Genetically encoded Ca2+ indicators; expanded affinity range, color hue and compatibility with optogenetics

Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs) such as derivatives of yellow cameleons (YCs) and GCaMPs/pericams (Miyawaki et al., 1997; Nakai et al., 2001; Nagai et al., 2001) are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photo-stability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca2+ affinity and Hill constant, have also contributed to increase the detectability of Ca2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca2+ indicators and bioluminescence-based Ca2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.


INTRODUCTION
Fluorescent protein-based indicators are invaluable tools for functional imaging of living cells and organisms. Genetically encoded calcium indicators (GECIs) such as derivatives of yellow cameleons (YCs) and GCaMPs/pericams (Miyawaki et al., 1997;Nagai et al., 2001;Nakai et al., 2001) are a highly advanced class of indicators. Continued efforts for improvement of the performance of GECIs have resulted in brighter indicators with better photostability and expanded dynamic range, thus improving the sensitivity of detection. Fine-tuning of other properties, including Ca 2+ affinity and Hill constant, have also contributed to increase the detectability of Ca 2+ dynamics. Emerging optogenetic technology has forced the spectrally compatible GECI color variants. In this opinion, we highlight the recent development of GECIs including photo-switchable Ca 2+ indicators and bioluminescence-based Ca 2+ indicator, mainly invented in our group, focusing especially on the parameters determining their performance in order to provide a guideline for the selection of appropriate GECI for a given experiment.

AFFINITY VARIANT
After the first reports regarding design concept of YCs and GCaMPs/pericams (Miyawaki et al., 1997;Nagai et al., 2001;Nakai et al., 2001), their properties have been modified in term of dynamic range of signal change, pH sensitivity and color hue, and so on. However, application of these GECIs had been still limited in certain experimental targets. One of the critical limitations of these GECIs was their relatively poor repertoire of affinity variants. Because the Ca 2+ concentration ([Ca 2+ ]) at resting state and the amplitude of [Ca 2+ ] change differ significantly within the subcellular locations, cell types, and organisms, a diverse set of affinity variants of GECIs covering dissociation constants (K d s) from nM to mM would be needed for studying a wide range of research targets.
While moderate-and low-affinity variants of YCs (K d > 0.1 μM) were developed successfully by either site-directed mutagenesis of the Ca 2+ binding domain in the indicator (Miyawaki et al., 1997) or by the rearrangement of the overall molecular structure of the indicator (Truong et al., 2001) (Table 1), there was no systematic way for engineering a high-affinity variant. In vitro analysis revealed that free calmodulin (CaM) and its binding peptide M13 had much higher Ca 2+ affinity (K d of 20 nM) than that of the CaM and M13 fusion protein linked with two amino acid linkers (K d of 80 nM) (Porumb et al., 1994). This suggested that steric hindrance might prevent efficient interaction of Ca 2+ -CaM with M13 in YCs. This possibility was examined by serial increment of the length of the linker from 2 to 5 amino acids. Flexible linkers with 3, 4, and 5 amino acids yielded K d s of 60 nM, 30 nM, and 15 nM, respectively. Linker elongation also worked for YC 3.60, yielding five YC variants covering K d s from 15 nM to 140 nM ( Table 1). These affinity variants of YCs called YC-Nano showed increased sensitivity and could detect subtle changes in [Ca 2+ ] in pyramidal neurons Yamada et al., 2011) (Table 1), becoming an ideal toolbox to efficiently monitor the novel Ca 2+ dynamics in cases where the concentration range of Ca 2+ is poorly described ( Table 1). Recent identification of Ca 2+ twinkle, which is a localized Ca 2+ transient in the fine astrocytic processes, is one of the examples (Kanemaru et al., 2014).
So far, high-affinity variants of the GCaMP and pericam families are not available (smallest K d of 160 nM for B-GECO) (Zhao et al., 2011) (Table 1). Although these single FP-based Ca 2+ indicators have distinct structural design unlike YCs, it will be interesting to examine whether elongation of linkers, which connect the sensor modules, contributes to increased Ca 2+ affinity, as in the case of YC-Nano.

POSSIBLE SIDE EFFECTS OF GECI
One might worry that the strong Ca 2+ chelating effect of YC-Nano would affect endogenous Ca 2+ homeostasis. Depletion of target molecules or ions by loaded indicators is often problematic for imaging of non-buffered signaling molecules, such as cyclic nucleotides and NO, but this is not the case for Ca 2+  dynamically through the balancing action of Ca 2+ buffers (i.e., Ca 2+ binding proteins), which exist in abundance within the cell. Of course, excessive loading of Ca 2+ indicator/chelator beyond the buffering capacity of these buffers does affect cellular Ca 2+ homeostasis. In cases where more than mM concentrations of EGTA were loaded to observe subcellularly localized Ca 2+ , Ca 2+ puffs and blips were generated (Cheng and Lederer, 2008). Moderate loading/expression at sub-μM concentration of YC-Nano never affected the viability of fish embryos including a set of neurons . On the other hand, functional interference of GECIs with endogenous Ca 2+ binding proteins and their targets could pose a problem. The CaM of YCs potentially trans-activate endogenous CaM targets. In vitro analysis reported that excessive amounts of CaM affect the dynamic range of conventional YCs in a dose-dependent manner (Palmer et al., 2006). To avoid these side effects, computational re-design of Ca 2+ sensing motifs was performed. Modified binding interface of the synthetic CaM and its target prevented intermolecular interaction. The resulting YCs, named D2/3/4cpV, have been demonstrated to be insensitive to large excesses of CaM, while maintaining a K d of 0.03-64 μM and a large dynamic range of 3.8-to 5.3-fold (Palmer et al., 2006) (Table 1).
An alternate way to avoid uncontrolled interaction of GECI with endogenous proteins is to employ a different Ca 2+ -binding motif. While CaM has a variety of downstream targets, troponin C (TnC), a skeletal and cardiac musclespecific Ca 2+ -binding protein, is known to limit its interaction to just troponin I and troponin T. Indicators incorporating TnC from avian skeletal muscle or human cardiac muscle were generated, based on molecular design similar to that of cameleon. The resulting TN-L15 and TN-hTnC displayed a moderate Ca 2+ affinity but a lower Ca 2+ specificity (due to its cross reactivity with Mg 2+ ) and a small signal change (due to the lack of its binding peptide which enhances the conformational change of sensor motif) (Heim and Griesbeck, 2004) ( Table 1). As with YCs, further improvements have been introduced to TN-L15. Mg 2+ reactivity was eliminated by site directed mutagenesis on TnC, and the dynamic range was increased by replacing the Citrine acceptor with its cp174 variant, eventually yielding TN-XL (Mank et al., 2006). Low affinity of TN-XL was improved in TN-XXL by replacing TnC moiety with a concatenate of its high affinity C-lobe (Table 1). Although the in vitro dynamic range of TN-XXL was small, its in vivo performance was acceptable, suggesting the advantages of using TnC with reduced interference (Mank et al., 2008) ( Table 1).

PHOTOACTIVATABLE GECI
To visualize Ca 2+ dynamics in specific cell types, tissues, or organs, targeted expression of GECI gene is imperative. Although many promoters for cell/tissue/organ-specific expression are available, they do not cover all types of cell/tissue/organ. Photoswitchable GECIs (PS-GECI) can help overcome this limitation. Fluorescence status of PS-GECIs can be switched through light irradiation in arbitrary cell/tissue/organ, enabling cell/tissue/organ-specific visualization of Ca 2+ dynamics. This "highlighted Ca 2+ imaging" is beneficial in elucidating the activity of a single cell in the convoluted cell population of the neuronal network. There are only two reported PS-GECIs: a photoactivatable GECI, PA-TNXL , and a photoconvertible GECI, GR-GECO (Hoi et al., 2013) (Supplementary Image 1). The PA-TNXL was developed by replacing the donor and the acceptor fluorescent proteins in the TN-XL with a PA-GFP (photoactivatable GFP) and a dim yellow fluorescent protein DimVenus, respectively (Supplementary Image 1A and Table 1). Fluorescence of PA-TNXL can be switched on by violet light (∼ 400 nm) irradiation. The fluorescence of the photoactivated PA-TNXL dims upon Ca 2+ binding. GR-GECO has a similar design as GCaMPs/pericams. It has an mMaple, which can change fluorescence wavelength from green to red on being irradiated with violet light (∼ 400 nm) (McEvoy et al., 2012;Hoi et al., 2013)

GECIs FOR OPTOGENETICS
In neuroscience, a paradigm shift has been brought about by optogenetics. Channel rhodopsin (ChR), a light-gated ion channel, and halorhodopsin (HR), a pump, allow us to control the activity of neural circuits with fine spatiotemporal resolution (Boyden et al., 2005;Zhang et al., 2007). As ChR and HR are activated by blue (400-500 nm) and yellow (500-600 nm) light, respectively; spectrally separated GECIs are necessary for combinatorial application of optogenetics with Ca 2+ imaging. GECOs are the first reported color variants of single-FP GECIs based on cpGFPs and cpmApple (Zhao et al., 2011). Large-scale screening carried out by utilizing bacterial periplasmic expression system helped identify blue and red color variants of GECOs, including green and ratio metric alternates. In addition to the expanded color pallet, GECOs are also show sizable signal change (111-fold for GEM-GECO1), sensitizing them for the detection of subtle Ca 2+ response. Structure guided evolution of GCaMP yielded BCaMP1c, CyCaM1a, YCaMP1b, and a series of RCaMPs (Akerboom et al., 2013). Compatibility of GECI color variants with optogenetic control was demonstrated by using CA3 pyramidal neuron co-expressing ChR2 and RCaMP1.07, which was in turn developed by the site-directed mutagenesis of R-GECO1 (Ohkura et al., 2012).
Bioluminescence-based Ca 2+ imaging is an alternate and ideal strategy that is highly compatible with optogenetics. As bioluminescent indicators do not require excitation with light, observation can be free from functional crosstalk between optogenetic actuators. The limitation of this indicator was their dim signal as in the case of Ca 2+ sensitive Aequorin and its emission-enhanced variants such as GA (Baubet et al., 2000). A considerable increase of emission signal was, however, achieved in BRAC and Nano-lantern (Ca 2+ ), the latest version of bioluminescence-based GECIs (Saito et al., 2010(Saito et al., , 2012  Nano-lantern (Ca 2+ ) was constructed by direct fusion of Venus with RLuc8, which is split by insertion of CaM-M13. Ca 2+ binding to CaM-M13 induces reconstitution of the split Rluc8. This leads to FRET from reconstituted Rluc to Venus, resulting in a large increase in emission signal (Supplementary Image 1C, right and Table 1). Thus, Ca 2+ dynamics is monitored as a total intensity change, from both RLuc8 and FRET-enhanced emission of Venus. In the state-of-the-art demonstration by using cultured hippocampal neurons, Ca 2+ , transiently triggered by photo-activated ChR2, were imaged at 10 Hz with high SNR, showing the good compatibility of bioluminescence imaging with optogenetics (Saito et al., 2012) (Supplementary Images 1D,E).

CONCLUSION AND PERSPECTIVE
GECIs are advantageous over synthetic Ca 2+ dyes in their targetability and reliability for chronic imaging. However, there remains room for further improvement on several parameters. Suboptimal kinetic property, non-linearity due to cooperativity, and pH sensitivity of single-FP based indicator should be improved to perform reliable detection of Ca 2+ . The future development of GECI is, regardless of the faults, promising, because of its evolvability. As a result of the past and the current attempts, not only have the basic property of GECIs been optimized, but also of new family of GECIs have been successfully developed, including color variants and self-illuminating GECIs. Future experiments will focus on improving the compatibility of GECIs with optogenetic tools. GECIs, used in collaboration with latest imaging platform such as a deep tissue imaging and ultra-fast and large-scale recording systems, will pave way to deepening our understanding of the supple ability our brain to learn and to memorize.