Light generation of intracellular Ca2+ signals by a genetically encoded protein BACCS

Ishii, Tomohiro; Sato, Koji; Kakumoto, Toshiyuki; Miura, Shigenori; Touhara, Kazushige; Takeuchi, Shoji; Nakata, Takao

doi:10.1038/ncomms9021

Download PDF

Article
Open access
Published: 18 August 2015

Light generation of intracellular Ca²⁺ signals by a genetically encoded protein BACCS

Tomohiro Ishii^1,2,
Koji Sato^3,4,5,6,
Toshiyuki Kakumoto^1,2,
Shigenori Miura^4,5,
Kazushige Touhara^6,7,
Shoji Takeuchi^4,5 &
…
Takao Nakata^1,2

Nature Communications volume 6, Article number: 8021 (2015) Cite this article

9427 Accesses
58 Citations
8 Altmetric
Metrics details

Subjects

Abstract

Ca²⁺ signals are highly regulated in a spatiotemporal manner in numerous cellular physiological events. Here we report a genetically engineered blue light-activated Ca²⁺ channel switch (BACCS), as an optogenetic tool for generating Ca²⁺ signals. BACCS opens Ca²⁺-selective ORAI ion channels in response to light. A BACCS variant, dmBACCS2, combined with Drosophila Orai, elevates the Ca²⁺ concentration more rapidly, such that Ca²⁺ elevation in mammalian cells is observed within 1 s on light exposure. Using BACCSs, we successfully control cellular events including NFAT-mediated gene expression. In the mouse olfactory system, BACCS mediates light-dependent electrophysiological responses. Furthermore, we generate BACCS mutants, which exhibit fast and slow recovery of intracellular Ca²⁺. Thus, BACCSs are a useful optogenetic tool for generating temporally various intracellular Ca²⁺ signals with a large dynamic range, and will be applicable to both in vitro and in vivo studies.

Temperature-responsive optogenetic probes of cell signaling

Article 22 December 2021

William Benman, Erin E. Berlew, … Lukasz J. Bugaj

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

Article Open access 21 December 2022

Rodrigo G. Fernandez Lahore, Niccolò P. Pampaloni, … Peter Hegemann

Selective optogenetic control of Gq signaling using human Neuropsin

Article Open access 01 April 2022

Ahmed Wagdi, Daniela Malan, … Tobias Bruegmann

Introduction

Precise spatial and temporal coordination of molecular events are the basis of many cellular functions. Recently, there has been growing interest in using optogenetic tools to investigate cellular functions, because light is non-invasive and can be easily controlled spatiotemporally. Optical control enables precise regulation of intracellular signals in the target cells or even in the local area of single cells^1,2. The most successful optogenetic tool, channelrhodopsin (ChR) from the green alga Chlamydomonas reinhardtii, modulates the membrane voltage in response to light and has been widely applied in the field of neurosciences³. More recently, in addition to natural photosensory proteins, there have been advances in the development of genetically encoded photosensory modules for investigating cellular functions. One such photosensory module is the Avena sativa (oat) LOV2-Jα domain^4,5, which has been fused with various effector domains of proteins to generate novel engineered light-controlled molecules^2,6,7,8.

Ca²⁺ is a ubiquitous second messenger in nearly all cells and regulates a wide variety of cell functions from cell division to cell death, including gene expression, cell migration, secretion, neural activities and muscle contraction. Ca²⁺ signals function over a wide timescale, from milliseconds for synaptic vesicle release to hours for gene expression leading to cell development and differentiation^9,10. Furthermore, Ca²⁺ signals play important roles at the subcellular level, such as in learning and memory at spiny dendrites and in neurotransmitter release at synaptic endings in a single neuron¹¹. A genetically encoded light-activated protein that regulates intracellular Ca²⁺ signals could be useful and has been eagerly awaited^12,13.

Compared with caged compounds¹⁴, genetically encoded light-sensitive proteins are more convenient to apply in vivo, because they usually do not require an external supply of chromophores to absorb photons¹⁵. ChR2 (refs 16, 17), opto-α1AR¹⁸, melanopsin¹⁹, LOVS1K²⁰ and PACR²¹ are currently available optogenetic tools to generate intracellular Ca²⁺ signals. ChR2 is a light-activated cation channel¹⁶, while opto-α1AR and melanopsin stimulate the phospholipase C cascade, thereby indirectly regulating Ca²⁺ signals. Therefore, these optogenetic tools are not specific for Ca²⁺ signals. LOVS1K is a fusion protein of a plant LOV2-Jα domain and a human stromal interaction molecule-1 (STIM1) fragment, and is a light-dependent regulator of Ca²⁺-selective ORAI1 channels²². Repeated photoactivation of LOVS1K co-expressed with ORAI1 was reported to induce a slow increase in global Ca²⁺, reaching an intracellular Ca²⁺ concentration of ∼500 nM after 40 min²⁰. PACR is a Ca²⁺-binding protein with an inserted LOV2-Jα domain, and releases Ca²⁺ in response to light. The amplitude of Ca²⁺ elevation by PACR is relatively small, and requires improvement to control many cellular events²¹. Engineering of an optogenetic tool that can control intracellular Ca²⁺ signals with high selectivity, good time resolution and large dynamic range is required for practical use.

Here we develop an optogenetic tool, BACCS, to generate intracellular Ca²⁺ signals. One of the BACCS variants induces an increase in intracellular global Ca²⁺ signals within 1 s. BACCS is tuneable for its recovery kinetics, enabling fast and slow recovery of intracellular Ca²⁺. We further demonstrate the application of BACCS in vitro and in ex vivo whole-mount preparations. Thus, BACCS is a useful optogenetic tool for regulating a wide variety of cellular events via intracellular Ca²⁺ signals in various cell types both in vitro and in vivo.

Results

Engineering of a photoswitch for a Ca²⁺-selective channel

We generated a series of fusion genes of the LOV2-Jα sequence from A. sativa phototropin 1 (refs 4, 5) as a photosensory module and the regulatory sequence for ORAI1 from human STIM1 (refs 23, 24, 25, 26) as a signal effector (Fig. 1a; Supplementary Fig. 1). We assumed that the closer the photosensor and the signal effector were, the more efficient steric hindrance of the STIM1 interaction would be, thereby enabling inhibition of the signalling function in the dark. The basic core unit of BACCS was selected by combining the following three screening steps (see Supplementary Fig. 1 for details). First, the minimal signal effector domain of the STIM1 fragment for ORAI1 activation was defined. Second, the minimal STIM1 fragment was fused to the C terminus of a deletion series of LOV2-Jα to find the structure causing steric hindrance of the STIM1 interaction by LOV2-Jα. Third, the candidate fusion proteins were expressed with nuclear factor of activated T cells (NFAT)::CFP (‘::’ represents a fusion) to examine light-induced NFAT translocation from the cytoplasm to the nucleus. It has been reported that elevation of intracellular Ca²⁺ (>200–300 nM) induces dephosphorylation of NFAT, followed by its nuclear translocation²⁷. LOV2-Jα (amino acids 404–538)::STIM1 (amino acids 347–448) was the most efficient photoswitch, displaying high sensitivity and low basal activity, and is designated human blue light-activated Ca²⁺ channel switch 1 (hBACCS1). hBACCS1 has the structural feature that a leucine residue (originally an isoleucine in LOV2-Jα) at the junction of the fusion protein, which is important for the function of both LOV2-Jα and STIM1, is shared between them (Fig. 1a). Therefore, the dark-state form of LOV2-Jα will block the function of STIM1 through steric hindrance over the leucine residue. Three variants of BACCS were designed (Fig. 1b): (1) hBACCS2, a dimer of hBACCS1; (2) ORAI1::hBACCS2, a fusion protein of human ORAI1 and hBACCS2; and (3) dmBACCS2, protein with the same structure as hBACCS2 except that Drosophila Stim was used instead of human STIM1. The designs of hBACCS2 and ORAI1::hBACCS2 were based on the observation that a tandem dimer of STIM1 (336–485) and its ORAI1 fusion protein can efficiently open ORAI1 channels²⁸.

**Figure 1: Schematic representation of BACCSs.**

Calcium response of ORAI1 activated by hBACCS

To evaluate the hBACCS function, we performed Ca²⁺ imaging in HEK293T cells expressing hBACCS2 (Fig. 2a). The widespread expression of ORAI proteins in most tissues^29,30,31 will enable the omission of additional introduction of ORAI proteins in most cell types. We compared the following four constructs: hBACCS1, hBACCS2, hBACCS2-IRES-ORAI1 and ORAI1::hBACCS2 (Fig. 2b,c; Supplementary Fig. 2a,b). The IRES sequence allows co-translation of hBACCS2 with ORAI1 from a bicistronic messenger RNA. More than 98% of cells expressing hBACCS2 or ORAI1::hBACCS2 showed a Ca²⁺ increase on light exposure, while only 46% of cells expressing hBACCS1 showed an increase (Supplementary Fig. 2a; Supplementary Table 1), suggesting that hBACCS2 and ORAI1::hBACCS2 are more efficient photoswitches than hBACCS1. The time courses of the intracellular Ca²⁺ increase revealed that hBACCS2 induced a faster intracellular Ca²⁺ increase than ORAI1::hBACCS2 (Fig. 2b,c). Exogenous expression of ORAI1 in addition to hBACCS2 (hBACCS2-IRES-ORAI1) did not enhance the Ca²⁺ response in HEK293T cells compared with expression of hBACCS2 alone (Fig. 2b). Two ORAI1::hBACCS2 variants with shorter and longer linkers were also examined, resulting in no apparent improvement in Ca²⁺ response (Supplementary Fig. 2c). Removal of Ca²⁺ from the extracellular Ringer’s solution prevented the light-induced increase in intracellular Ca²⁺ in cells expressing hBACCS2 (Fig. 2b), suggesting that the Ca²⁺ increase occurs through influx of extracellular Ca²⁺, rather than release of Ca²⁺ from intracellular organelles such as the endoplasmic reticulum or mitochondria. The Ca²⁺ indicator dye Fura-2/AM was used to measure the intracellular Ca²⁺ concentrations. The geometric means of the basal Ca²⁺ level in cells expressing ORAI1::hBACCS2 or hBACCS2 in the dark were both around 100 nM, slightly higher than that in control cells but within the range of physiological basal intracellular Ca²⁺ concentrations (Fig. 2d). In cells expressing hBACCS2, the intracellular Ca²⁺ concentration was elevated ∼4.5-fold after 30 s and to beyond 1 μM (>10-fold) after 60 s in response to ultraviolet light (Fig. 2e). The efficiency of photoactivation of hBACCS2 was light intensity dependent. The magnitude of the Ca²⁺ response increased with increasing intensity in the range 0.6–180 μW mm⁻² of continuous 470-nm light emitting diode (LED) illumination (Supplementary Fig. 2d). Activation of hBACCS2 was reversible (Supplementary Fig. 2e), and repeated photoactivation resulted in similar levels of Ca²⁺ elevation each time (Supplementary Fig. 2f).

**Figure 2: Characterization of BACCSs expressed in HEK293T cells.**

Using whole-cell patch-clamp recording, inward currents were recorded at a holding potential of −60 mV in HEK293T cells expressing ORAI1::hBACCS2 or hBACCS2 on blue light exposure (Supplementary Fig. 3a,c). Inward currents were also recorded in Jurkat cells (Supplementary Fig. 3e), which are known to have significant endogenous Ca²⁺ release-activated Ca²⁺ currents. The inward rectification of the light-induced current (Supplementary Fig. 3b,d,f) is consistent with the characteristic of ORAI1 channels^32,33,34,35. Furthermore, photostimulation did not generate outward currents representing efflux of Na⁺ or K⁺ in depolarized HEK293T cells expressing hBACCS2 (Supplementary Fig. 3d), suggesting that hBACCS2 does not activate non-selective cation channels, such as Trpc (transient receptor potential canonical) channels, which give large outward cationic currents at depolarized potential³⁶, and that Ca²⁺ is the only charge carrier of photoactivated currents under the whole-cell current recording condition. These results provide further indications that light-activated hBACCS2 can open ORAI channels in the plasma membrane and induce influx of extracellular Ca²⁺.

Characterization of dmBACCS2

To examine the applicability of hBACCS2 to other species, we performed Ca²⁺ imaging in Drosophila S2 cells expressing hBACCS2. However, we found no intracellular Ca²⁺ increase on photoactivation (Supplementary Fig. 4a), suggesting that interaction between hBACCS2 and Drosophila Orai (dOrai) was absent or insufficient. Since currents through a store-operated Ca²⁺ channel were recorded³⁷ and dOrai was identified as a gene involved in store-operated Ca²⁺ entry in S2 cells by genome-wide RNA interference screens^33,34,38, dOrai should be expressed in S2 cells. Therefore, we performed Ca²⁺ imaging in S2 cells expressing dmBACCS2, a Drosophila version of BACCS2, and found that intracellular Ca²⁺ signals were generated in a subset of cells after stimulation with a 488-nm laser (Supplementary Fig. 4b). The time course of the Ca²⁺ changes in S2 cells revealed that the concentration of intracellular Ca²⁺ reached its maximum ∼50 s after illumination (Supplementary Fig. 4c). When Ca²⁺ imaging was performed in HEK293T cells expressing dmBACCS2 alone, intracellular Ca²⁺ was not elevated by photoactivation (Fig. 2b), suggesting that dmBACCS2 does not activate human ORAI1. As dmBACCS2 appeared to activate dOrai according to the Ca²⁺ imaging experiments in S2 cells, we examined HEK293T cells expressing dmBACCS2-IRES-dOrai. Surprisingly, activation of dmBACCS2 in dOrai-expressing HEK293T cells caused fast and robust intracellular Ca²⁺ elevation (Fig. 2a–c). Removal of Ca²⁺ from the extracellular Ringer’s solution prevented the light-induced increase in intracellular Ca²⁺ in these cells (Fig. 2b), suggesting that the Ca²⁺ increase occurred through influx of extracellular Ca²⁺. Intracellular Ca²⁺ elevation was observed in 98.0% of cells expressing dmBACCS2-IRES-dOrai (Supplementary Table 1). To closely observe the Ca²⁺ changes, the cells were photoactivated with a 488-nm laser and observed every 0.1 s. Intracellular Ca²⁺ elevation was clearly observed within 1 s (Fig. 2f,g). Activation of dmBACCS2 was reversible, and repeated activation resulted in similar levels of intracellular Ca²⁺ elevation each time (Supplementary Fig. 2e,f). We performed whole-cell patch-clamp recording in HEK293T cells expressing dmBACCS2-IRES-dOrai, and confirmed the stimulus-dependent response amplitude and the inward rectification (Fig. 2h,i), consistent with the electrical characteristics of store-operated calcium channels in S2 cells³⁷. LOVS1K (ref. 20), which is composed of LOV2-Jα (404–546) and STIM1 (233–450) with mRFP, was also analysed for comparison. Photoactivation of LOVS1K in HEK293T cells resulted in slightly faster intracellular Ca²⁺ elevation compared with that due to hBACCS2 in our system (Fig. 2b). However, the speed of the Ca²⁺ elevation and amplitude of the Ca²⁺ increase by dmBACCS2 were significantly greater than those induced by LOVS1K or hBACCS2 (Fig. 2c). The geometric mean of the basal Ca²⁺ level in cells expressing dmBACCS2-IRES-dOrai was 119.8 nM (Fig. 2d), higher than that in control cells but almost within the physiological range. It tended to be elevated with increases in the expression level (Supplementary Fig. 5a). We also examined NFAT::GFP subcellular localization in the dark, an alternative physiological assessment of the basal intracellular Ca²⁺ level (Supplementary Fig. 5b,c). We observed that NFAT::GFP was distributed in the cytoplasm, but not in the nucleus, in cells expressing hBACCS2 or dmBACCS2-IRES-dOrai in the dark, indicating a low basal intracellular Ca²⁺ level. In contrast, a large population (64.8%) of cells expressing LOVS1K showed nuclear localization of NFAT::GFP without light illumination, suggesting that the basal Ca²⁺ concentration in cells expressing LOVS1K was high.

Generation of Ca²⁺ signals in various cell types

To examine the applicability to various types of cells, hBACCS2 or dmBACCS2-IRES-dOrai was expressed in other cell types, namely COS-7 cells (derived from African green monkey kidney), mouse cultured hippocampal neurons and HIT-T15 hamster pancreatic β-cells, and Ca²⁺ imaging was performed (Fig. 3). Efficient elevation of intracellular Ca²⁺ was observed in most COS-7 cells (Fig. 3a,b) and mouse hippocampal neurons (Fig. 3c,d) on photoactivation. The extracellular solution for the hippocampal neurons contained tetrodotoxin, a sodium channel blocker, thus excluding any effect of the action potential. In general, excitable cells such as neuronal cells express Orai proteins, but they provide little contribution to any Ca²⁺ influx³⁹. Our results suggest that hBACCS2 can be applied to both excitable and non-excitable cells. In HIT-T15 cells, a comparison of hBACCS2 expression and intracellular Ca²⁺ elevation revealed that higher expression of hBACCS2 resulted in a smaller response, while lower expression resulted in a larger response (Fig. 3e,f). In contrast, a Ca²⁺ response was observed in a large population of HIT-T15 cells expressing hBACCS2-IRES-ORAI1 or dmBACCS2-IRES-dOrai regardless of the expression level (Fig. 3e,f). These results indicate that the level of endogenous Orai expression could vary substantially among HIT-T15 cells.

**Figure 3: Activation of hBACCS2 and dmBACCS2 in various cell types.**

These findings suggest that our strategy can generate Ca²⁺ signals in most mammalian cell types under blue light illumination, and that dmBACCS2 induces faster and larger intracellular Ca²⁺ elevation compared with hBACCS2 regardless of the cell types involved.

Spatiotemporal generation of Ca²⁺ signals

To examine whether Ca²⁺ signals could be generated with spatial specificity, Ca²⁺ imaging was performed in HEK293T cells expressing hBACCS2 with focused light illumination on a specific population of cells. In this experiment, near-infrared fluorescent protein (iRFP) was used as a marker for transfected cells, thereby making it possible to image hBACCS2-expressing cells with simultaneous blue light stimulation and Ca²⁺ imaging. Five cells loaded with the Ca²⁺ indicator dye Rhod-3/AM were sequentially illuminated with a 488-nm laser, and intracellular Ca²⁺ elevation was specifically observed in the targeted cells (Fig. 4a). Next, mouse cultured hippocampal neurons were examined for subcellular local photoactivation. We confirmed that hBACCS2 and ORAI1 were detected throughout the neuronal structure, including the dendrites and axon, by fluorescence imaging and immunostaining (Fig. 4b,c). We then carried out Fluo-4/AM Ca²⁺ imaging of neurons expressing hBACCS2. The photoactivation was initially focused on the tip of the axon and then focused on a dendrite. Ca²⁺ signals were clearly elevated in the axon terminal first and then in the dendrite (Fig. 4d,e). The Ca²⁺ elevation was restricted to a small area and the signal did not show long-distance transmission (<20 μm) (Fig. 4f,g). We also performed a subcellular light illumination in hippocampal neurons expressing dmBACCS2-IRES-dOrai, and observed much faster local intracellular Ca²⁺ elevation than that for hBACCS2 (Fig. 4h).

**Figure 4: Local generation of intracellular Ca²⁺ signals.**

Control of cellular events

To evaluate BACCS for cell biological applications, we first tested the light-activated switch in transcriptional regulation. NFAT is a transcription factor that regulates the expressions of cytokines, growth factors and their receptors, proteins involved in cell–cell interactions and many microRNAs⁴⁰; elevation of intracellular Ca²⁺ induces dephosphorylation of NFAT, followed by its nuclear translocation²². HEK293T cells expressing hBACCS2-IRES-NFAT::mCherry or dmBACCS2-IRES-dOrai-IRES-NFAT::mCherry were stimulated with a 488-nm laser every 2 min. Live-cell imaging of NFAT::mCherry revealed that NFAT molecules, which were initially distributed in the cytoplasm, translocated to the nucleus within 10 min (Fig. 5a). To examine whether BACCS photoactivation could induce gene expression, HEK293T cells were co-transfected with dmBACCS2-IRES-dOrai and pHY41 (P_NFAT-YFP-pA)¹⁹ encoding yellow flurescent protein (YFP) under the control of a promoter with an NFAT response element. After 12 h of blue light exposure, YFP expression was induced (Fig. 5b). To quantify the level of induction, we performed luciferase assays. hBACCS2, LOVS1K and dmBACCS2-IRES-dOrai were compared (Fig. 5c,d). Significant luciferase induction was observed for hBACCS2 after 6 h of light exposure. Low, but slightly elevated, basal luciferase activity in the dark was observed in cells expressing hBACCS2. This could reflect a small population of cells with an elevated basal Ca²⁺ level. LOVS1K induced luciferase activity at a similar level to that of hBACCS2 on light exposure, but showed high basal activity in the dark, consistent with a higher basal Ca²⁺ level. dmBACCS2 induced robust luciferase activity on light exposure compared with hBACCS2 and LOVS1K. dmBACCS2 also showed basal activity in the dark, which decreased with lower amounts of transfected DNA. The dynamic ranges of the induced luciferase activity for hBACCS2 and dmBACCS2 in this assay were much higher than that for LOVS1K.

**Figure 5: Induction of cellular responses by manipulating intracellular Ca²⁺ signals.**

We also used hBACCS2 to initiate the formation of blebs, spherical membrane protrusions, in HEK293 cells in combination with Lyn-CaRQ⁴¹, an engineered membrane-anchored Ca²⁺-sensitive RhoA protein that becomes activated in response to Ca²⁺ signals. Plasma membrane blebs have been implicated in apoptosis, cytokinesis and cell migration⁴². Cells expressing both hBACCS2 and Lyn-CaRQ were photoactivated with a 488-nm laser. Blebs were induced in a stimulation-dependent manner (Fig. 5e), and the number of induced blebs reached the maximum level within 3.5 min (Fig. 5f).

Thus, BACCS can be applied to the control of cellular events such as NFAT-dependent transcription and cell morphological changes.

Electrophysiological response in mouse olfactory epithelium

To examine whether BACCS is applicable to mouse tissues, we generated a recombinant adenovirus for expression of hBACCS2-IRES-mem::tdTomato, and infected the adenovirus into the olfactory epithelium (Fig. 6a). We recorded local field potentials from the surface of the olfactory epithelium in an ex vivo whole-mount preparation, the electro-olfactogram (EOG)⁴³. Figure 6c shows the waveforms of the average EOG responses to blue light and the odorant amyl acetate as a positive control. Significant responses to both blue light and amyl acetate were recorded at the infected surface of the olfactory epithelium on blue light exposure, while the olfactory epithelium of wild-type mice only showed responses to amyl acetate (Fig. 6c,d), suggesting that hBACCS2 was photoactivated in olfactory sensory neurons (OSNs).

**Figure 6: EOG analyses of odorant- and light-induced responses.**

To further consolidate the EOG results, we generated a transgenic mouse strain possessing a subset of OSNs that expressed ORAI1::hBACCS2. A DNA fragment containing the odorant receptor gene M71 (also known as Olfr151) was used to generate the transgenic mouse strain⁴⁴, in which neurons expressing the transgene M71 co-expressed ORAI1::hBACCS2 and mem::tdTomato (Supplementary Fig. 6a). The M71 transgene was expressed in a subset of OSNs (Fig. 6b), and their axonal projection to the olfactory bulb and neural survival appeared normal (see Supplementary Fig. 6b–f for details). We carried out EOG using the transgenic mice, and observed electrophysiological responses in these mice (Fig. 6c,d). The responses in the transgenic mice were smaller, but less variable, than those in the adenovirus-infected mice, suggesting that the specific and stable expression of BACCS in transgenic animals enabled the acquisition of more stable EOG responses.

Thus, BACCS can be used in the mouse olfactory epithelium and will be applicable to in vivo studies.

Fast and slow recovery kinetic mutants of dmBACCS2

dmBACCS2 is an efficient Ca²⁺ photoswitch in mammalian cells, but still suffers from slow recovery kinetics (time constant of the whole-cell current decay phase) (Fig. 2h)⁴⁵, rendering it difficult to precisely control its temporal signalling activities. Recently, mutational analysis of the LOV2 domain was conducted to obtain fast- and slow-cycling LOV2 variants^46,47. We introduced such mutations into the LOV2 domain of dmBACCS2; N425S and V416L⁴⁶ resulted in fast and slow switch-off phenotypes, respectively. We performed Ca²⁺ imaging in HEK293T cells expressing dmBACCS2-IRES-dOrai, dmBACCS2-N425S (dmBACCS2NS)-IRES-dOrai or dmBACCS2-V416L (dmBACCS2VL)-IRES-dOrai, and compared the Ca²⁺ responses. The amplitude of the Ca²⁺ increase in cells expressing the dmBACCS2NS construct was smaller than those in cells expressing the dmBACCS2 or dmBACCS2VL constructs (Fig. 7a–c). To analyse the recovery kinetics, the intracellular Ca²⁺ decrease was observed every 5 s after 170 s of photostimulation (Fig. 7d). Notably, dmBACCS2NS had faster recovery kinetics and dmBACCS2VL had slower recovery kinetics than dmBACCS2 (Fig. 7e,f). Whole-cell patch-clamp recording revealed the precise time courses of dOrai activation and inactivation for dmBACCS2 and the two mutants (Fig. 8a). The results for three kinetic parameters, amplitude, time to peak and recovery time constant (Fig. 8b,c) were consistent with the Ca²⁺ imaging observations. The recovery time constant for dmBACCS2NS is less than one-half of that for dmBACCS2, while the time constant for dmBACCS2VL was >100 times larger than that for dmBACCS2.

**Figure 8: Characterization of light-activated whole-cell inward currents recorded from HEK293T cells expressing mutant *dmBACCS2-IRES-dOrai*.**

Thus, the recovery kinetics of BACCS are tuneable, and both fast and slow recovery switches can be developed for specific experimental purposes.

Discussion

We have engineered BACCS, an optogenetic tool for generating intracellular Ca²⁺ signals. Compared with previously reported Ca²⁺ optogenetic tools, some variants of BACCS were endowed with features of fast and large response induction and better off-recovery efficiency, thereby making BACCS a more useful tool for precisely generating Ca²⁺ signals.

The four BACCS variants, hBACCS1, hBACCS2, ORAI1::hBACCS2 and dmBACCS2, had different properties in their Ca²⁺ responses. Although the stoichiometry of ORAI1 and STIM1 remains controversial, Li et al.²⁸ proposed a model in which one STIM1 dimer can interact with one ORAI1 subunit in the pore formed by tetrameric ORAI1 and octameric STIM1 to maximally open the ORAI1 channel. Consistent with this model, hBACCS2, a tandem dimer of hBACCS1, was a more efficient photoswitch than hBACCS1. A fusion protein of hBACCS2 with the C terminus of ORAI1 was also functional as a switch in HEK293T cells, but the photoactivation time course of ORAI1::hBACCS2 was slower than that of hBACCS2. Although we observed no drastic differences in Ca²⁺ responses among the three ORAI1–hBACCS2 fusion constructs with different linker lengths, the location and length of the linker might still influence the function. Alternatively, endogenous ORAI1 and ORAI1::hBACCS2 might form a heterogeneous tetramer, resulting in insufficient maximal activation of the channel. dmBACCS2 was a much more efficient photoswitch but required coexpression of dOrai in mammalian cells. We observed that the intracellular Ca²⁺ increase occurred within 1 s in HEK293T cells expressing dmBACCS2-IRES-dOrai, although the Ca²⁺ response was not as fast in Drosophila S2 cells expressing dmBACCS2. Mammalian cells may lack negative regulatory mechanisms for dOrai function. Although we cannot exclude the possibility that the fast Ca²⁺ response arose through functionally better stoichiometry of dmBACCS2 and dOrai in cells expressing dmBACCS2-IRES-dOrai, this seems unlikely because expression of hBACCS2-IRES-ORAI1 did not result in faster or larger intracellular Ca²⁺ elevation compared with expression of hBACCS2 alone. It has been reported that Orai1-interacting STIM1 domains, CAD²⁴ and SOAR²⁵, can interact with voltage-gated Ca²⁺ Ca_v1.2 channels^48,49 and Trpc channels⁵⁰, although the domains themselves do not activate these channels. Therefore, hBACCS2, which contains a similar sequence to CAD/SOAR, may interact with such molecules, but should generate no signals through them.

We examined the basal Ca²⁺ level by two methods, comprising direct measurement by Fura-2/AM and indirect measurement by the NFAT::GFP assay. Although the Fura-2 method measures exact intracellular Ca²⁺ concentrations, we cannot exclude the possibility that BACCS could be slightly activated in the darkroom because of the difficulty in achieving complete darkness during the experiments. However, the NFAT::GFP assay would not be affected by this problem because NFAT::GFP was fixed in the darkroom before possible NFAT::GFP translocation, if any. The results for both the NFAT::GFP assay and the measurement by Fura-2/AM indicated that the mean basal Ca²⁺ levels in hBACCS2-expressing cells and dmBACCS2-IRES-dOrai-expressing cells were within the physiological range. However, BACCS-expressing cells, especially dmBACCS2-dOrai-expressing cells, contained a population of cells with a high basal Ca²⁺ level, probably because of intrinsic activity of BACCS or the difficulty in achieving complete darkness for the experiments, and these cells are likely to have influenced the results of the luciferase assay. The basal Ca²⁺ concentration could be controlled and kept at a lower level by avoiding high-level expression of the BACCS gene, at least partially, as demonstrated in the luciferase assay findings.

In what kinds of cells can BACCS be used? dmBACCS2-dOrai was successfully used in Drosophila S2 cells, mammalian HEK293T cells, COS-7 cells, HIT-T15 cells and hippocampal neurons, suggesting that it can be used in a wide variety of species and cell types. hBACCS2 also functioned in all mammalian cell types examined, and can be used to photoactivate endogenous Orai. The efficiency of intracellular Ca²⁺ signal elevation by light relies on the level of endogenous Orai expression in each cell. The cytosolic STIM1 C terminus has been reported to stimulate not only human ORAI1, but also ORAI2 and ORAI3 (ref. 51), indicating that hBACCS2 can activate all ORAI proteins by light illumination. On the basis of previous observations that Orai proteins are widely expressed in most mouse and human tissues^29,30,31, hBACCS2 expression alone is likely to be sufficient for the generation of Ca²⁺ signals through endogenous Orai in most mammalian cells on light illumination. Although Stim and Orai proteins are evolutionarily conserved across metazoans^52,53, direct cross-species interactions between Stim and Orai molecules appear to be inefficient between humans and Drosophila.

Two Ca²⁺-selective photoswitches have been reported, PACR (ref. 21) and LOVS1K (ref. 20). PACR is composed of calmodulin fused with the M13 peptide and LOV2-Jα, and releases Ca²⁺ upon light exposure. The expression can be restricted to a particular organelle, thereby allowing the control of Ca²⁺ signals at the organelle level. PACR induces Ca²⁺ concentration changes of 10–90 nM in HeLa cells, which are not sufficient to control many Ca²⁺-regulated cellular events because micromolar changes in Ca²⁺ concentration are observed in many biological responses. LOVS1K and hBACCS are both composed of the same photosensory module, LOV2-Jα, and effector domain, STIM1, with different sizes of each fragment: LOV2-Jα (404–538) and STIM1 (347–448) for hBACCS and LOV2-Jα (404–546) and STIM1 (233–450) for LOVS1K. Pham et al.²⁰ reported that repeated photoactivation of LOVS1K co-expressed with ORAI1 gradually caused intracellular Ca²⁺ accumulation to ∼500 nM over 40 min. However, it can be estimated from our experiments that intracellular Ca²⁺ elevation to ∼500 nM can be achieved in 30 s in cells expressing LOVS1K alone (Fig. 2b,e), probably reflecting differences in the experimental setup. One non-negligible defect of LOVS1K is its high basal activity, which was observed in our NFAT::GFP assay and luciferase assay in the dark. The basal Ca²⁺ level, which affects NFAT-dependent transcription, could lead to considerable interference with cell function.

BACCS has the following features and advantages. hBACCS2 can be used to induce intracellular Ca²⁺ elevation to ∼500 nM in 30 s and >1 μM in 60 s, and its basal activity in the dark is relatively low compared with LOVS1K. hBACCS2 functions in the mouse olfactory system. In M71 transgenic mice, OSNs expressing ORAI1::hBACCS2 appeared normal in terms of cell survival and axonal projection⁵⁴, suggesting that the basal Ca²⁺ concentration induced by ORAI1::hBACCS2 was sufficiently low to have no effect on the development of olfactory neurons. The relatively low basal activity and high Ca²⁺ response (micromolar change) make hBACCS2 suitable for application to many Ca²⁺-dependent biological events both in vitro and in vivo. dmBACCS2-dOrai is by far the most efficient and robust Ca²⁺ photoswitch in mammalian cells. Although basal activity in the dark can be observed when dmBACCS2-dOrai is expressed at a high level, the dynamic range of the Ca²⁺ response is wide, and fast Ca²⁺ intracellular elevation can be induced. We observed that both its magnitude and speed of Ca²⁺ elevation were better in all types of tested cells expressing dmBACCS2-IRES-dOrai than in cells expressing hBACCS2, suggesting that dmBACCS2-dOrai is a more useful tool to precisely induce Ca²⁺ responses. Furthermore, as dmBACCS2 does not appear to interact with human ORAI, the dmBACCS2-dOrai system is likely to function without interacting with other endogenous molecules in mammalian cells. Therefore, hBACCS2 and dmBACCS2 are alternative candidates for practical use in mammalian cells (Supplementary Fig. 7). hBACCS2 will be an appropriate tool to control the activity of endogenous Orai and Orai-mediated cellular events, such as induction of T-cell activation²², turning of growth cones⁵⁵ and cell migration⁵⁶. The dmBACCS2-dOrai system may be used with better temporal resolution for controlling general Ca²⁺-dependent cellular events without interfering with the functions of other endogenous molecules.

Although the activation time was improved by using dmBACCS2, the recovery kinetics would be a limiting factor for inter-trial frequency. In cases of synaptic transmission or cardiac contraction, the Ca²⁺ effector systems respond to pulses within the microsecond to millisecond range⁹. The photocycles of PACR, LOVS1K, hBACCS2 and dmBACCS2 all depend on LOV2-Jα and their recovery kinetics will be similar; LOV2-Jα recovers spontaneously with half-times of ∼50 s (ref. 45). As an effort to tune the recovery kinetics, we introduced mutations implicated in the photocycle of LOV2-Jα (refs 46, 47) into dmBACCS2 to obtain various recovery kinetic mutants (Supplementary Fig. 7). The fast recovery mutant showed significantly faster Ca²⁺ recovery in the dark than wild-type dmBACCS2. This improvement will help substantially towards the manipulation of cellular events controlled by oscillations of Ca²⁺ transients on the second to minute timescale, including growth-cone migration/turning, axonal growth, cell migration, muscle development and release of cytokines⁹, although this mutant still falls short for the control of synaptic transmission or cardiac contraction. Further mutagenesis in the LOV2-Jα domain will produce improved dmBACCS2 with faster reversibility. The slow recovery kinetic mutant showed prolonged intracellular Ca²⁺ elevation after the lights were turned off. It will require less light illumination for sustained activation of the photoswitch, which could avoid undesirable phototoxicity to cells and unfocused activation through drifting of the target sample. The fact that the introduction of the mutations for the fast- and slow-cycling LOV2-Jα into dmBACCS2 resulted in fast and slow recovery kinetics of dmBACCS2-mediated dOrai responses, respectively, is consistent with the idea that these mutations changed the LOV2-Jα kinetics, resulting in the tuning of the binding of dmBACCS2 to dOrai.

One of the advantageous features of photoactivatable molecules is the ability to control their activity with high spatial resolution. By focused illumination of a blue light laser, we successfully activated local Ca²⁺ signals at axon terminals and dendrites of hippocampal neurons. Ca²⁺ microdomains are sites with localized high Ca²⁺ concentrations near Ca²⁺ channels at the cell surface or internal stores¹¹. Ca²⁺ microdomains enable different subcellular domains to execute many distinct cellular processes with specificity in single cells. For example, Ca²⁺ microdomains in spiny/aspiny dendrites, soma, mossy fibres and synaptic endings are responsible for distinct neuronal functions of learning and memory, gene transcription and transmitter release¹¹. Thus, light manipulation of distinct Ca²⁺ microdomains in single cells will contribute to the elucidation of subcellular events with high spatial resolution.

The ability of BACCS to generate global and subcellular Ca²⁺ signals, and our application of BACCS to various cell types and transgenic animals suggest that BACCS will be a useful optogenetic tool for generating intracellular Ca²⁺ signals both in vitro and in vivo. Precise and non-invasive exploration of intracellular Ca²⁺ functions at the subcellular level by light illumination will contribute towards a deeper understanding of the numerous Ca²⁺-dependent physiological events.

Methods

DNA constructs

HA-NFAT1 (1–460)-GFP, human STIM1-CFP and Orai1-YFP were kind gifts from Anjana Rao (Addgene plasmids #11107, #19755 and #19756); pLyn-CaRQ and LOVS1K from Kevin Truong (Addgene plasmids #34559 and #31981); M71-TV from Peter Mombaerts (Addgene plasmid #15635); pTriEx-mVenus-PA-Rac1, pTriEx-mVenus-PA-Rac1-C450A and pTriEx-PA-Rac1-I539E from Kraus Hahn (Addgene plasmids #22007, #22021 and #22026); Mem::tdTomato (tdTomato with a membrane localization signal) from Kazunari Miyamichi, iRFP⁵⁷ from Michiyuki Matsuda; and P_NFAT-YFP (pHY41) from Martin Fussenegger. For details of each construct used in this study, please see the Supplementary Methods. Plasmids and sequence information for hBACCS2, ORAI1::hBACCS2, dmBACCS2-IRES-dOrai, dmBACCS2NS-IRES-dOrai, dmBACCS2VL-IRES-dOrai and mRFP::dmBACCS2 will soon be available from Addgene (http://www.addgene.org).

Cell culture and transient transfection

Cells were maintained in the following media: for HEK293T (obtained from TaKaRa), HEK293 (RIKEN Cell Bank) and COS-7 cells⁵⁸, minimal essential medium (MEM; Nacalai Tesque) supplemented with 10% (v v⁻¹) fetal bovine serum (FBS); for HIT-T15 cells (DS Pharma Biomedical Co. Ltd.), F-12K medium (WAKO Chemical Co.) supplemented with 10% (v v⁻¹) horse serum and 2.5% (v v⁻¹) FBS; for Jurkat cells (RIKEN Cell Bank), RPMI1640 (Nacalai Tesque) supplemented with 10% (v v⁻¹) FBS; for Drosophila S2 cells (RIKEN Cell Bank), Schneider’s medium (Life Technologies) containing 10% (v v⁻¹) FBS. Two days before experiments, HEK293T, HEK293 and COS-7 cells were seeded on glass-bottomed dishes (Greiner) coated with collagen (Cellmatrix Type IC; Nitta Gelatin Inc.), and HIT-T15 and S2 cells were seeded on non-coated glass-bottomed dishes. On the next day, plasmid transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were analysed 24–36 h after transfection. Jurkat cells were seeded and transfected with plasmids using Lipofectamine 2000 according to the manufacturer’s instructions. After 4 h, phorbol 12-myristate 13-acetate (PMA; Wako Chemical Co.), a specific activator of protein kinase C, was added (final concentration: 50 ng ml⁻¹) to enhance the cytomegalovirus promoter activity and gene expression in Jurkat cells. The cells were analysed 8–12 h after transfection.

Primary hippocampal neurons derived from mouse embryos were cultured in accordance with the method of Kaech and Banker⁵⁹ with some modifications⁶⁰. Briefly, neurons were subjected to plasmid transfection using Lipofectamine 2000 after 3 or 6 days of culture for Ca²⁺ imaging and immunohistochemistry, respectively. After 2 further days, the transfected neurons were analysed.

Immunostaining

mCherry::hBACCS2 was transfected into hippocampal cells 6 days after dissection. The cells were fixed with 4% paraformaldehyde/PBS for 20 min, permeabilized with 0.1% Triton X-100/PBS for 15 min and incubated in 5% bovine serum albumin (BSA)/PBS for 15 min. The cells were then incubated with a mouse anti-Tau1 monoclonal antibody (PC1C6; Millipore; 1:4,000 dilution) in 2% BSA/PBS for 1 h. After blocking with 5% BSA/PBS for 15 min, the cells were incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen; 1:500 dilution) in 2% BSA/PBS for 30 min. Images were obtained using an LSM510 META laser scanning microscope (Carl Zeiss).

Ca²⁺ imaging

Cells were loaded with Fluo-4/AM, Fluo-8/AM (2.5 μM for both; Invitrogen) or Rhod-3/AM (10 μM; Invitrogen) according to the manufacturer’s instructions. Fluo-4/Fluo-8 and Rhod-3 were used for different wavelengths, and there were no specific reasons underlying the use of Fluo-4 and Fluo-8 for individual experiments. Lasers with wavelengths of 488 and 458 nm were used for photostimulation depending on the apparatus. The standard extracellular Ringer’s solution contained 138 mM NaCl, 5.6 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 9.4 mM D-glucose, 5 mM HEPES and 2 mM sodium pyruvate (pH 7.4, adjusted with NaOH) for mammalian cells, and 150 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 4 mM MgCl₂ and 10 mM HEPES (pH 7.2, adjusted with NaOH) for S2 cells. Intracellular Ca²⁺ was monitored using an MRC-1024 confocal laser scanning microscope (Bio-Rad Laboratories) on an Axiovert 100 inverted microscope (Carl Zeiss) equipped with a × 40 C-Apochromat objective lens (Carl Zeiss), a LSM510 META laser scanning microscope on an Axiovert 200 M microscope (Carl Zeiss) equipped with a × 40 × C-Apochromat objective lens (Carl Zeiss) or an FV1200 laser scanning microscope (Olympus) on an IX83 microscope (Olympus) equipped with a × 40 × UPlanSApo objective lens. A laser line (488 nm) was used to photoactivate BACCS and image Fluo-4/AM and Fluo-8/AM in all cell types, except for local photoactivation of hippocampal neurons. Two laser lines (488 and 559 nm) were used to photoactivate BACCS and image Rhod-3/AM, respectively. Photostimulation was performed at a scan rate of 10 μs per pixel (pixel size: 0.615 μm per pixel) with a laser line (488 nm; 10%) for Fluo-8/AM and Fluo-4/AM imaging (0.2 Hz), a scan rate of 8 μs per pixel (pixel size: 1.230 μm per pixel) with a laser line (488 nm; 20%) for Fluo-8/AM imaging (10 Hz) or a scan rate of 20 μs per pixel (pixel size: 0.615 μm per pixel) with a laser line (488 nm; 20%) for Fluo-8 imaging of reversible and repeated photoactivation experiments. For light dose-response analyses, a custom-made LED (470 nm; OSUB5111A-ST; SEMITEC) and laser line (568 nm) were used to photoactivate BACCS and image Rhod-3/AM, respectively. For Ca²⁺ imaging of hippocampal neurons, tetrodotoxin was added to the extracellular solution to exclude any effect of the action potential. For local photoactivation of hippocampal neurons, Fluo-4/AM was loaded according to the manufacturer’s instructions. Laser light (458 nm; 10%; 0.2 Hz) was used to photoactivate BACCS at a scan rate of 2.56 μs per pixel (pixel size: 0.64 μm per pixel; iterations: 3), and 633-nm and 514-nm laser lines were used to image iRFP and Fluo-4/AM, respectively. It should be noted that BACCS was not activated with a 514-nm laser. Image analyses were performed using ImageJ software (NIH). In all experiments, a positive Ca²⁺ response was defined as a stimulus-dependent deviation in Ca²⁺ fluorescence that exceeded twice the s.d. of the mean baseline fluorescence noise. All Ca²⁺ imaging experiments were performed at room temperature.

The number of experimental replicates is mentioned in each figure or figure legend except for the following Ca²⁺ imaging experiments: local activation of mouse hippocampal neurons expressing hBACCS2-IRES-mem::iRFP (n=27 cells from six independent experiments) or dmBACCS2-IRES-dOrai-IRES-iRFP (n=13 cells from five independent experiments).

No data points were excluded, except for cells with high Ca²⁺ concentrations before photoactivation, which were excluded from all analyses (Supplementary Table 1) and cells beyond the ranges of the Ca²⁺ indicator dyes after photoactivation, which were excluded from data analyses. For the generation of time-course curves of fluorescence changes (Figs 2b,g, 3b,d and 7a,e; Supplementary Figs 2b–f and 4c), cells that showed no Ca²⁺ response were excluded from the analyses (Supplementary Table 1), except for data in the absence of extracellular Ca²⁺, data for dmBACCS2 in Fig. 2b, and data for the control experiments in Figs 2g and 3d.

In most of the Ca²⁺ imaging experiments, an apparent decrease in ΔF/F was observed during the first few seconds, probably through bleaching of the Ca²⁺ indicators.

Live-cell imaging and photoactivation

Time-lapse images of NFAT translocation were obtained using the FV1200 laser scanning microscope. hBACCS2-IRES-NFAT::mCherry or dmBACCS2-IRES-dOrai-IRES-NFAT::mCherry was transfected into HEK293T cells. The cells were photoactivated every 2 min at a scan rate of 20 μs per pixel (pixel size: 0.307 μm per pixel) with a laser line (488 nm; 20%) for NFAT translocation, and the NFAT::mCherry localization was imaged using the 568-nm laser every 2 min (n=3 independent experiments for hBACCS2-IRES-NFAT::mCherry; n=7 independent experiments for dmBACCS2-IRES-dOrai-IRES-NFAT::mCherry). Time-lapse images of bleb formation were obtained using the LSM510 META laser scanning microscope. Lyn-CaRQ (containing YFP) and hBACCS2-IRES-iRFP were co-transfected into HEK293 cells. hBACCS2-transfected cells were verified by iRFP expression. The cells with YFP fluorescence were first imaged using the 514-nm laser (no photoactivation), and then photoactivated and imaged with the 488-nm laser. Cells that showed bleb formation before photoactivation were excluded from the analyses for both Lyn-CaRQ/hBACCS2-IRES-iRFP double-transfected cells and Lyn-CaRQ single-transfected cells (control). These experiments were performed at room temperature.

NFAT translocation assay

For screening of BACCS constructs, NFAT::CFP and tdTomato::LOV::STIM1 were co-transfected into HEK293T cells. Cells were either illuminated with a 470-nm LED light (cat. no. NF-1050-B; NIHON EIDO) or not illuminated, for 20 min in a darkroom. The cells were then fixed with 4% paraformaldehyde.

For analyses of the basal NFAT location, NFAT::GFP, hBACCS2-IRES-NFAT::GFP, dmBACCS2-IRES-NFAT::GFP or LOVS1K-IRES-NFAT::GFP were transfected into HEK293T cells, and the cells were fixed with 4% paraformaldehyde in the darkroom 24 h after transfection. The cells were counterstained with 4,6-diamidino-2-phenylindole (Nacalai Tesque) to visualize the nuclei. Images were obtained using the LSM510 META laser scanning microscope. Cells with brighter NFAT::CFP (or GFP) fluorescence in the nucleus compared with that in the cytoplasm were defined as cells with nuclear NFAT.

Luciferase assay and YFP induction

For the luciferase assay, HEK293T cells in a 96-well format were transfected with 50 ng per well P_NFAT-luc2P (pGL4.30[luc2P/NFAT RE/]; Promega), 50 ng per well P_SV40-hRluc (pRL-SV40; Promega) and 0–10 ng per well various Ca²⁺ photoswitch constructs, and incubated for 23 h. hRluc (Renilla luciferase) activity was used as an internal control for cell viability and transfection efficiency. After the medium was replaced with MEM supplemented with 10% FBS containing 10 ng ml⁻¹ PMA, the cells were either illuminated with a 470-nm LED light (60 μW mm⁻²) or not illuminated, for 6 h in CO₂ incubators. The firefly and Renilla luciferase activities were quantified using a Dual-Glo Luciferase assay system (Promega). Luminescence was measured using an ARVO X5 (PerkinElmer). For YFP induction, HEK293T cells in a glass-bottomed 35-mm dish with four compartments (Greiner Bio-One) were transfected with 1 μg per well pHY41 and 500 ng per well dmBACCS2-IRES-dOrai, and incubated for 24 h. The medium was then replaced with MEM supplemented with 10% FBS containing 10 ng ml⁻¹ PMA, and the cells were either illuminated with a 470-nm LED light (60 μW mm⁻²) for 12 h or not illuminated, in CO₂ incubators. YFP expression was observed using the FV1200 laser scanning microscope.

Recombinant adenovirus infection

The recombinant cosmid vector was packaged with Gigapack III Plus Packaging Extract (Agilent) according to the manufacturer’s instructions and amplified. The vector was digested with BspT1041, purified, and transfected into HEK293 cells using a CalPhos Transfection Kit (Clontech). The amplified recombinant adenovirus was used for in vivo infection. A total of 6–12 μl of the adenovirus solution was injected by pipetting into a mouse nostril.

Animals

To generate M71-IRES-ORAI1::hBACCS2-IRES-mem::tdTomato, the transgene sequence was excised by PmeI digestion, purified and microinjected into the pronuclei of C57BL/6 mice. The transgenic mice were maintained in a C57BL/6 background or crossed with the M71-IRES-tauGFP mouse strain (a kind gift from Peter Mombaerts) to generate a transgenic mouse strain with homozygous M71-IRES-tauGFP. Mice of either sex were used. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University.

Whole-mount imaging and cell counting

The surface of the olfactory epithelium from M71 transgenic mice with homozygous M71-IRES-tauGFP at 2- and 8-week old was observed in a whole-mount preparation using the FV1200 laser scanning microscope on the IX83 microscope. The numbers of cells labelled with intrinsic GFP and tdTomato fluorescence on ectoturbinates II, II′ and III were counted. The mean counts between the left and right epithelia were used as the M71 cell numbers for each mouse. Whole-mount imaging of the olfactory bulb was performed using the LSM510 META laser scanning microscope on the Axiovert 200 M microscope (n=4 mice).

Electrophysiology

For whole-cell patch-clamp recording in HEK293T cells, BACCS expression vectors were transiently transfected using the FuGENE HD Transfection Reagent (Promega). Whole-cell currents were amplified with a patch-clamp amplifier (Axopatch 200B; Molecular Devices), and then digitized with PowerLab (AD Instruments). The extracellular solutions contained 140 mM NaCl, 5 mM CsCl, 10 mM HEPES, 10 mM CaCl₂, 1.2 mM MgCl₂ and 30 mM D-glucose (pH 7.4). The electrode solution contained 140 mM CsCl, 10 mM NaCl, 10 mM HEPES, 2.84 mM CaCl₂, 2.47 mM MgCl₂ and 5 mM EGTA-2K (pH 7.4). The calculated free Ca²⁺ concentration in this solution was 100 nM. The data were low-pass filtered at 2 kHz and sampled at 10 kHz. Kinetic analysis of patch-clamp data was performed in a blinded fashion.

For perforated patch recording in Jurkat cells, cells transiently transfected with hBACCS2-IRES-mCherry were suspended in extracellular buffer solution containing 130 mM Na-methanesulfonate, 10 mM HEPES, 10 mM Ca(OH)₂, 1 mM MgCl₂ and 10 mM D-glucose (pH 7.4), and loaded on Cell-TAK-coated glass-bottomed dishes. Micropipettes of 5–10 MΩ were manufactured from borosilicate glass using a puller (PC-10; Narishige). The electrode solution contained 140 mM CsCl, 10 mM NaCl, 10 mM HEPES, 2.84 mM CaCl₂, 2.47 mM MgCl₂ and 5 mM EGTA-2Cs (pH 7.4). Nystatin was dissolved in methanol containing 100 mM N-methyl-D-glucamine, 100 mM methanesulfonic acid and 10 mM phenol red to a concentration of 5 mg ml⁻¹. Subsequently, 50 μl of this nystatin solution was evaporated, and the precipitate was dissolved in 1 ml of electrode solution. The solution was loaded into the electrode through a 0.22-μm syringe filter. After GΩ seal formation by manual suction, 10–15 min was allowed for stable perforation.

For EOG recording, mice at 5–7 weeks of age were anaesthetized with diethyl ether and euthanized by decapitation. The heads of the mice were split along the midline with a scalpel, and the nasal septum was removed so that the olfactory turbinate could be exposed. The head was placed on a recording agar chamber. Slow transepithelial potential changes were recorded from the olfactory epithelium using a glass pipette (0.1–0.5-MΩ tip resistance; filled with 140 mM NaCl, 5.6 mM KCl, 5 mM HEPES, 2 mM pyruvic acid sodium salt, 1.25 mM KH₂PO₄, 2 mM CaCl₂, 2 mM MgCl₂, 9.4 mM D-glucose and 2 wt% agar, adjusted to pH 7.4), which was connected by an Ag/AgCl wire to a DC-differential amplifier (DP-301; Warner). A second Ag/AgCl wire was connected to another glass pipette inserted into the agar chamber and served as a reference electrode. Electrical signals were band-passed at DC-100 Hz, stored on an Apple computer using a 1-kHz sampling recorder, and processed with a 30-Hz low-pass digital filter. Odorant solutions were prepared in 25 ml of water as 0.1 wt% solutions. Vapour-phase odorant stimuli were generated by collection of the headspace above 25 ml of odorant solution in a sealed 100-ml glass bottle. Vapour stimuli were injected into a stream of air under the control of a combination of custom-made transistor–transistor logic-controlled electric valves (Cole-Parmer). hBACCS2 was photoactivated with an LED and a 438±24-nm band filter (SPECTRA light engine; Lumencor). The timing of the light stimulation was controlled by a custom-made transistor–transistor logic signal controller.

Measurement of intracellular Ca²⁺ concentration

Transfected HEK293T cells were loaded with 2.5 μM Fura-2/AM for 30 min. Fluorescence was measured with an Aquacosmos Ca²⁺ imaging system and a CCD camera (ORCA-ER; Hamamatsu Photonics). Fura-2/AM was excited by a Xenon lamp (Lambda DG-4 PLUS; Sutter Instruments). For calibration of the intracellular Ca²⁺ concentration after completion of the experiments, ionomycin Ca²⁺ salt (5 μM) and the Ca²⁺ chelator EGTA (10 mM) were added to chelate the Ca²⁺ in the chamber and obtain the minimal Fura-2 fluorescence. CaCl₂ (10 mM) was then added to the imaging chamber to obtain the maximal Fura-2 fluorescence.

The intracellular free Ca²⁺ concentration was calculated from the Fura-2 fluorescence using the following equation:

where K_d is the dissociation constant of the calcium complex, β is the minimum/maximum fluorescence ratio of Fura-2 excited at the reference wavelength of 380 nm, and R is the fluorescence ratio of 340/380 nm.

Statistical analysis

Statistical analyses were performed using Excel software (Microsoft) for cell counts (t-test) and HIT-T15 cell analyses (Pearson correlation coefficient), and StatView (Hulinks) for EOG and whole-cell current analyses (t-test) and measurements of intracellular Ca²⁺ concentrations (one-way analysis of variance and post hoc Fisher’s Protected Least Significant Difference (PLSD) test). All tests were two sided.

Additional information

How to cite this article: Ishii, T. et al. Light generation of intracellular Ca²⁺ signals by a genetically encoded protein BACCS. Nat. Commun. 6:8021 doi: 10.1038/ncomms9021 (2015).

References

Gorostiza, P. & Isacoff, E. Y. Optical switches for remote and noninvasive control of cell signaling. Science 322, 395–399 (2008).
Article ADS MathSciNet CAS PubMed PubMed Central Google Scholar
Pathak, G. P., Vrana, J. D. & Tucker, C. L. Optogenetic control of cell function using engineered photoreceptors. Biol. Cell 105, 59–72 (2013).
Article CAS PubMed Google Scholar
Fenno, L., Yizhar, O. & Deisseroth, K. The development and application of optogenetics. Annu. Rev. Neurosci. 34, 389–412 (2011).
Article CAS PubMed PubMed Central Google Scholar
Christie, J. M., Salomon, M., Nozue, K., Wada, M. & Briggs, W. R. LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc. Natl Acad. Sci. USA 96, 8779–8783 (1999).
Article ADS CAS PubMed PubMed Central Google Scholar
Harper, S. M., Neil, L. C. & Gardner, K. H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003).
Article ADS CAS PubMed Google Scholar
Lee, J. et al. Surface sites for engineering allosteric control in proteins. Science 322, 438–442 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Strickland, D., Moffat, K. & Sosnick, T. R. Light-activated DNA binding in a designed allosteric protein. Proc. Natl Acad. Sci. USA 105, 10709–10714 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Berridge, M. J., Bootman, M. D. & Roderick, H. L. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529 (2003).
Article CAS PubMed Google Scholar
Berridge, M. J., Lipp, P. & Bootman, M. D. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21 (2000).
Article CAS PubMed Google Scholar
Berridge, M. J. Calcium microdomains: organization and function. Cell Calcium 40, 405–412 (2006).
Article CAS PubMed Google Scholar
Stierl, M. et al. Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. J. Biol. Chem. 286, 1181–1188 (2011).
Article CAS PubMed Google Scholar
Liu, X. & Tonegawa, S. Optogenetics 3.0. Cell 141, 22–24 (2010).
Article CAS PubMed Google Scholar
Ellis-Davies, G. C. R. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4, 619–628 (2007).
Article CAS PubMed PubMed Central Google Scholar
Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 9–34 (2011).
Article CAS PubMed Google Scholar
Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl Acad. Sci. USA 100, 13940–13945 (2003).
Article ADS CAS PubMed PubMed Central Google Scholar
Kleinlogel, S. et al. Ultra light-sensitive and fast neuronal activation with the Ca²⁺-permeable channelrhodopsin CatCh. Nat. Neurosci. 14, 513–518 (2011).
Article CAS PubMed Google Scholar
Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H. & Deisseroth, K. Temporally precise in vivo control of intracellular signalling. Nature 458, 1025–1029 (2009).
Article ADS CAS PubMed Google Scholar
Ye, H., Daoud-El Baba, M., Peng, R.-W. & Fussenegger, M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, 1565–1568 (2011).
Article ADS CAS PubMed Google Scholar
Pham, E., Mills, E. & Truong, K. A synthetic photoactivated protein to generate local or global Ca²⁺ signals. Chem. Biol. 18, 880–890 (2011).
Article CAS PubMed Google Scholar
Fukuda, N., Matsuda, T. & Nagai, T. Optical control of the Ca²⁺ concentration in a live specimen with a genetically encoded Ca²⁺-releasing molecular tool. ACS Chem. Biol. 9, 1197–1203 (2014).
Article CAS PubMed Google Scholar
Hogan, P. G., Lewis, R. S. & Rao, A. Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28, 491–533 (2010).
Article CAS PubMed PubMed Central Google Scholar
Muik, M. et al. A cytosolic homomerization and a modulatory domain within STIM1 C terminus determine coupling to ORAI1 channels. J. Biol. Chem. 284, 8421–8426 (2009).
Article CAS PubMed PubMed Central Google Scholar
Park, C. Y. et al. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136, 876–890 (2009).
Article CAS PubMed PubMed Central Google Scholar
Yuan, J. P. et al. SOAR and the polybasic STIM1 domains gate and regulate Orai channels. Nat. Cell Biol. 11, 337–343 (2009).
Article CAS PubMed PubMed Central Google Scholar
Kawasaki, T., Lange, I. & Feske, S. A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels. Biochem. Biophys. Res. Commun. 385, 49–54 (2009).
Article CAS PubMed PubMed Central Google Scholar
Kim, M.-S. & Usachev, Y. M. Mitochondrial Ca²⁺ cycling facilitates activation of the transcription factor NFAT in sensory neurons. J. Neurosci. 29, 12101–12114 (2009).
Article CAS PubMed PubMed Central Google Scholar
Li, Z. et al. Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits. Cell Res. 21, 305–315 (2011).
Article ADS CAS PubMed Google Scholar
Gwack, Y. et al. Biochemical and functional characterization of Orai proteins. J. Biol. Chem. 282, 16232–16243 (2007).
Article CAS PubMed Google Scholar
Takahashi, Y. et al. Essential role of the N-terminus of murine Orai1 in store-operated Ca²⁺ entry. Biochem. Biophys. Res. Commun. 356, 45–52 (2007).
Article CAS PubMed Google Scholar
Gross, S. A. et al. Murine ORAI2 splice variants form functional Ca²⁺ release-activated Ca²⁺ (CRAC) channels. J. Biol. Chem. 282, 19375–19384 (2007).
Article CAS PubMed Google Scholar
Prakriya, M. et al. Orai1 is an essential pore subunit of the CRAC channel. Nature 443, 10–13 (2006).
Article Google Scholar
Feske, S. et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179–185 (2006).
Article ADS CAS PubMed Google Scholar
Vig, M. et al. CRACM1 is a plasma membrane protein essential for store-operated Ca²⁺ entry. Science 312, 1220–1223 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Yeromin, A. V. et al. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443, 226–229 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Zeng, W. et al. STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol. Cell 32, 439–448 (2008).
Article CAS PubMed PubMed Central Google Scholar
Yeromin, A. V., Roos, J., Stauderman, K. A. & Cahalan, M. D. A store-operated calcium channel in Drosophila S2 cells. J. Gen. Physiol. 123, 167–182 (2004).
Article CAS PubMed PubMed Central Google Scholar
Zhang, S. L. et al. Genome-wide RNAi screen of Ca²⁺ influx identifies genes that regulate Ca²⁺ release-activated Ca²⁺ channel activity. Proc. Natl Acad. Sci. USA 103, 9357–9362 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Cahalan, M. D. Cell biology. How to STIMulate calcium channels. Science 330, 43–44 (2010).
Article CAS PubMed PubMed Central Google Scholar
Crabtree, G. R. & Schreiber, S. L. SnapShot: Ca²⁺-calcineurin-NFAT signaling. Cell 138, 210 210.e1 (2009).
Article CAS PubMed PubMed Central Google Scholar
Mills, E. & Truong, K. Ca²⁺-mediated synthetic biosystems offer protein design versatility, signal specificity, and pathway rewiring. Chem. Biol. 18, 1611–1619 (2011).
Article CAS PubMed Google Scholar
Fackler, O. T. & Grosse, R. Cell motility through plasma membrane blebbing. J. Cell Biol. 181, 879–884 (2008).
Article CAS PubMed PubMed Central Google Scholar
Cygnar, K. D., Stephan, A. B. & Zhao, H. Analyzing responses of mouse olfactory sensory neurons using the air-phase electroolfactogram recording. J. Vis. Exp pii: 1850, doi:10.3791/1850 (2010).
Rothman, A., Feinstein, P., Hirota, J. & Mombaerts, P. The promoter of the mouse odorant receptor gene M71. Mol. Cell. Neurosci. 28, 535–546 (2005).
Article CAS PubMed Google Scholar
Swartz, T. E. et al. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J. Biol. Chem. 276, 36493–36500 (2001).
Article CAS PubMed Google Scholar
Kawano, F., Aono, Y., Suzuki, H. & Sato, M. Fluorescence imaging-based high-throughput screening of fast- and slow-cycling LOV proteins. PLoS ONE 8, e82693 (2013).
Article ADS PubMed PubMed Central Google Scholar
Zayner, J. P. & Sosnick, T. R. Factors that control the chemistry of the LOV domain photocycle. PLoS One 9, e87074 (2014).
Article ADS PubMed PubMed Central Google Scholar
Park, C. Y., Shcheglovitov, A. & Dolmetsch, R. The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channels. Science 330, 101–105 (2010).
Article ADS CAS PubMed Google Scholar
Wang, Y. et al. The calcium store sensor, STIM1, reciprocally controls Orai and CaV1.2 channels. Science 330, 105–109 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Lee, K. P. et al. Molecular determinants mediating gating of Transient Receptor Potential Canonical (TRPC) channels by stromal interaction molecule 1 (STIM1). J. Biol. Chem. 289, 6372–6382 (2014).
Article CAS PubMed PubMed Central Google Scholar
Frischauf, I. et al. Molecular determinants of the coupling between STIM1 and Orai channels: differential activation of Orai1-3 channels by a STIM1 coiled-coil mutant. J. Biol. Chem. 284, 21696–21706 (2009).
Article CAS PubMed PubMed Central Google Scholar
Cai, X. Molecular evolution and functional divergence of the Ca²⁺ sensor protein in store-operated Ca²⁺ entry: stromal interaction molecule. PLoS ONE 2, e609 (2007).
Article ADS PubMed PubMed Central Google Scholar
Cai, X. Molecular evolution and structural analysis of the Ca²⁺ release-activated Ca²⁺ channel subunit, Orai. J. Mol. Biol. 368, 1284–1291 (2007).
Article CAS PubMed Google Scholar
Serizawa, S. et al. A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting. Cell 127, 1057–1069 (2006).
Article CAS PubMed Google Scholar
Mitchell, C. B., Gasperini, R. J., Small, D. H. & Foa, L. STIM1 is necessary for store-operated calcium entry in turning growth cones. J. Neurochem. 122, 1155–1166 (2012).
Article CAS PubMed Google Scholar
Tsai, F.-C. et al. A polarized Ca²⁺, diacylglycerol and STIM1 signalling system regulates directed cell migration. Nat. Cell Biol. 16, 133–144 (2014).
Article CAS PubMed PubMed Central Google Scholar
Filonov, G. S. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 29, 757–761 (2011).
Article CAS PubMed PubMed Central Google Scholar
Takemura, R. et al. Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau. J. Cell Sci. 103, 953–964 (1992).
CAS PubMed Google Scholar
Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).
Article CAS PubMed Google Scholar
Kakumoto, T. & Nakata, T. Optogenetic control of PIP3: PIP3 is sufficient to induce the actin-based active part of growth cones and is regulated via endocytosis. PLoS One 8, e70861 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank Atsushi Sato, Takuya Tamura and Hitoshi Okazawa for plasmids and Drosophila reagents, Hirofumi Nishizumi for advice on adenovirus infection, Junko Kurokawa and Akihiro Inoue for helpful discussion, Satoko Nakamura for general assistance and Haruto Makiguchi for technical help. This work was supported by JSPS KAKENHI Grant Numbers 22700410, 24500456 and 15K06735 to T.I., a grant from the Takeda Science Foundation to T.I., JSPS KAKENHI Grant Number 25293042 to T.N., JSPS KAKENHI Grant Number 21680035 to K.S., JSPS KAKENHI Grant Number 19677002 and ERATO Touhara Chemosensory Signal Project to K.T. and a Grant-in-Aid for Scientific Research on Innovative Areas ‘Fluorescence Live Imaging’ of The Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was partly supported by the Takeuchi Biohybrid Innovation Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology (JST), Japan. A part of this work was supported by the Joint Studies Program (2014–2015) of the Okazaki Institute for Integrative Bioscience.

Author information

Authors and Affiliations

Department of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
Tomohiro Ishii, Toshiyuki Kakumoto & Takao Nakata
The Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
Tomohiro Ishii, Toshiyuki Kakumoto & Takao Nakata
Division of Bioregulatory Signaling, Okazaki Institute for Integrative Bioscience, 5-1 Higashiyama, Myodaijichou, Okazaki, 444-8585, Aichi, Japan
Koji Sato
Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan
Koji Sato, Shigenori Miura & Shoji Takeuchi
ERATO Takeuchi Biohybrid Innovation Project, Japan Science and Technology Agency, Tokyo, 153-8904, Japan
Koji Sato, Shigenori Miura & Shoji Takeuchi
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
Koji Sato & Kazushige Touhara
ERATO Touhara Chemosensory Signal Project, JST, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
Kazushige Touhara

Authors

Tomohiro Ishii
View author publications
You can also search for this author in PubMed Google Scholar
Koji Sato
View author publications
You can also search for this author in PubMed Google Scholar
Toshiyuki Kakumoto
View author publications
You can also search for this author in PubMed Google Scholar
Shigenori Miura
View author publications
You can also search for this author in PubMed Google Scholar
Kazushige Touhara
View author publications
You can also search for this author in PubMed Google Scholar
Shoji Takeuchi
View author publications
You can also search for this author in PubMed Google Scholar
Takao Nakata
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

T.I. and T.N. conceived and designed the entire project; K.S. designed and performed the experiments (Fura-2 Ca²⁺ imaging, patch-clamp recording, EOG analysis), analysed the data, discussed the results and helped write the manuscript; T.K. helped with the experiments on mouse hippocampal neurons and discussed the results; S.M. helped with the experiments performed by K.S.; K.T. and S.T. supported the experiments performed by K.S.; K.T. discussed the results; T.I. performed all other experiments, analysed the data and wrote the manuscript; T.N. edited the manuscript.

Corresponding author

Correspondence to Takao Nakata.

Ethics declarations

Competing interests

T.I. and T.N. are inventors of the patent-pending BACCS constructs (polypeptide, isolated nucleic acid, recombinant vector, gene transfer kit, transformant and method for regulating intracellular calcium signalling; laid-open disclosure number: WO2012/111772). The remaining authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-7, Supplementary Table 1, Supplementary Methods and Supplementary References (PDF 1932 kb)

Rights and permissions

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

About this article

Cite this article

Ishii, T., Sato, K., Kakumoto, T. et al. Light generation of intracellular Ca²⁺ signals by a genetically encoded protein BACCS. Nat Commun 6, 8021 (2015). https://doi.org/10.1038/ncomms9021

Download citation

Received: 27 May 2015
Accepted: 09 July 2015
Published: 18 August 2015
DOI: https://doi.org/10.1038/ncomms9021

This article is cited by

Development of an optogenetics tool, Opto-RANK, for control of osteoclast differentiation using blue light
- Aiko Takada
- Toshifumi Asano
- Tomohiro Ishii
Scientific Reports (2024)
Cardiac optogenetics: shining light on signaling pathways
- Siri Leemann
- Franziska Schneider-Warme
- Sonja Kleinlogel
Pflügers Archiv - European Journal of Physiology (2023)
RCN2 promotes Nasopharyngeal carcinoma progression by curbing Calcium flow and Mitochondrial apoptosis
- Hui Yao
- Siyu Zhang
- Bo You
Cellular Oncology (2023)
Light-controlled calcium signalling in prostate cancer and benign prostatic hyperplasia
- Vipin Sharma
- Rita Rana
- Manish Nivsarkar
Future Journal of Pharmaceutical Sciences (2020)
Optogenetic manipulation of calcium signals in single T cells in vivo
- Armelle Bohineust
- Zacarias Garcia
- Philippe Bousso
Nature Communications (2020)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.