Organelle Optogenetics: Direct Manipulation of Intracellular Ca2+ Dynamics by Light

As one of the ubiquitous second messengers, the intracellular Ca2+, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca2+-dependent signals: influx from the extracellular space and release from the intracellular Ca2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca2+ release from the stores under high spatiotemporal precision, we established a new method termed “organelle optogenetics.” That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca2+-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGRER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGRER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGRER; (3) an increase of fluorometric Ca2+ was evoked by the optical stimulation (OS) in the cells expressing ChRGRER in a manner independent on the extracellular Ca2+ concentration ([Ca2+]o); (4) the ΔF/F0 was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and (5) the store-operated Ca2+ entry (SOCE) was induced by the OS in the ChRGRER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca2+ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca2+ dynamics under spatiotemporal precision.


INTRODUCTION
The intracellular Ca 2+ , as one of the second messengers, plays a pivotal role in any kind of cell by conducting information (Berridge et al., 1998;Bagur and Hajnóczky, 2017). The Ca 2+ signals that emerge from the external input, such as extracellular signaling molecules, are subsequently transduced for the activation of various signaling molecules to coordinate a wide variety of cell functions (Kakiuchi and Yamazaki, 1970;Berridge et al., 2003). When a murine myoblast, C2C12, was compelled to express one of the chimeric channelrhodopsins, channelrhodopsin-green receiver (ChRGR), a patterned optical stimulation (OS) induced an oscillation of membrane potential and accelerated the assembly of sarcomere, the smallest contractile unit in muscle fibers in a manner dependent on the increase of intracellular Ca 2+ ([Ca 2+ ] i ) (Asano et al., 2015). Therefore, the cyclic [Ca 2+ ] i elevation is assumed to be necessary for the sarcomere assembly. However, it has yet to be elucidated whether it is a sufficient condition without Ca 2+ influx through a plasma membrane.
So far, the mobilization of intracellular Ca 2+ has been investigated through pharmacological methods using drugs or caged compounds (Morad et al., 1988;Kaplan and Somlyo, 1989;Adams et al., 1997). However, these methods are limited in the spatiotemporal resolution because of the rapid diffusion of reagents in the cytoplasm. On the other hand, the optical control of Ca 2+ signaling in mammalian cells would render two major advantages over conventional approaches: high spatiotemporal resolution and tunability of the magnitude in a manner dependent on the light energy. The Ca 2+ signaling could be initiated through either influx from the extracellular space or efflux from the internal Ca 2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR) (Bagur and Hajnóczky, 2017). Here, we established our state-of-theart method "organelle optogenetics." This method manipulates the Ca 2+ release from Ca 2+ stores under high spatiotemporal precision. In fact, one of the light-responsive cation channels (ChRGR) was specifically targeted to ER/SR, and subsequently illuminated to induce Ca 2+ release from these organelles as well as their depletion.

Plasmids
A cDNA fragment encoding ChRGR-Venus (Wen et al., 2010) was amplified by PCR and subcloned into EcoRI sites in pCAGGS by In-Fusion cloning (Takara Bio, Shiga, Japan). To generate a ChRGR with an ER-retention motif (ChRGR ER ), a cDNA encoding Gln 4765 -Ile 4866 of mouse ryanodine receptor 2 (NM_023868) was inserted in-frame between ChRGR and Venus using the AgeI restriction site by In-Fusion cloning. The construct was verified by DNA sequencing.

Cell Culture and Transfection
The current-voltage (I-V) relationship of the ChRGR photocurrent was assessed using the ND 7/23 cell-a hybrid cell lines derived from neonatal rat dorsal root ganglia neurons fused with the mouse neuroblastoma (Wood et al., 1990). ND 7/23 cells were grown on a poly-L-lysine (Sigma-Aldrich, St Louis, MO)-coated coverslip in Dulbecco's modified Eagle's medium (DMEM, Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) under a 5% CO 2 atmosphere at 37 • C. The cells were maintained for no more than ten passages and grown to 80-90% confluence in the culture dish. The expression plasmids were transiently transfected in ND 7/23 cells using Effectene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The medium was supplemented with 2.5 µM all-trans retinal at 6 h after transfection. Electrophysiological recordings were then conducted 24-48 h after the transfection. Successfully transfected cells were identified by the presence of Venus fluorescence.
The C2C12 cell is a myoblast line derived from mouse skeletal muscle (RIKEN Cell Bank, Tsukuba, Japan), which has been used as one of model systems of skeletal muscle development and differentiation. The cells were maintained for no more than fifteen passages and kept at 37 • C with a 5% CO 2 atmosphere in Dulbecco's Modified Eagle's Medium (DMEM, Wako Pure Chemical Industries), which was supplemented with 20% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich). C2C12 myoblasts were grown to 80-90% confluence on a collagencoated coverslip, transfected with plasmids using either Effectene or Lipofectamine 2000 (Invitrogen) and used for patch clamp and calcium imaging experiments.

Real Time Ca 2+ Imaging
Cells were transfected with ChRGR ER and the red fluorescent Ca 2+ probe R-CaMP1.07 (Ohkura et al., 2012) plasmids simultaneously using Lipofectamine 2000. After 24 h, the red fluorescent signal of R-CaMP1.07 was acquired using a highspeed laser-scanning confocal microscopy system (A1R, Nikon, Tokyo, Japan) equipped with 16 × water-immersion objectives (0.8 NA), a 561-nm DPSS laser, a 405/488/561/640-nm dichroic mirror and a 580 ± 23 nm bandpass filter. The OS was given by high power 7-unit LED (450 ± 10 nm, LXML-PR01, Lumileds Lighting, USA). The images were sampled at 30 fps (resonant scan; 512 × 128 pixels) and analyzed using ImageJ software while regions of interest (ROIs) were each set to cover a single cell under visual identification. The fluorescence intensity in each ROI was sampled as the time series of digits and analyzed with Excel software (Microsoft Japan, Tokyo, Japan). The fluorescence change was defined as F/F 0 = (F t -F 0 )/F 0 , where F t is the fluorescence intensity at time t, and F 0 is the average baseline fluorescence 1 s before the stimulation. The averaged F/F 0 within 300 ms during the stimulation was used as the magnitude of the Ca 2+ transient. The imaging experiment was performed under superfusion with normal extracellular Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, 4 NaOH, and 11 glucose (pH 7.4 adjusted with HCl), then switched to the Ca 2+ -free extracellular Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 5 MgCl 2 , 10 HEPES, 4 NaOH, 1 Na 2 EGTA and 11 glucose (pH 7.4 adjusted with HCl). The store depletion and the subsequent store-operated Ca 2+ entry (SOCE) were investigated for the ChRGR ER -expressing C2C12 myoblasts co-transfected with R-GECO1, a red-shifted fluorescent Ca 2+ -sensor (Zhao et al., 2011), under superfusion with the Ca 2+ -free Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 3.75 MgCl 2 , 10 HEPES, 4 NaOH, and 11 glucose (pH 7.4 adjusted with HCl), then switched to the standard Tyrode's solution containing (in mmol): 138 NaCl, 3 KCl, 2.5 CaCl 2 , 1.25 MgCl 2 , 10 HEPES, 4 NaOH and 11 glucose (pH 7.4 adjusted with HCl). In some experiments thapsigargin (TG, 2-5 µM, Tocris Bioscience, Bristol, UK) was included in the Ca 2+ -free solution. For the store depletion experiments, images of R-GECO1 fluorescence were acquired using a laser-scanning confocal microscopy system (A1R, Nikon) equipped with 16× water-immersion objectives (0.8 NA). A fiber-coupled 451-nm laser source (Optohub, Saitama, Japan) was used for the optical stimulation. The free end of the optic fiber (core diameter; 50 µm, Doric Lenses, Quebec City, Canada) was placed close to the C2C12 cell. The power of light was directly measured at the free end of the optic fiber, and was 18.7 µW. For the SOCE experiments, images of R-GECO1 fluorescence were acquired every 10 s on a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40×UPlanSApo objective lens using the 559 nm excitation and 590 nm emission long pass filter sets. The OS (475 ± 10 nm, 2.8 mWmm −2 ) were applied at 20 Hz with 10 ms duration and 100 pulses between imaging sequences. The store-operated Ca 2+ entry (SOCE) was monitored using Ca 2+ add-back protocol after treating with either OS or TG in a Ca 2+ -free Tyrode's solution. The F/F 0 after Ca 2+ add-back was expressed by averaging 20 s of peak value after changing [Ca 2+ ] o .

Statistics
All data in the text and figures are expressed as the mean ± SEM and evaluated using the Mann-Whitney U-test for the unpaired data, the Wilcoxon signed rank test for the paired data, and the one-way Kruskal-Wallis test by ranks for multi-group data to determine statistical significance, unless stated otherwise. It was judged as statistically insignificant when P > 0.05.

Characterization of ER-Targeting Channelrhodopsin
It was expected that selective control of the intracellular Ca 2+ dynamics could be achieved by the specific targeting of lightsensitive actuators to the ER/SR membrane. Expanding the optogenetic toolbox enabled us to choose the optimal lightsensitive actuator depending on the experimental context (Mattis et al., 2011;Schneider et al., 2013). One of the chimeric channelrhodopsins, ChRGR, was characterized by the red-shifted light absorbance spectrum and lower desensitization over ChR2   Wen et al., 2010), and it effectively facilitated myogenesis in a manner dependent on light when expressed in C2C12 myoblasts (Asano et al., 2015). As shown in Figures 1A,B, when ChRGR was expressed in ND 7/23 cells, which are optimal for the voltage control with negligible dye coupling (Hososhima et al., 2015),  (Figure 1C). Therefore, ChRGR would be suitable for optogenetic manipulation of the Ca 2+ dynamics. We then tested the ER/SR localization of ChRGR, connecting a sequence consisting of the 3rd-4th transmembrane helices of mouse ryanodine receptor 2 at its C-terminus end (hereafter FIGURE 6 | Organelle optogenetics. In the present study, the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR)-targeted channelrhodopsin, ChRGR ER was selectively expressed in the ER/SR membrane. The light absorption by this molecule triggers release of Ca 2+ from the intracellular store, which activates various Ca 2+ signaling cascades such as calcium-induced calcium release (CICR) or store-operated calcium entry (SOCE). Nav1, voltage-dependent sodium channel 1; VDCC, voltage-dependent calcium channel; NCX, Na + -Ca 2+ exchanger; RyR, ryanodine receptor; IP 3 R, IP 3 receptor; SERCA, sarco/endoplasmic reticulum Ca 2+ -ATPase.
called ChRGR ER ). The expression patterns of ChRGR and the ChRGR ER were visualized by Venus, which was tagged at their C-terminus (Figures 2A,B). The signal of ChRGR ER -Venus was confined in the peri-nucleus region in contrast to the conventional ChRGR-Venus, which localized in the plasma membrane with the original membrane targeting property (Wen et al., 2010;Asano et al., 2015). To further examine the precise localization, ChRGR ER -expressing C2C12 cells were immunostained for ER/SR-marker KDEL and subsequently observed under the STED super-resolution microscopy (Figures 2C-F). As shown in Figure 2F and Movie S1, the Venus fluorescence was mostly co-localized with the KDEL signal.
Leaked expression of ChRGR ER in the plasma membrane caused by miss-targeting or overexpression would hamper the precise regulation of the intracellular Ca 2+ dynamics. To determine whether functional ChRGR ER was incorporated in the plasma membrane, we performed patch clamp experiments under the whole-cell voltage clamp configuration. Cyan light (505 ± 15 nm; 1.58 mWmm −2 ; 1 s) evoked a photocurrent of 428 ± 65 pA (n = 6) in the ChRGR-expressing cells ( Figure 3A). On the other hand, the photocurrent was negligible for the ChRGR ER -expressing cells (n = 7) even with the same light stimulation (Figure 3B), with significant difference (Figure 3C).

Functional Operation of ChRGR ER in the Cell
To determine whether the ChRGR ER was fully functional in the ER/SR, we performed real-time Ca 2+ imaging using the red fluorescent Ca 2+ indicator R-CaMP1.07 (Ohkura et al., 2012). The double positive cells with ChRGR ER -Venus and R-CaMP1.07 were identified and stimulated with a train of pulses (5 ms at 20 Hz for 1 s) of blue LED light (4.5 mWmm −2 at 450 nm). In the standard extracellular milieu containing 2 mM Ca 2+ , the ChRGR ER -expressing cells responded to the light by 7.1 ± 1.4% (n = 11) red fluorescence F/F 0 (Figures 4A,B). When the perfusing solution was switched to the [Ca 2+ ] ofree solution containing 1 mM EGTA, the F/F 0 signal was significantly enhanced to 8.8 ± 1.8% by the same light. However, the light-dependent F/F 0 was reduced with the repetitive OS when the Ca 2+ entry was suppressed by the external EGTA and nifedipine and the ER/SR uptake were blocked by thapsigargin (Figures 4C,D). In fact, the F/F 0 ranged from −15 to −11% of the initial value at the end of the repetition (10-50 cycles, n = 3).
Generally, the depletion of Ca 2+ from the ER/SR induced Ca 2+ entry through the plasma membrane and facilitated the subsequent uptake of Ca 2+ (store-operated Ca 2+ entry, SOCE) by the coupling between STIM and Orai proteins (Prakriya and Lewis, 2015). Indeed, when the Ca 2+ store in a C2C12 cell was depleted by treatment with thapsigargin (5 µM) in a [Ca 2+ ] ofree milieu, the entry of Ca 2+ was evident from the [Ca 2+ ] i increase upon the add-back of [Ca 2+ ] o to 2.5 mM (Figure 5A). Similarly, a significant Ca 2+ entry was observed when the OS was repetitively applied before the [Ca 2+ ] o add-back (Figure 5B), yet was negative in a [Ca 2+ ] o -free milieu ( Figure 5C). In summary, a significant SOCE was observed after OS of the ChRGR ERexpressing C2C12 cells ( Figure 5C).

DISCUSSION
This paper demonstrated for the first time specific control of intracellular Ca 2+ dynamics by light with the application of organelle optogenetics using an ER/SRtargeted channelrhodopsin, ChRGR ER , that includes the 4th transmembrane helix of ryanodine receptor as an ERretention motif (Bhat and Jianjie, 2002). This conclusion was supported by the following evidences: (1) the localization pattern of ChRGR ER was 3-dimensionally merged with the ER markers even when examined under the super-resolotion microscopy; (2) whole cell patch clamp recording detected the photocurrent in the ChRGR-expressing C2C12 cells but not in the ChRGR ER -expressing ones; (3) the fluorometric Ca 2+ signal ( F/F 0 ) was induced by the OS in the ChRGR ER -expressing cells even in the absence of [Ca 2+ ] o (see also Movie S2). It was even larger than that in the presence of [Ca 2+ ] o , probably due to the reduction of basal [Ca 2+ ] i ; (4) the F/F 0 was sensitive to the SERCA inhibitor (thapsigargin). (5) the SOCE was induced by the OS in the ChRGR ER -expressing cells. The organelle optogenetics (Figure 6) would thus enable the regulation of not only the intracellular Ca 2+ release from ER/SR but also the subsequent activation of other internal Ca 2+ signaling cascades such as calcium-induced calcium release (CICR) (Endo et al., 1977) or SOCE (Soboloff et al., 2012;Bagur and Hajnóczky, 2017). Although prevalent in every cell, tissue, and organ of any living organism, the consequences of the spatiotemporal dynamics of intracellular Ca 2+ have not been extensively studied because of technical limitations in differentiating between the two major sources of Ca 2+ mobilization: the extracellular milieu and the internal Ca 2+ stores. Therefore, the present optogenetic manipulation of ER/SR would represent a breakthrough in elaborating its physiological significance, such as the triggering of the sarcomere assembly (Ferrari et al., 1996(Ferrari et al., , 1998Li et al., 2004). Accumulating evidence has indicated that intracellular Ca 2+ stores similarly have an important functional role in neurons; for example, somatodendritic signaling, synaptic transmission and plasticity (Simpson et al., 1995;Bouchard et al., 2003;Collin et al., 2005;Korkotian, 2014, 2015) in addition to being involved in neurodegenerative diseases such as Alzheimer's (Villegas et al., 2014;Zhang et al., 2016). Further improvements in optogenetic molecular tools, targeting and expression techniques, and optical systems will enable the precise manipulation of intracellular Ca 2+ in neuroscience.

AUTHOR CONTRIBUTIONS
TA, HI, TI, and HY conceived, designed, and performed the experiments, and analyzed the data. HI, TI, and HY contributed reagents, materials, and analysis tools. All authors drafted and reviewed the manuscript. and ER-marker KDEL (red) were merged for the same ROI shown in Figure 1C. Scale, 2 µm.
Movie S2 | Representative movies of light-induced intracellular Ca 2+ increase in a ChRGR ER -expressing C2C12 cell. (A,B) The R-GECO1 signals were sampled at 2 Hz and the fluorescence intensity (F) was imaged during an optical stimulation (OS: 451 nm; duration, 20 ms; 10 Hz for 5 s) in the presence (A) and absence (B) of extracellular Ca 2+ ([Ca 2+ ] o ). The images were sampled at 2 Hz and pseudocolor-displayed at 20 Hz. Scale bar, 10 µm. (C,D) The changes ( F/F 0 ) of R-GECO1 fluorescence of ROIs above the cells shown in (A,B), respectively. Each OS was indicated as a cyan stripe.