Generation of a cGMP Indicator with an Expanded Dynamic Range by

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Generation of a cGMP Indicator with an Expanded Dynamic Range by Optimization of Amino Acid Linkers between a Fluorescent Protein and PDE5# Shogo Matsuda, Kazuki Harada, Motoki Ito, Mai Takizawa, Devina Wongso, Takashi Tsuboi, and TETSUYA KITAGUCHI ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00582 • Publication Date (Web): 26 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Generation of a cGMP Indicator with an Expanded Dynamic Range by Optimization of Amino Acid Linkers between a Fluorescent Protein and PDE5α Shogo Matsuda,†,a Kazuki Harada,‡,a Motoki Ito,† Mai Takizawa,‡ Devina Wongso,§ Takashi Tsuboi,†,‡,* and Tetsuya Kitaguchi§,#,* †

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan, ‡Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan, §Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABIOS), 11 Biopolis Way #05-02 Helios, Singapore 138667, Singapore, #Comprehensive Research Organization, Waseda University, #304, Block 120-4, 513 Wasedatsurumaki-cho, Shinjuku, Tokyo 162-0041, Japan. Supporting Information Placeholder ABSTRACT: Here we describe the development of a single fluorescent protein (FP)-based cGMP indicator, Green cGull, based on the cGMP binding domain from mouse phosphodiesterase 5α. The dynamic range of Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP binding by optimization of the amino acid linkers between the cGMP binding domain and FP. Green cGull has excitation and emission peaks at 498 and 522 nm, respectively, and specifically responds to cGMP in a dose-dependent manner. Live cell imaging analysis revealed that addition of a nitric oxide (NO) donor induced different cGMP kinetics and was cell-type dependent. We also found that the NO donor induced an increase of intracellular cGMP, while intracellular Ca2+ exhibited a complex profile, as revealed by dual-color imaging of cGMP and Ca2+. The results suggest that Green cGull sheds new light on understanding the complex interactions between various signaling molecules by multi-color imaging and that our systematic strategy for expanding the dynamic range of single-FP-based indicators is valuable to generate indicators for molecules of interest. KEYWORDS: biosensors, fluorescent protein, cGMP, mutant screening, live cell imaging Cyclic 3′,5′-monophosphate (cGMP) serves as an important second messenger in a variety of physiological functions.1–4 The intracellular cGMP concentration ([cGMP]i) is strictly controlled through production by two types of guanylyl cyclases (GCs: the transmembrane, peptide hormone-activated type and the soluble, nitric oxide (NO)-activated type) and degraded by phosphodiesterases (PDEs).2 The balance between activation and expression levels of these proteins generates complex dynamics of cGMP in living cells. However, a precise spatiotemporal regulation mechanism of [cGMP]i remains largely unknown. Currently, two types of genetically-encoded fluorescent cGMP indicators have been developed for visualizing intracellular cGMP dynamics.5–11 First, Förster resonance energy transfer (FRET)based ratiometric proteins have been developed.5–8,10 Since FRET imaging requires measurement at two different wavelengths, this limits the flexibility and expandability of simultaneous imaging of the dynamics for different signaling molecules and/or subcellular compartments with multi-color imaging. To overcome this drawback, secondly single fluorescent protein (FP)-based,

intensiometric indicators have been developed. However, they suffer from small dynamic ranges.9,11 Single FP-based indicators utilize the exchange of ionization states in the chromophore of FPs. Since the fluorescence intensity of FPs critically depends on the proton environments surrounding the chromophore, an environment change caused by a conformational change in response to a specific molecule enables us to monitor the level of molecules as a change of fluorescence intensity.12 On the other hand, recent studies have revealed that linker lengths and amino acid sequences joining the FP and binding domains play a crucial role in defining the characteristics of indicators, such as turn-on/off types, ratiometric/intensiometric types and dynamic ranges.12–17 Based on our cumulative data,16,17 we aimed to establish a systematic and universal scheme to develop single FP-based indicators. In the present study, we developed a single FP-based cGMP indicator named Green cGull (cGMP visualizing fluorescent protein). Green cGull is composed of the cGMP-binding domain of cGMP-specific mouse phosphodiesterase 5α (PDE5α) inserted in the vicinity of the chromophore of a green FP variant, Citrine. Upon binding to cGMP, conformational changes to the cGMPbinding domain increase the fluorescence intensity. Because there are many physiological events accompanied by spatiotemporal dynamics of cGMP with other molecules in cells, Green cGull will be a useful and powerful tool to monitor these molecules with cGMP by multi-color imaging. We designed a genetically-encoded cGMP indicator by simply inserting a cGMP-binding domain from mouse PDE5α into a single FP, Citrine (Figure 1A). Insertion-type single FP-based indicators are composed of two separated FP fragments, a binding domain for the target molecule and two linkers that join the FP fragments and the binding domain. To obtain successful single FP-based fluorescent indicators, not only must a conformational change to the binding domain occur, but the insertion point for the binding domain must be optimal, and the linker length and character are also important. Based on our previous studies,16,17 we inserted the binding domain into Citrine at Tyr145, which is within the vicinity of the chromophore. We selected the GAFa domain of mouse PDE5α,18 because this domain exhibits a large conformational change specifically upon binding to cGMP.19,20 We first generated a prototype indicator by inserting the amino acids 164–298 of PDE5α (i.e., cGMP binding domain) with

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linkers LRG at the N-terminus and EF at the C-terminus, which were created by SacII and EcoRI recognition DNA sequences (Figure S1A). Subsequently, we created various variants, which had additional linkers or shorter fragments of PDE5α. We chose a leucine zipper NZ as a basis of the linker at the N- and C-termini based on previous studies. 16,17,21 When we first optimized the length of the N-terminus, the candidate with the LRG linker and two amino acid deletions in PDE5α showed the strongest response (N – 2; Figure S1A). Then, we used N – 2 as a template and optimized the length of the C-terminus. The candidate with the EF linker and no deletion in PDE5α was most responsive (C ± 0; Figure S1B). Interestingly, variants with high responses were located at certain length points. After optimization of the linker lengths, we introduced point mutations primarily around the Citrine-PDE5α boundaries. As a result, a variant with nine mutations exhibited the highest dynamic range, and we termed this mutant Green cGull (cGMP visualizing fluorescent protein) (Figure 1). These results demonstrate that optimal selection for linker length and amino acid sequence substantially improve dynamic ranges.

Figure 2. Spectral properties of Green cGull. (A and B) Excitation/emission (A) and absorption (B) spectra of Green cGull in the presence (solid line) and absence (dashed line) of cGMP. (C) Dose-response curves of Green cGull to cGMP (●, green line) and cAMP (■, gray line). Data are shown as means ± SD. (D) pH titration curves of Green cGull in the presence (●, solid line) and absence (○, dashed line) of cGMP.

Figure 1. Schematic drawing of Green cGull. (A) Diagrams for Citrine, mouse PDE5α and resultant Green cGull. Asterisks indicate the mutations. (B) Schematic 3D image of Green cGull bound and unbound to cGMP. Images were created using structural graphics for Citrine (PDB_3PDW), PDE5α (cGMPbound PDB_2K31 and unbound PDB_3MF0). We next investigated the in vitro properties of Green cGull using bacterially expressed proteins. Fluorescence excitation and emission spectra showed that Green cGull has excitation and emission peaks at 498 and 522 nm, respectively (Figure 2A). When added with a saturating dose of cGMP (100 µM), the fluorescence intensity of Green cGull showed a 7.5-fold increase, which is substantially higher than that of previous single FP-based cGMP indicators (Figure S2A, B).9,11,22 Citrine possesses absorption peaks at ~400 and 500 nm, and these peaks correspond to the protonated and deprotonated states of the chromophore, respectively.23 Absorption spectra of 10 µM Green cGull in the presence and absence of 1 mM cGMP revealed a decrease of the absorption peak at ~421 nm and an increase of the peak at ~500 nm (Figure 2B). These results suggest that exchange between protonated and deprotonated states of the chromophore for Green cGull upon cGMP binding results in an increase in the fluorescence intensity, as shown in Figure 2A.

Based on dose-response curves of Green cGull for cGMP and cAMP, the dissociation constant (Kd) values calculated by the Hill equation were 1.09 µM for cGMP and 1.56 mM for cAMP (Figure 2C). Hill coefficients for cGMP and cAMP were 1.00 and 1.06, respectively. Since many cGMP indicators had similar affinity (around 1 µM) and successfully monitored intracellular cGMP dynamics,7,9,11,24 Green cGull should be applicable at physiological cGMP levels and specifically react to cGMP without allosteric effects. Meanwhile, previous studies have developed several cGMP indicators with higher and lower affinities, and these sensors can be used to observe various physiological events with different reaction thresholds for cGMP.10,24 Thus, Green cGull with different affinities would be useful for further extensive studies. pH titration curves for Green cGull in the presence and absence of 100 µM cGMP showed an increase in the fluorescence intensity according to pH elevation (Figure 2D). pH sensitivity is a common characteristic of many single FP-based indicators,12,13,16,17 and thus Green cGull also needs careful analysis and evaluation during pharmacological stimulation. To examine intracellular dynamics of cGMP, we applied Snitroso-N-acetyl-D, L-penicillamine (SNAP, NO donor), 3isobutyl 1-methylxanthine (IBMX, PDE inhibitor), or 8-Br-cGMP (membrane permeable cGMP analogue) to Green cGullexpressing HEK293 cells. While application of SNAP and 8-BrcGMP induced >6-fold increase of the fluorescence intensity, IBMX had little effect (Fig. 3A, B). Consistently, previous reports using other cGMP indicators including FRET-type also displayed the stimulatory effect of IBMX on [cGMP]i only in the presence of NO donors applied beforehand.6,22 However, there are only a few reports with different cells and hypoxic HEK293 cells that show an increase in [cGMP]i in the presence of IBMX alone.25,26 Thus, the effect of PDE inhibition on [cGMP]i may vary depending on the expression levels of PDEs in different cell types and conditions. When compared with the previous cGMP

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indicator δ-FlincG, Green cGull showed a much higher response to SNAP, reflecting its high dynamic range (Figure S2C, D).

Figure 3. cGMP dynamics in Green cGull-expressing cells. (A) Sequential images of Green cGull-expressing HEK293 cells following the application of 300 µM SNAP. (B) Time courses of fluorescence intensity in Green cGull-expressing HEK293 cells in response to SNAP, IBMX and 8-Br-cGMP. Green traces show the average responses from at least three different experiments. (C) Sequential images of Green cGull-expressing GLUTag cells following the application of 300 µM SNAP. (D) Time courses of fluorescence intensity in Green cGull-expressing GLUTag, C6 and COS7 cells in response to SNAP. Green traces show the average responses from three independent experiments. Cells were stimulated 2 min after initiating image acquisition. Data are shown as means ± SD. We next investigated the effect of SNAP on three different cell lines: GLUTag (mouse enteroendocrine L cell), C6 (rat glioma), and COS7 (monkey kidney) cells. Interestingly, while application of SNAP resulted in a 1.8 ± 0.2-fold increase of fluorescence intensity in GLUTag cells, it had minimal effect on the other two cell lines (Fig. 3C, D). Because SNAP only activates soluble GCs, different responses to SNAP application would be attributed to expression levels of soluble GCs in these cell lines. Previous studies showing different expression and activity levels of soluble GCs in various cells and tissues support this concept.27,28 An advantage of single FP-based indicators is the application for dual-color imaging. Because interplay between cGMP and Ca2+ is important for many physiological events,2,29–31 we attempted to simultaneously monitor dynamics of both cGMP and Ca2+ in HEK293 cells, using Green cGull and red Ca2+-sensitive dye Rhod2. Application of SNAP triggered a rapid increase in fluorescence intensity of Green cGull. In contrast, the fluorescence intensity of Rhod2 exhibited a complex profile for mixture of a transient decrease and a gradual increase (Figure 4A, B). The complex Ca2+ dynamics may reflect the involvement of

cGMP in many pathways related to intracellular Ca2+ concentration ([Ca2+]i). For instance, cGMP activates Ca2+ATPase pumping and opens Ca2+-activated K+ channels for decrease of [Ca2+]i. On the other hand, cGMP activates cyclic nucleotide-gated (CNG) channels and ryanodine receptors for increase of [Ca2+]i.32 When we removed extracellular Ca2+, increase of fluorescence intensity for Rhod2 by SNAP was significantly suppressed during [cGMP]i elevation (Figure 4C, D). These results suggest that the increase of [Ca2+]i by SNAP resulted from the Ca2+ influx from extracellular space caused by [cGMP]i elevation. Interestingly, the increase of fluorescence intensity for Green cGull by SNAP was significantly enhanced by removal of extracellular Ca2+. This result could be explained by the activity of soluble GCs and PDE1, which are modulated by Ca2+ negatively and positively, respectively. Meanwhile, Rhod2 slightly accumulates in mitochondria,33 thus the complex profile may also reflect different [Ca2+]i kinetics between cytosol and mitochondria.

Figure 4. Dual-color imaging of [cGMP]i and [Ca2+]i in HEK293 cells. (A) Sequential images of Rhod2-loaded and Green cGullexpressing HEK293 cells following the application of 400 µM SNAP. These images are representative images derived from three different experiments. (B, C) Time courses of fluorescence intensities in response to SNAP (B), and those under the depletion of extracellular Ca2+ by EGTA (C). Green and red traces show the average responses. Cells were stimulated 1 min after initiating image acquisition. (D) Comparison of the maximum fluorescence intensity in Green cGull and Rhod2. N ≥ 5 cells from three different experiments. Data are shown as means ± SD. **, P < 0.01; ****, P < 0.0001. In conclusion, we successfully developed an insertion-type single FP-based cGMP indicator using amino acid linker insertions and mutations. With this simple but systematic strategy, further single FP-based sensors with different colors, or for other signaling molecules, will be developed easily to broaden our perspective for multi-color imaging in living cells.

ASSOCIATED CONTENT Supporting Information Experimental procedures and comparison of dynamic ranges between Green cGull and a previous genetically encoded cGMP indicator δ-FlincG (PDF).

AUTHOR INFORMATION Corresponding Author * Tetsuya Kitaguchi, Ph.D. E-mail: [email protected] * Takashi Tsuboi, Ph.D. E-mail: [email protected]

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a

These authors contributed equally.

Notes The authors declare no competing financial interests.

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ACKNOWLEDGMENTS We thank Dr. Daniel Drucker for kindly providing GLUTag cells and Daryl Koh for technical assistance. This work was partly supported by a Grant-in-aid for Scientific Research (26460289 for TT and 16K01922 for TK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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