glucose addition. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of threalase as an indication of PKA activation [21], that however is an indirect way and may give experimental bias.
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Please cite this article in press as: S. Colombo, et al., Detection of cAMP and of PKA activity in Saccharomyces cerevisiae single cells using Fluorescence Resonance Energy Transfer (FRET) probes, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/ 10.1016/j.bbrc.2017.04.097