Development and application of fluorescent protein-based indicators ...

Journal of Oral Biosciences 57 (2015) 54–60

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Journal of Oral Biosciences journal homepage: www.elsevier.com/locate/job

Review

Development and application of fluorescent protein-based indicators for live cell imaging Akihiko Tanimura n Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 15 January 2015 Received in revised form 30 January 2015 Accepted 2 February 2015 Available online 7 April 2015

Background: Developments in fluorescent molecular imaging techniques have enabled characterization of the localization and dynamics of particular molecules at the cellular and subcellular levels. Highlight: Advances in imaging devices (fluorescence microscopy) and fluorescent indicators have contributed to the development of fluorescence imaging techniques. In particular, cloning of green fluorescent protein from the bioluminescent jellyfish Aequorea victoria and the subsequent development of other fluorescent proteins have revolutionized imaging technologies and accelerated their application in molecular and cell biological studies. There are two types of genetically encoded fluorescent indicators, namely, fluorescence resonance energy transfer-based ratiometric indicators and single fluorescent protein (FP)-based intensiometric indicators. The principal advantages of FP-based indicators are their superior sensitivities and specificities for molecular and physiological events, which are achieved by the incorporation of naturally evolved protein sensor domains. Consequently, numerous fluorescent indicators that enable monitoring of intracellular signaling, enzyme activities, apoptosis, cell cycle progression, and other cellular events have been developed. FP-based indicators can be expressed in specific cells in a temporally controlled manner; hence, they are favorable for long-term in vitro and in vivo experiments. Conclusion: The advantages of FP-based indicators extend their application to understanding the functions of particular molecules and cellular events during physiological responses in live animals. & 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Keywords: Fluorescent protein Imaging Fluorescence resonance energy transfer Intracellular messenger Genetically encoded fluorescent indicator

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Fluorescent proteins . . . . . . . . . . . . . . . . . . . . 3. Fluorescent protein-based indicators . . . . . . . 4. Genetically encoded calcium indicators . . . . . 5. Genetically encoded fluorescent indicators of 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approvals . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Abbreviations: CaM, calmodulin; cAMP, cyclic AMP; CFP, cyan fluorescent protein; Epac, exchange protein activated by cAMP; FP, fluorescent protein; FRET, fluorescence resonance energy transfer; GECI, genetically encoded Ca2 þ indicator; GFP, green fluorescent protein; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; M13, CaM-binding domain of myosin light chain kinase; PKA, protein kinase A; PLC, phospholipase C; YFP, yellow fluorescent protein n Tel.: þ 81 133 23 2431; fax: þ 81 133 23 1399. E-mail address: [email protected]

Fluorescent molecular imaging can be used to visualize biological processes in live cells, tissues, and animals. This method enables characterization of the localization and dynamics of particular molecules at the cellular and subcellular levels, and has become an indispensable technique for understanding many biological processes [1–3]. The development of sensitive imaging

http://dx.doi.org/10.1016/j.job.2015.02.006 1349-0079/& 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

A. Tanimura / Journal of Oral Biosciences 57 (2015) 54–60

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Fig. 1. Live cell imaging of YFP-STIM1 in HSY-EA1, a human parotid ductal cell line. HSY-EA1 cells expressing YFP-STIM1 were stimulated with 10 mM ATP in Ca2 þ -free medium. The YFP signals were obtained at 5- s intervals via confocal laser scanning microscopy. The images shown are 160 s (A), 180 s (B), 340 s (C), and 440 s (D) after the stimulation. The smaller images show the area indicated in panel A at 10- s intervals. Scale bar: 10 mm.

Fig. 2. The structure of GFP. The overall structure of the β-barrel of GFP is shown from the side (A) and bottom (B). The chromophore is shown in a space-filling representation. Data from Protein Data Bank Japan (PDBj).

devices such as confocal laser scanning and two-photon fluorescence excitation microscopes has contributed to recent advances in fluorescence imaging. In addition, the generation of optimized image detectors such as cooled charge-coupled cameras, electron multiplying charge-coupled cameras, and complementary metal oxide semiconductors has aided the evolution of conventional fluorescence microscopy to total internal reflection microscopy, which can be used for single molecule imaging. More recently, photoactivated localization microscopy techniques have been developed for super-resolution imaging [3]. Improvements in fluorescent indicators have also been crucial to the development of effective fluorescence imaging techniques. In the 1990s, fluorescent indicators were developed mainly using organic chemistry; thus, the targets of imaging studies were limited by the scope of the available fluorescent indicators such as Fura-2, which detects Ca2 þ [4]. However, the discovery and subsequent cloning of green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria revolutionized the visualization of biological systems [1,2,5]. One of the most important and attractive features of GFP as a biological marker is that it generates fluorescence in the absence of additional cofactors in a variety of species, including vertebrates, invertebrates, plants, fungi, and bacteria [3,6]. The simplest application of GFP is as a passive marker fused to a target protein of interest, which enables visualization of its spatiotemporal distribution in live cells (Fig. 1). In addition, molecular biology-based engineering of fluorescent proteins (FPs), such as circular permutations and fluorescence resonance energy transfer

(FRET) between two differently colored FPs, has allowed researchers to design and create a variety of fluorescent indicators capable of detecting ions, intracellular messengers, phosphorylation, cell cycle stages, cell death, and other molecules or events [1–3,7–9]. This review focuses on FP-based fluorescent indicators that detect concentrations of intracellular messenger molecules.

2. Fluorescent proteins GFP from the jellyfish A. victoria comprises 238 amino acids and has a cylindrical structure with a diameter of 30 Å and a length of 40 Å. The fluorophore of GFP consists of residues Ser65, dehydroTyr66, and Gly67, which form a tripeptide located in the geometric center of the cylinder (Fig. 2). This folding motif, which contains an alpha helix inside a beta sheet structure, was named the beta-can [3,10]. Considerable efforts have been made to develop GFP variants with altered excitation and emission wavelengths, enhanced brightness, and improved pH resistance [11,12]. The growing popularity of GFP and its variants prompted researchers to search for new alternatives and resulted in the discovery and cloning of a number of distinct FPs from marine organisms [3]. FPs can be applied to a wide variety of studies related to various aspects of biological systems, and the range of these applications is expanding continuously. Placing FPs under the control of a promoter of interest allows the characterization of promoter activities in specific gen-

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Fig. 3. The basic design of FRET-based fluorescent indicators. (A) Intermolecular FRET-based indicators. Two different proteins (x and y) are fused with a donor or acceptor FP. Binding of x and y increases the FRET efficiency. (B, C) Intramolecular FRET-based indicators. A conformationally responsive sensor domain (B) or a cleavable linker (C) is fused between a FRET pair. (B) Changes in the conformation of the sensor domain can increase or decrease FRET efficiency. (C) Cleavage of the linker causes FRET to disappear.

etic environments, cells or tissues, as well as their responses to external influences [3]. Fusion with an FP does not usually affect the proper localization or function of proteins; therefore, FP fusion constructs can be used to study the subcellular localization of proteins of interest as well as their real-time expression, translocation, interaction, and degradation in living systems.

3. Fluorescent protein-based indicators FPs represent a unique basis for the development of genetically encoded fluorescent indicators. FP-based indicators have several advantages over chemically synthesized fluorescent indicators such as their superior sensitivity and specificity for molecular and physiological events; these features are achieved by the incorporation of naturally evolved protein sensor domains [2]. FP-based indicators are delivered to the target cell or tissue as genetic material and are produced either transiently or stably by cells; hence, they are not liable to suffer from leakage and are particularly suitable for long-term experiments. In addition, the use of FPs located downstream of tissue- or stage-specific inducible promoters enables researchers to drive the selective expression of fluorescent indicators in a cell-type specific or temporally controlled manner [3]. Two types of genetically encoded fluorescent indicators have been developed, namely FRET-based ratiometric indicators and single FP-based intensiometric indicators. The ratiometric response is independent of the indicator concentration, which is an important advantage of FRET-based indicators. On the other hand, single FP-based indicators are advantageous for multi-color imaging in combination with other fluorescent indicators because they require measurement at only a single wavelength. FRET is the distance- and orientation-dependent radiationless transfer of excitation energy from a donor fluorophore to an acceptor chromophore [2,7]. FRET is detected by quenching of the donor emission and/or sensitized emission from the acceptor. Thus, molecular interactions between proteins fused with different colored FPs can be detected by changes in the fluorescence ratio of the donor and acceptor (Fig. 3). In addition, fusion of two differently colored FPs at an appropriate site of a target molecule

enables monitoring of changes in the molecular structure. More efficient energy transfer is achieved by a greater extent of the spectral overlap between the donor emission and acceptor excitation profiles; based on this requirement, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) are typically selected as a suitable pair of FPs for FRET. In addition, GFP is fairly tolerant of a variety of dramatic structural manipulations, including (i) the genetic insertion of a second protein [13,14], (ii) circular permutation, where two portions of the polypeptide are flipped around the central site, and the original N- and C-terminus are connected with a short linker [13–15], or (iii) splitting into two polypeptide chains that are able to fold into a functional FP when brought into close proximity [16,17]. Single FP-based indicators can be generated using circularly permuted FPs containing sensor proteins inserted between the original N- and C-terminus or fused to the new N- and C-terminus (Fig. 4). Based on these properties of FPs, a number of protein-based fluorescent indicators were designed.

4. Genetically encoded calcium indicators The first FP-based FRET indicators were the cameleons; these Ca2 þ indicators comprise the Ca2 þ -binding protein calmodulin (CaM), the CaM-binding domain of myosin light chain kinase (M13), and a pair of FPs, usually enhanced blue fluorescent protein (EBFP) and enhanced GFP (EGFP), or enhanced CFP (ECFP) and enhanced YFP (EYFP) [18]. Binding of Ca2 þ to this chimeric protein causes intramolecular binding of CaM to M13 and alters the distance and orientation of the two FPs, resulting in a change in the fluorescence ratio. Improved versions of cameleons were developed by substituting EYFP with pH-stable EYFP [19]. GCaMPs and pericams are single FP-based Ca2 þ indicators that contain circularly permuted FPs with M13 and CaM fused to the new N- and C-terminus, respectively [13,20,21]. Binding of Ca2 þ to these chimeric proteins causes the M13 and CaM domains to interact and leads to an increase in fluorescence due to an interaction between the chromophore and R377 of CaM [22]. Unlike chemically synthesized Ca2 þ indicators, genetically encoded calcium indicators (GECIs) are particularly suitable for long-term imaging studies because they are not liable to suffer from leakage. In


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Fig. 4. The basic design of single FP-based fluorescent indicators. (A) A sensor domain (X) is inserted into an FP. The change in conformation of the inserted domain can modulate the fluorescence intensity of the FP. (B) Interactive proteins or peptides (x and y) are fused to the amino (N) and carboxyl (C) termini of an FP or circularly permuted FP (cpFP). The interaction between the fused domains can modulate the fluorescence intensity of the FP. In the cpFP, n0 and c0 are the original amino and carboxyl termini, respectively.

Fig. 5. Detection of acetylcholine-induced Ca2 þ responses in the submandibular gland of a live rat expressing G-GECO, a green FP-based indicator. The fluorescent images were obtained 36 h after retrograde ductal injection of an adenovirus vector expressing G-GECO. The fluorescence intensity was increased rapidly by the direct administration of acetylcholine (100 mM). (A) G-GECO fluorescence before the stimulation. (B–E) G-GECO fluorescence at 1 s (B), 2 s (C), 3 s (D), and 15 s (E) after the stimulation. Scale bar: 1 mm.

addition, GECIs can be targeted to specific cellular components such as the ER, mitochondria, nucleus, or cytoplasm [3,18]. Until recently, the use of GECIs was limited by their relatively small dynamic range; however, this limitation was overcome by recent advancements in their development. The dynamic range of cameleons can be improved by substituting EYFP with bright variants such as Citrine or Venus, or by using circularly permuted YFP [22,23]. Furthermore, ultrasensitive Ca2þ indicators, such as the yellow cameleon-Nano series of proteins, which have Kd values of 15.8, 31.2, 52.5, 64.8, 93.5, and 140.5 nM, were developed by engineering the Ca2þ -sensing domain of a GECI [24]. These enhanced GECIs have been used to monitor Ca2þ responses in salivary glands, pancreatic acinar cells [25], astrocytes [26], and neurons [27]. The potential applications of GECIs were expanded further by the development of multiple color variants of single FP-based Ca2 þ indicators with extended dynamic ranges and enhanced affinities and kinetics [28]. In conjunction with multiple FPs or indicators, color variants are beneficial for Ca2 þ imaging. In particular, R-GECO, a red FP-based indicator [29], can be used with ATeam, a FRETbased indicator, to detect Ca2 þ and ATP, respectively. In addition, virus-mediated expression of G-GECO and GCaMPs can be used to image Ca2 þ responses in the heart [30], salivary glands (Fig. 5), and neural activities in live rodents and fishes [28,31–34] Some newly developed GECIs have comparable dynamic ranges and even better sensitivities than chemically synthesized

Ca2 þ indicators. In addition, the advantages of GECIs for long-term studies and tissue- or stage-specific inductions are favorable for the visualization of Ca2 þ responses in live animals. According to this trend, chemiluminescent indicators that allow imaging without excitation light have been developed [35].

5. Genetically encoded fluorescent indicators of inositol 1,4,5trisphosphate and cyclic AMP Like Ca2þ , cyclic AMP (cAMP) and inositol 1,4,5-trisphosphate (IP3) are important intracellular messengers; however, fluorescent indicators for these molecules have not been developed using organic chemistry. However, the fusion of naturally evolved protein sensor domains to FPs allowed the generation of various indicators capable of monitoring cellular events, including the dynamics of IP3 and cAMP signaling. IP3 is the intracellular messenger that releases Ca2þ from intracellular stores via IP3 receptors (IP3Rs) [36]. Binding of agonists to G protein-coupled receptors leads to the activation of phospholipase C β (PLCβ) through interactions with G proteins (Gq and/or G11). PLC cleaves phosphatidylinositol 1,4-bisphosphate to produce diacylglycerol and the soluble signaling molecule IP3. In immune cells such as T and B lymphocytes, IP3-mediated release of Ca2 þ is linked with PLCγ through the activation of T cell or B cell receptors [37].

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Fig. 6. Simultaneous monitoring of IP3 and Ca2 þ . (A) IP3 dynamics during Ca2 þ oscillations in COS-7 and HSY-EA1 cells. Cells expressing LIBRAvIIS were loaded with Fura-2, a chemically synthesized Ca2 þ indicator, and sequentially stimulated with various concentrations of ATP. (a) COS-7 cells showed no detectable fluctuation in the intracellular concentration of IP3 ([IP3]i; black line) during ATP-induced Ca2 þ oscillations. (b) HSY-EA1 cells showed repetitive IP3 spikes (black line) during ATP-induced Ca2 þ oscillations. The red lines show the excitation ratio of Fura-2. Note that the IP3 spikes (black arrowheads) are preceded by Ca2 þ spikes (red arrowheads). Data from Tanimura et al. [42]. (B) Mechanical stimulation-induced IP3 responses in HSY-EA1 cells. The upper panels show fluorescent images (a) and normalized emission ratio (R/R0) images (b–e) of LIBRAvIII after the mechanical stimulation. The lower panels show the Fura-2 fluorescence at 380 nm excitation (f) and the normalized fluorescence ratio (F0/F) images (g–j) of Fura-2 after the mechanical stimulation. The arrowhead shows the position of the mechanical stimulation. The images were obtained before (b and g), and 1 s (c and h), 4 s (d and i), or 15 s (e and j) after the stimulation. Scale bar: 5 mm. Data from Nezu et al. [45].

Metabolism of IP3 proceeds via two distinct pathways: (1) sequential dephosphorylation to 1,4-IP2, IP1, and inositol, or (2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate, followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, IP1, and inositol. The specific recognition of IP3 among these inositol phosphates is an essential requirement for IP3 indicators. The IP3R is a naturally occurring tetrameric IP3-gated Ca2 þ release channel that selectively recognizes IP3. At least three types of IP3Rs (types 1–3) are derived from distinct genes in mammals [38,39]; IP3R binds IP3 within its N-terminal 600 amino acids, independently of tetramer formation [40]. LIBRA, the first IP3 indicator developed, comprises the IP3-binding domain of the rat type 3 IP3R (amino acid residues 1–604) fused between the wellestablished FRET pair ECFP and EYFP, and preceded by the plasma membrane-targeting signal GAP43 (Fig. 2A) [41]. Several improved variants of LIBRA were developed by introducing amino acid substitutions within the IP3-binding domains [42]. Several different groups also developed similar IP3 indicators [43,44], which

have been used to monitor IP3 dynamics during Ca2 þ oscillations [42] and Ca2 þ waves [45] (Fig. 6), and have been applied to the development of novel ligands for IP3Rs [46–48]. As with many other biosensors, the major drawback of LIBRA and other IP3 sensors is their small dynamic range. Recently, an IP3-evoked conformational change in the amino-terminal region of IP3R1 was reported; this structural information could be useful for improving the dynamic range of IP3 indicators. The intracellular messenger cAMP is produced by the activation of adenylyl cyclases through the stimulation of Gs-coupled receptors. The cellular effects of cAMP are achieved via activation of three different types of effectors [49], namely, the cAMP-dependent protein kinase PKA, cyclic nucleotide-gated channels, and exchange proteins directly activated by cAMP (Epacs). The first FP-based indicator for cAMP was created using PKA; in this indicator, the regulatory and catalytic subunits of PKA are labeled with FPs, and the cAMP-induced dissociation of these subunits leads to elimination of the FRET signal [50]. Other types of cAMP indicators were also generated using Epacs


or the cyclic nucleotide binding domains of these proteins fused between YFP and CFP (Epac1, Epac2, and Epac-camp) [51,52]. Recently, single FP-based indicators of cAMP (Flamindo and Flamindo2) were also developed by inserting linker peptides and the cyclic nucleotide binding domain of Epac1 into the YFP variant Citrine [53].

6. Conclusions The development of sophisticated light microscopy techniques and fluorescent indicators has allowed the visualization of dynamic processes in living cells. These imaging methods are particularly suitable for studying Ca2 þ signaling and have contributed to the discovery of spatially and temporally organized responses such as Ca2þ waves and oscillations [54]. This trend has been accelerated by the advent of FPs and the subsequent development of numerous fluorescent indicators over the past 10 years. These FP-based indicators enable monitoring of intracellular signaling, enzyme activities, apoptosis, cell cycle development, and other cellular processes. Although many FP-based indicators have relatively small dynamic ranges, some newly developed GECIs have comparable dynamic ranges and better sensitivities than chemically synthesized Ca2 þ indicators and should be useful for the further development of improved FP-based indicators. FP-based indicators are delivered to target cells or tissues as genetic material and are subsequently produced by cells either transiently or stably; hence, they are favorable for long-term experiments both in vitro and in vivo. This advantage of FP-based indicators extends their application and contributes to understanding the functions of particular molecules and cellular events during physiological responses in live animals.

Ethical approvals Experimental procedures performed on animals were approved by the Animal Ethics and Research Committee of the Health Sciences University of Hokkaido (approval number 20).

Conflict of interest The author has no potential conflicts of interest.

Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research (No. 23390425 to A.T.), by the “High-Tech Research Center” Project for Private Universities 2007–2012, with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a Grant-in-aid for the 2012–2013 Research Project of the Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido. References [1] Miyawaki A. Visualization of the spatial and temporal dynamics of intracellular signaling. Develop Cell 2003;4:295–305. [2] Zhang J, Campbell RE, Ting AY, Tsien RY. Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 2002;3:906–18. [3] Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 2010;90:1103–63. [4] Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2 þ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440–50.

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