High-performance optical control of GPCR signaling by bistable animal opsins MosOpn3 and LamPP in a molecular property–dependent manner

Edited by Paul Sternberg, California Institute of Technology, Pasadena, CA; received March 11, 2022; accepted October 5, 2022
November 23, 2022
119 (48) e2204341119

Significance

Optogenetics for GPCR signaling is highly valuable but is still developing and requires effective tools like ChR2. We demonstrated that two animal opsins, mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP), are available in optogenetics equivalently to ChR2 in terms of retinal requirement. Furthermore, MosOpn3 introduced into nociceptor neurons of C. elegans exhibited ~7,000 times higher sensitivity than ChR2 in the light-induced avoidance behavior. LamPP introduced into motor neurons induced violet light-dependent stop and green light-dependent go, demonstrating color-dependent control of behavior using LamPP. In addition, our molecular engineering extended the usability of MosOpn3 and LamPP to different signaling cascades and kinetics. The current findings provide numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.

Abstract

Optical control of G protein-coupled receptor (GPCR) signaling is a highly valuable approach for comprehensive understanding of GPCR-based physiologies and controlling them precisely. However, optogenetics for GPCR signaling is still developing and requires effective and versatile tools with performance evaluation from their molecular properties. Here, we systematically investigated performance of two bistable opsins that activate Gi/Go-type G protein (mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP)) in optical control in vivo using Caenorhabditis elegans. Transgenic worms expressing MosOpn3, which binds 13-cis retinal to form photopigments, in nociceptor neurons showed light-induced avoidance responses in the presence of all-trans retinal, a retinal isomer ubiquitously present in every tissue, like microbial rhodopsins and unlike canonical vertebrate opsins. Remarkably, transgenic worms expressing MosOpn3 were ~7,000 times more sensitive to light than transgenic worms expressing ChR2 in this light-induced behavior, demonstrating the advantage of MosOpn3 as a light switch. LamPP is a UV-sensitive bistable opsin having complete photoregenerative ability by green light. Accordingly, transgenic worms expressing LamPP in cholinergic motor neurons stopped moving upon violet light illumination and restored coordinate movement upon green light illumination, demonstrating color-dependent control of behavior using LamPP. Furthermore, we applied molecular engineering to produce MosOpn3-based tools enabling light-dependent upregulation of cAMP or Ca2+ levels and LamPP-based tool enabling clamping cAMP levels color dependently and context independently, extending their usability. These findings define the capacity of two bistable opsins with similar retinal requirement as ChR2, providing numerous strategies for optical control of various GPCR-based physiologies as well as GPCR signaling itself.
G protein–coupled receptors (GPCRs) are transmembrane receptors, which are involved in various cellular and physiological functions including neural responses, cell metabolisms, and hormonal responses (1, 2). GPCRs generally bind a variety of chemical ligands such as odorants, hormones, and neurotransmitters and transduce these extracellular signals into intracellular responses (GPCR signaling) via heterotrimeric G proteins. The GPCR signaling varies mainly depending on the subtype of G protein alpha subunit engaged and includes upregulation and downregulation of cAMP, by Gs- and Gi-type G proteins, respectively, and phosphoinositol signaling for Ca2+ elevation mediated by Gq-type G protein. Most animals have hundreds of GPCR genes, and humans, for example, have ~800 GPCR genes, indicating the importance of GPCR signaling for biological activities (3). Although the structure–function relationships of many GPCRs have been well investigated so far, for comprehensive understanding of GPCR-based physiologies as well as controlling them precisely, optical control of GPCR signaling would be one of the ultimate approaches because of high temporal resolution of light stimulus.
Animal rhodopsins (opsin-based pigments), which underlie vision and nonvisual functions such as circadian photoentrainment in varied animals, consist of a protein moiety, opsin, and a chromophore (11-cis retinal in many cases) and basically serve as light-sensitive GPCRs (4). Therefore, opsins have been considered promising tools for optical control of GPCR signaling, and indeed, such optogenetic application has succeeded to some extent using vertebrate visual opsins (57). However, the fact that relatively small number of papers on optogenetic research using animal opsins have been published until now compared with that using microbial rhodopsins such as ChR2 for optical control of neural activities suggests that there is still room for improvement. The point would be related to molecular properties of vertebrate visual opsins: 1) they basically form photopigments by binding to 11-cis retinal, which is abundant in photoreceptor tissues like the eyes but not in other tissues (11-cis retinal requirement), and 2) after light absorption, their photoproducts immediately release chromophore (bleach) to be functionless (bleaching property), both of which are unfavorable characteristics for high-performance optical control of GPCR signaling, especially in vivo (4). To overcome the problems associated with the molecular properties, utilization of animal opsins that is more suitable for optogenetic tools should be required.
To date, thousands of opsins have been identified from a wide range of animals from cnidarians to vertebrates, and they are phylogenetically divided into eight or more groups, which is almost consistent with the classification based on G protein selectivity and activation manner (4, 8). With respect to the photochemical property, opsins are basically classified into two types: bleaching opsins like vertebrate visual opsins and bleach-resistant or bistable opsins, which convert to stable active states upon light absorption, and in many cases, the active states revert to the original inactive state by subsequent light absorption (4, 9), like invertebrate visual opsins (1013). The bistable nature appears to be suitable for sustained optical control of GPCR signaling. In fact, a bistable opsin melanopsin (OPN4) was applied to optical control of some physiologies including restoration of vision (1416). In addition to melanopsin, we have identified many kinds of bleach-resistant/bistable opsins from both invertebrates and vertebrates (10, 1723). Among them, we particularly focused on optogenetic potentials of Opn3 and parapinopsin because of their interesting molecular properties, which are recently receiving increasing attention (24).
Opn3 was first identified from the mammalian brain and therefore originally called encephalopsin (25). Then, its homologues were identified from many animals and revealed to be expressed in their various tissues including the brain, suggesting their involvement in photoreception in “nonphotoreceptive” tissues. We have previously succeeded in functional analyses of members of the Opn3 group (22, 26) and found that one of the members mosquito Opn3 (MosOpn3) has a unique property; it forms a photopigment that light dependently activates Gi- and Go-type G proteins when bound to 13-cis retinal as well as 11-cis retinal (22, 27). Since 13-cis retinal is thermally equilibrated with all-trans retinal, a retinal isomer ubiquitously present in animals, we have proposed the idea that the Opn3 is applicable to anywhere in the body as an optogenetic tool. In fact, mammalian cultured cells expressing the MosOpn3 exhibited a light-induced decrease in the intracellular cAMP level probably mediated by the Gi activation with all-trans retinal addition or even without retinal addition in the presence of only a small amount of retinoid in the serum (22). The idea was also supported by a recent report showing optogenetic silencing of neurotransmitter release with the MosOpn3 in vitro and in vivo (28).
Another promising bistable opsin parapinopsin was first identified from catfish pineal and parapineal organs (29) and thereafter from many lower vertebrate pineal-related organs (17, 23, 30, 31). Spectroscopic and biochemical analyses revealed that the lamprey parapinopsin (LamPP) is an ultraviolet (UV)-sensitive bistable opsin, which activates transducin and Gi-type G protein upon UV light absorption (3234). Notably, parapinopsins convert to a stable active state having an absorption maximum at ~500 nm (in the green region), which is largely distinct from that of the inactive dark state (32). The large spectral difference between the inactive and active states allows selective illumination of the active state, resulting in its complete recovery to the inactive state (17). The same is true for the signal transduction level. The G protein activation by parapinopsin was up- and down-regulated by UV and green light illumination, respectively, in vitro and in vivo (3338), demonstrating its optogenetic potential for color-deendent on and off of GPCR signaling.
Here, to evaluate performances of MosOpn3 and LamPP in optical control of GPCR signaling in vivo based on their molecular properties, we focused on Caenorhabditis elegans, in which the relationships between GPCR signaling and behaviors have been well defined. Importantly, in the case of optogenetics in C. elegans, isomeric forms of chromophore retinal can be controlled by exogenously adding specific retinal isomers (6, 39), which is an irreplaceable advantage in testing the chromophore requirement of opsin for functioning in vivo, in 11-cis form poor condition. In this paper, we showed that MosOpn3 functions in vivo under the absence of 11-cis retinal with much higher sensitivity compared with ChR2, which is the most used optogenetic tool. We also succeeded in color-dependent control of C. elegans behavior using LamPP. Together with our demonstration of introducing G protein selectivity of particular GPCRs into MosOpn3 and LamPP, the current findings provide versatile and powerful optogenetic tools for controlling GPCR signaling and various physiologies based on the molecular properties of the two opsins.

Results

Optical Control of C. elegans Behavior Using the Mosquito Opn3.

We evaluated the performance of mosquito Opn3 (MosOpn3) for its molecular property–based optical control of GPCR signaling in vivo using C. elegans. Since MosOpn3 is a Gi/o-coupled opsin (22), we focused on ASH neurons, a kind of nociceptors, in which chemoreceptors trigger Gi/o-like G protein (ODR-3)–mediated signal transduction upon ligand binding to induce avoidance behavior of C. elegans (40, 41) (Fig. 1A). MosOpn3 was introduced into ASH neurons under the control of the sra-6 promoter, a chemosensory receptor mainly expressed in ASH neurons (42) (Fig. 1B). We obtained several lines of transgenic (Tg) worms that express MosOpn3 in ASH neurons (MosOpn3-worms) with the aid of the pharynx expression of mCherry introduced as a selection marker together with MosOpn3. The expression of MosOpn3 in ASH neurons was confirmed by the expression of GFP, which was designed to be bicistronically expressed with MosOpn3 in ASH neurons. We performed behavioral experiments (Fig. 1C) for a light-induced avoidance of MosOpn3-worms that were fed 11-cis retinal–containing Escherichia coli (MosOpn3/11-worms). As a result, MosOpn3/11-worms exhibited clear avoidance responses by illumination of white light (Fig. 2A upper panels, Movie S1A). On the other hand, MosOpn3-worms without a supply of retinal (MosOpn3/NoRet-worms) did not exhibit the light-induced avoidance responses at all (Movie S1B), which is consistent with previous observations that the addition of retinal is required for functioning of rhodopsins in C. elegans (6, 39). The retinal requirement demonstrated that the light-induced avoidance responses of MosOpn3-worms were not caused by the endogenous light sensor protein, lite-1 (43, 44), but indeed by MosOpn3 provably through Gi/o-mediated signaling. Collectively, the results ensure the validity of our experimental conditions including light intensity for investigating the functionality of heterologously expressed animal opsins in C. elegans. Importantly, when MosOpn3-worms were fed all-trans retinal (MosOpn3/AT-worms), they also exhibited the light-induced avoidance responses (Fig. 2A, Lower Panels and Movie S1C) like the case of MosOpn3/11-worms, which can be explained by the unique molecular property of MosOpn3, the pigment formation ability with 13-cis retinal thermally generated from all-trans form as observed in our previous in vitro experiment (22).
Fig. 1.
Examination of the functionality of MosOpn3 in ASH neurons of C. elegans. (A) The construction for introducing MosOpn3 with EGFP under the sra-6 promoter. A selection marker mCherry was also introduced under the myo-2 promoter. (B) GPCR signaling in ASH neurons. Activation of ODR-3, a Gi-like protein eventually causes avoidance behavior of C. elegans. (C) Experimental setup. Worms were continuously monitored with infrared light. Light stimuli were supplied using a dichroic mirror, and the intensities were regulated with ND filters.
Fig. 2.
Functionality of MosOpn3 in ASH neurons of C. elegans. (A) Snapshot images at 0.7, 1.6, 2.5, 3.4, 4.3, and 5.2 s after recording start, showing light-induced avoidance responses of MosOpn3-expressing Tg worms in the presence of 11-cis (Upper) and all-trans (Lower) retinal. The horizontal bar and mCherry signals emitted by the light illumination indicate the timing of white light stimulus. Dotted lines indicate the position of the frontal tip of worms when they stopped moving forward. (B and C) White light intensity–response probability relationships for MosOpn3-worms (magenta squares and curve) and BovRh-worms (green squares and curve) in the minimum necessary amount (1/100 of the standard amount of retinal) 11-cis retinal (B, n = 6) and all-trans retinal (C, n = 3). Both Tg worms exhibited a similar light sensitivity in the case of 11-cis retinal, whereas only MosOpn3-worms responded to light in the presence of all-trans retinal. The light intensity–response probability relationship for MosOpn3/11-worms is also indicated in (C) for a comparison (black open squares and dashed curve), showing a similar performance of MosOpn3/11- and MosOpn3/AT-worms in the light-induced avoidance behavior. (D) White light intensity–response probability relationships for MosOpn3/11-worms (black triangles) and MosOpn3/AT-worms (blue triangles) in the presence of 1/1,000 of the standard amount of retinal (n = 3). (EG) Response probabilities of Tg worms at critical light intensities for B (E, I = 3 × 10−4I0), C (F, I = 3 × 104I0), and D (G, I = 3 × 103I0) were evaluated (n = 10). The data for MouSWS1- and HumLWS-worms in the presence of the standard amount of 11-cis retinal are also shown. I0 = ~0.8 mW/mm2 (BG).
Movie S1
Movies showing typical light-induced avoidance behaviors of Tg C. elegans expressing MosOpn3 or BovRh in ASH neurons. (A) MosOpn3/11-worm. (B) MosOpn3/NoRet-worm. (C) MosOpn3/AT-worm. (D) BovRh/11-worm. (E) BovRh/NoRet-worm. (F) BovRh/AT-worm. Timings of light illumination were indicated by emergence of white frames.

Comparison of the Functionality and Efficiency In Vivo between MosOpn3 and BovRh.

To evaluate the advantage of MosOpn3 in optical control of GPCR signaling in vivo, we also investigated the light-induced avoidance behaviors of Tg C. elegans expressing a bleaching opsin, bovine rhodopsin (BovRh), which activates Gi/o-type G protein (45) like MosOpn3 and does not bind 13-cis retinal or all-trans retinal unlike MosOpn3. Tg worms established to express BovRh in ASH neurons (BovRh-worms) exhibited the avoidance behavior by white light illumination when they were fed 11-cis retinal–containing E. coli (Movie S1 D and E). On the other hand, BovRh-worms did not respond to light when they were fed all-trans retinal–containing E. coli (Movie S1F), which is different from the case of MosOpn3/AT-worms, showing good agreement with molecular properties of respective opsins. We then compared the performances of these two animal opsins in optical control of GPCR signaling in vivo quantitatively. We investigated the relationships between light intensity and light-induced avoidance response probabilities of MosOpn3- and BovRh-worms with various amounts of 11-cis retinal to find the necessary amount of 11-cis retinal for functioning in ASH neurons to avoid side effects caused by an excess amount of retinal. The light intensity–response probability relationships revealed that MosOpn3-worms exhibited a similar light sensitivity even when the amount of 11-cis retinal was reduced to 1/100, and the sensitivity was slightly decreased under 1/1,000 amount of 11-cis retinal (SI Appendix, Fig. S1A). In the case of BovRh-worms, the light sensitivity was also similar even when the amount of 11-cis retinal was reduced to 1/100, but the sensitivity was largely decreased under 1/1,000 amount of 11-cis retinal (SI Appendix, Fig. S1B). These results indicate that the 1/100 amount is necessary to induce maximum performance for both opsins in this behavior. We then compared performances of MosOpn3- and BovRh-worms in the presence of the minimum necessary amount of retinal. We found that light sensitivities of MosOpn3/11- and BovRh/11-worms are similar, which suggests a similarity in performance of 11-cis–binding MosOpn3 and BovRh in the ASH neurons (Fig. 2 B and E). On the other hand, in the same amount of all-trans retinal, MosOpn3/AT-worms exhibited the avoidance responses by illuminations of 1/10,000~1/1,000 of the maximum intensity (I0) of light, whereas BovRh/AT-worms did not exhibit any avoidance responses even by illuminations of the maximum intensity of light (Fig. 2 C and F), demonstrating that the sensitivity of MosOpn3/AT-worms is more than 1,000 times higher than that of BovRh/AT-worms. Remarkably, the sensitivity of MosOpn3/AT-worms is comparable with that of MosOpn3/11-worms (Fig. 2C). In addition, when the amounts of 11-cis and all-trans retinal added to worms were reduced to 10 times lower level (1/1,000 of the standard amount), the sensitivities of MosOpn3-worms in the presence of 11-cis and all-trans retinal decreased similarly (Fig. 2 C, D, and G). Since the decreases of sensitivity are explained by those of formed photopigment amount in C. elegans, the similarities in sensitivity between MosOpn3/11- and MosOpn3/AT-worms under varied retinal amounts suggest that MosOpn3 bound to 11-cis retinal and MosOpn3 bound to 13-cis retinal, which is thermally generated from all-trans retinal, can function with similar efficiency in ASH neurons. These results clearly demonstrated that MosOpn3 performs efficiently in ASH neurons to evoke avoidance responses even in the presence of all-trans retinal like microbial rhodopsins.

Engineering MosOpn3 for Light-Dependent Upregulation of Intracellular cAMP and Ca2+ Levels.

MosOpn3 forms a photopigment by binding 13-cis retinal to activate Gi- and Go-type G proteins in a light-dependent manner (22), which leads to a decrease in cAMP. To expand the scope of optogenetic application of MosOpn3, we engineered MosOpn3 to up-regulate cAMP and Ca2+ levels, through Gs- or Gq-type G protein, respectively, by exchanging the cytoplasmic region(s) including the third cytoplasmic loop, the major determinant of G protein selectivity for Class A GPCRs (4551). We chose the ß2-adrenergic receptor (ß2AR) and α1-adrenergic receptor (α1AR), which are generally considered to selectively activate Gs- and Gq-type G proteins, respectively, as donor GPCRs. We generated a series of MosOpn3-ß2AR and MosOpn3-α1AR chimeras, in which the cytoplasmic region(s) were replaced with those of ß2AR or α1AR according to previous reports (5, 50), and investigated functional conversion. In the case of MosOpn3-ß2AR chimeras, we measured light-induced increases of the cAMP level in cultured cells expressing each chimera using the GloSensor cAMP assay and revealed that all chimeras induced obvious cAMP increases light dependently in contrast to the cAMP decrease in the wild type (WT) even without addition of retinal (Fig. 3A), indicating successful conversion of cAMP regulation from down- to upregulation by MosOpn3 bound to endogenous retinal in the culture medium. Any MosOpn3-ß2AR chimeras exhibited larger cAMP increases upon light absorption than the previously reported MosOpn3 chimera containing the third cytoplasmic loop of jellyfish Gs-coupled opsin (20, 26). Among all, the chimera in which all cytoplasmic regions were replaced with those of ß2AR (MosOpn3-ß2ARiL123C) exhibited the largest cAMP increase, and the chimera containing the second and third cytoplasmic loops, and C-terminal region of ß2AR (MosOpn3-ß2ARiL23C) was comparable. An interesting finding is that introducing the first cytoplasmic loop of ß2AR accelerates the reversion of increased cAMP levels to the basal level, which provides choices of different kinetics of cAMP changes based on the chimera species (Fig. 3A).
Fig. 3.
Engineering MosOpn3-based tools to activate Gs- or Gq-type G protein. (A) GloSensor cAMP assay with HEK293 cells expressing various MosOpn3-ß2AR chimeras, showing light-induced increases of intracellular cAMP levels. MosOpn3 chimera containing the third cytoplasmic loop of jellyfish Gs-coupled opsin (MosOpn3-JelOpiL3) is also shown for a comparison. The light-induced cAMP decrease in HEK293 cells expressing MosOpn3 WT is shown in Inset. Vertical lines indicate light illumination for 5 s. (B) Aequorin Ca2+ assay with HEK293 cells expressing MosOpn3-α1ARiL3 chimeras having varied lengths of the third cytoplasmic loop of α1AR, showing light-induced increases of intracellular Ca2+ levels (Left). Light-induced changes of intracellular cAMP levels of HEK293 cells expressing the MosOpn3-α1AR chimeras are also shown (Right). Note that other MosOpn3-α1AR chimeras did not show significant improvement of light-induced Ca2+ elevation. Vertical lines indicate light illumination for 1 s (Left) and 5 s (Right).
Regarding the regulation of Ca2+ level, aequorin luminescence-based calcium assay revealed that MosOpn3 WT originally exhibited a light-induced Ca2+ increase in cultured cells (Fig. 3B), although it also induced a decrease in the cAMP level as described previously (22) (Fig. 3 A and B). Then, we evaluated specific regulation of Ca2+ by MosOpn3-α1AR chimeras in cultured cells expressing each chimera. Contrary to the case of MosOpn3-ß2AR chimeras, only the MosOpn3-α1AR chimera having the third cytoplasmic loop of α1AR (MosOpn3-α1ARiL3) exhibited a light-induced Ca2+ increase under the condition without 11-cis retinal addition, although the Ca2+ increase by the chimera is less than that that by the WT, and the chimera still exhibited a cAMP decrease (Fig. 3B). To improve the specificity of the MosOpn3-α1ARiL3 for a Ca2+ increase, we further engineered the chimera by fine-tuning of the third cytoplasmic loop to be exchanged by shifting the donor/acceptor boundaries by two or four amino acids. In all possible combinations, we found that chimeras generated by reduction of two amino acids at the N-terminal and addition of four amino acids at the C-terminal of the third cytoplasmic loop of α1AR (MosOpn3-α1ARiL3(−2/+4)) exhibited the highest functionality in light-induced Ca2+ elevation, which is ~2.5-fold higher than the case of WT without a cAMP decrease. The light-induced cAMP increases observed in the case of MosOpn3-α1ARiL3(−2/+4) and MosOpn3-α1ARiL3(−2/+2) were presumably caused by Ca2+ elevation (5254). These results suggest the practical strategy for creating on-demand optogenetic tools based on MosOpn3 for controlling intracellular cAMP and Ca2+ levels probably through optimizing G protein signaling preference.

Parapinopsin for Color-Dependent Control of GPCR Signaling.

In cases of visible light–sensitive bistable opsins including MosOpn3, the dark (inactive) and photoproduct (active) states have largely overlapped absorption spectra in the visible light region. On the contrary, in the case of parapinopsin, the absorption spectra of the UV-sensitive inactive state and visible light–sensitive active state are distinct from each other, enabling to illuminate only the active state with visible light, which leads to complete regeneration of the inactive state. To evaluate the contribution of the photoregeneration ability in optical control of GPCR signaling in vivo, we focused on another C. elegans behavior, coordinated movement regulated by acetylcholine. Using the unc-17 promoter, we introduced LamPP into cholinergic motor neurons, in which activation of Go-mediated signal transduction down-regulates acetylcholine releases to lead uncoordinated movement including coiling of C. elegans (55, 56) (Fig. 4 A and B). Tg worms expressing LamPP in cholinergic motor neurons (LamPP-worms) were obtained with the same procedure as that for MosOpn3-worms. When we illuminated LamPP-wormsfed 11-cis retinal–containing E. coli with violet light, the Tg worms stopped moving and coiled as expected, and upon subsequent green light illumination, the Tg worms restarted coordinated movement (Fig. 4 C and D and Movie S2). The violet light–induced coiling of LamPP-worms sustained for 30 min after the turnoff of light, and subsequent green light illumination restored the movement (Fig. 4E). The behavioral switching between coiling and moving upon violet and green light stimuli occurred repeatedly (Movie S2), demonstrating color-dependent control of the C. elegans behavior using LamPP. In other words, the result suggested that introducing LamPP into cells containing Gi/o-mediated signaling could render physiologies color dependency. Furthermore, LamPP-wormsfed all-trans retinal under the red light (600 nm) exhibited violet light–induced stop and green light–induced recovery like the case of LamPP-wormsfed 11-cis retinal (Fig. 4D). Since retinal hardly absorbs red light, the phenomena can be explained by the complete photoregeneration ability of LamPP; LamPP bound all-trans retinal to form the active state (LamPP* in Fig. 4B) directly, and the active state completely converted to the 11-cis retinal–binding inactive state by red light absorption.
Fig. 4.
Color-dependent control of Tg C. elegans expressing LamPP in cholinergic motor neurons. (A) The construction for introducing LamPP with EGFP under the unc-17 promoter. (B) A schematic drawing of the relationship between status of LamPP and C. elegans behavior caused by violet and green light illumination. LamPP* indicates the active state of LamPP. (C) Snapshot images of the behavior of C. elegans expressing LamPP in cholinergic neurons in the dark, after violet illumination, and subsequent green light illumination, showing moving forward, coiling, and restart moving, respectively (Movie S2). (D) Quantitative evaluation of violet light–induced coiling and subsequent green light–induced recovery of LamPP-worms (n = 5). LamPP-worms fed E. coli without retinal (gray circles), with 11-cis retinal (magenta circles) in the dark, and with all-trans retinal (blue circles) under the red light were illuminated with violet light for 5 s (violet line), kept in the dark for 1 min, and illuminated with green light for 30 s (green line). (E) Violet light–induced coiling of LamPP-worms fed 11-cis retinal sustained for 30 min after turnoff of the violet light. All worms exhibited immobility and/or coiling for 30 min. Upon green illumination, all worms restarted moving.
Movie S2
The movie showing color-dependent control of behaviors of Tg C. elegans expressing LamPP in cholinergic motor neurons. A LamPP-worm was moving before illumination, stopped moving and coiled upon violet light illumination, and restarted movement upon green light illumination, which occurred repeatedly. Timings of violet and green light illumination were indicated by emergence of violet and green frames, respectively. Signals derived from mCherry were also detected by green light illumination.
Again, we engineered LamPP, which originally activates Gi/o-type G protein, to activate Gs-type G protein to up- and down-regulate intracellular cAMP levels with violet and visible light absorption, respectively, which is in the opposite direction caused by the WT. We generated a series of LamPP chimeras, in which the cytoplasmic region(s), including the third cytoplasmic loop, was replaced with those of ß2-adrenergic receptor as in the case of engineering Gs-coupled MosOpn3. Light-induced changes of the cAMP level in cultured cells expressing each chimera were measured using the GloSensor cAMP assay. We found that only the cells expressing the chimera containing the third cytoplasmic loop alone (LamPP-ß2ARiL3) exhibited a significant violet light–induced increase in the cAMP level, which in turn decreased to the basal level by subsequent green light illumination (Fig. 5A). The color-dependent upregulations and downregulations of cAMP levels occurred repeatedly. Notably, the cAMP increases induced by activation of the chimera were composed of two phases; upon violet light illumination, cAMP levels rapidly increased to a higher level and immediately decreased to a lower level (acute phase), and the lower level sustained for more than 1 h until green light illumination (chronic phase). Interestingly, when LamPP-ß2ARiL3–expressing cells were illuminated with blue light, the cAMP level was set at a level between levels caused by violet and green light illumination (Fig. 5B). Furthermore, the cAMP levels caused by blue light illumination were almost constant regardless of the levels just before illumination. The color dependency of the sustained cAMP level could be explained in part by color-dependent photoequilibrium of the inactive and active states of LamPP (17, 35). We also tested the performance of an additional LamPP chimera containing the third cytoplasmic loop of the jellyfish Gs-coupled opsin, LamPP-JelOpiL3. The LamPP-JelOpiL3 exhibited much higher amplitude of violet light–induced cAMP increases compared with LamPP-ß2ARiL3, whereas the increased cAMP level gradually decreased (SI Appendix, Fig. S2), unlike the case of LamPP-ß2ARiL3. The increase and decrease in the cAMP level by violet light and green light illumination also occurred repeatedly for LamPP-JelOpiL3. Collectively, these Gs-coupled LamPPs enable to control Gs-mediated signal transduction in a color-dependent manner, showing successful expansion of LamPP as tools for color-dependent control of GPCR signaling.
Fig. 5.
Color-dependent control of cAMP level by LamPP-ß2ARiL3 through the activation of Gs-type G protein. (A) Violet light illumination increased intracellular cAMP levels in HEK293 cells expressing LamPP-ß2ARiL3, and subsequent green light illumination decreased the cAMP level to the basal level, which occurred repeatedly. (B) Blue light illumination generated a particular cAMP level. The cAMP levels generated by i) the first illumination, ii) the subsequent illumination, iii) the illumination after violet light illumination, iv) the subsequent illumination, v) the illumination after green light illumination, and vi) the subsequent illumination of blue light were almost constant, showing color-dependent cAMP clamping. Vertical lines indicate light illumination for 5 s.

Discussion

In this study, we demonstrated high performances of two bistable opsins, MosOpn3 and LamPP, in controlling GPCR signalings in vivo. Tg worms expressing MosOpn3 in ASH neurons exhibited light-induced avoidance behaviors in the presence of 11-cis retinal and all-trans retinal with a similar sensitivity, which is comparable with the case of Tg worms expressing bovine rhodopsin (BovRh) in the presence of 11-cis retinal (Figs. 1 and 2). We also tested the performance of Tg worms expressing mouse SWS1 opsin (MouSWS1) and human LWS opsin (HumLWS), which are known opsin-based tools having Go activation ability (57), in the presence of 11-cis retinal, but the performances are less than that of BovRh (Fig. 2E). On the other hand, BovRh-worms did not exhibit light-induced avoidance behaviors in the presence of all-trans retinal as shown in the previous report examining the functionality of BovRh in other type of neurons of C. elegans, in which 9-cis retinal addition induced light-dependent neural responses, but all-trans retinal addition did not (6). Since all-trans retinal is basically present in every tissue, the performance of MosOpn3 in the presence of all-trans retinal comparable with that of BovRh under the 11-cis retinal suggests that MosOpn3 has a crucial advantage in optical control of GPCR signaling in vivo. It is widely accepted that microbial rhodopsins such as ChR2 are powerful tools for use in optogenetics, especially in optical control of neural activities (5861). One of the reasons for the popularity is that microbial rhodopsins form photosensitive pigments by binding all-trans retinal to function in every tissue, and the 11-cis retinal requirement of bleaching opsins is the main reason for unpopularity of animal opsins. In terms of this point, the current study demonstrated that the availability of MosOpn3 could be equivalent to that of microbial rhodopsins. Moreover, the sensitivity of the avoidance response light dependently induced by MosOpn3 in ASH neurons was revealed to be ~0.0002 mW/mm2 (white light) in the presence of all-trans retinal (Fig. 2C), which is ~7,000 times higher than that induced by ChR2 in ASH neurons (~1.48 mW/mm2, blue light) reported in the previous study (62). In fact, we generated Tg worms expressing ChR2 in ASH neurons and tested light-induced avoidance behaviors with the same protocol as in cases of MosOpn3 and BovRh, but no reproducible avoidance response was observed by illuminating with ~0.8 mW/mm2 of white light, the highest light intensity of our light source. These data demonstrated that MosOpn3 could function as a light-sensitive switch to provide much higher photosensitivity to any tissue with much lower light–induced toxicity and/or temperature increase compared with the case of ChR2. There was a potential concern that the basal activity of 13-cis retinal–bearing MosOpn3 pigment might affect cellular conditions intrinsically in vivo because the activity of G protein by the 13-cis retinal–bearing pigment in the dark was higher than that of the 11-cis retinal–bearing pigment (22). However, MosOpn3-wormsfed all-trans retinal–containing E. coli, in which 13-cis retinal–bearing MosOpn3 pigments formed, normally behaved in the dark (Fig. 2, Movie S1 C). The data suggest that intracellular conditions in ASH neurons were somehow maintained to be normal, even with the partial G protein activation by 13-cis retinal–bearing MosOpn3 pigment. Then, we established ß2AR (Gs-coupled receptor) and α1AR (Gq-coupled receptor) versions of MosOpn3 by replacing the cytoplasmic region(s) with those of ß2AR and α1AR, respectively, to expand the applicability of MosOpn3 (Fig. 3). Analysis of a series of MosOpn3 chimeras revealed that fine-tuning of replaced regions improves the G protein activation efficiency and specificity (α1ARiL3) and alters the kinetics of chimeras (ß2AR), which could be a guide for making various MosOpn3 chimeras with other GPCRs of interest for appropriate purposes.
We also demonstrated color-dependent control of GPCR signaling by LamPP to lead behavioral switching between coiled and moving of C. elegans upon violet and green light stimuli, respectively (Fig. 4 D and E). The switching is consistent with the molecular behavior of LamPP by UV and visible light absorption (17), which suggests that the photoregeneration ability of LamPP contributes to the mode change of animal behaviors. Interestingly, we recently reported that in the zebrafish pineal photoreceptor cells, the parapinopsin alone generates color opponency; UV and orange lights induce hyperpolarization and depolarization, respectively, based on the photoequilibrium levels between the inactive and active states of the parapinopsin (35). The idea was also supported by the current results by heterologous expressions of LamPP in C. elegans neurons, showing that introducing LamPP is sufficient for color-dependent behavioral switching. Furthermore, we demonstrated that LamPP-wormsfed all-trans retinal under the red light (600 nm) responded to light stimuli (Fig. 4E), indicating that LamPP could function as a light-sensitive switch in the presence of all-trans retinal, in other words, in every tissue with red light preillumination. Accordingly, with red light, LamPP is optogenetically available in every tissue, which is practically equivalent to MosOpn3 as well as microbial rhodopsins.
The color-dependent control of Gi/o-type G protein by the wild-type LamPP has also been expanded to that of Gs-type G protein by LamPP-ß2ARiL3 and LamPP-JelOpiL3, which enables color-dependent control of intracellular cAMP levels in the reverse direction to the reactions by the WT (Fig. 5). Interestingly, LamPP-ß2ARiL3 showed molecular properties for not only color-dependent switch on and switch off of GPCR signaling but also color-dependent and context-independent maintenance of the intracellular cAMP level (Fig. 5). The results indicate that LamPP-ß2ARiL3 achieves clamping intracellular cAMP levels depending on color of light. We also emphasize that the cAMP levels were maintained in the dark after a light flash, depending on its color, suggesting an alternative strategy for “light” regulation of the cAMP level. On the other hand, the “cAMP clamping” was not achieved in the case of LamPP-JelOpiL3 (SI Appendix, Fig. S2). The clamped cAMP level could relate to shut off manners of the active state of LamPP-ß2ARiL3 based on unknown effects of the third cytoplasmic loop of ß2AR including phosphorylation and/or arrestin binding, which provides a new insight into prolongation of GPCR signalings.
In this study, the availability of two bistable animal opsins has shown to be equivalent to that of microbial rhodopsins including ChR2 in terms of retinal requirement, which would accelerate research by optical control of GPCR signaling. In controlling GPCR signaling, chemical control, namely chemogenetics using chemoreceptors such as designer receptor exclusively activated by designer drugs (DREADD), has been widely used (6365), which is in contrast to the situation of optogenetics with animal opsins, mainly bleaching opsins. The low popularity of optical control of GPCR signaling was mainly due to the absence of effective and robust tools, but the situation has been recently changing with bistable opsins, especially MosOpn3 and LamPP (28, 3638), and advantages of them and their derivatives were systematically demonstrated based on their molecular properties in this paper. The main advantage of chemogenetics is that stimuli (chemicals) can be reliably delivered deep inside the body. On the other hand, temporally precise control of GPCR signaling in vivo is basically unachievable by chemogenetics but achievable by optogenetics. In particular, temporally precise termination of G protein signaling as demonstrated by LamPP (Figs. 4 and 5) is a significant advantage of optogenetics, which enables controlling durations, intervals, and numbers of the stimuli. Together with chemical control, the optical control with bistable animal opsins would greatly facilitate comprehensive and deeper understating of GPCR-based physiologies as well as GPCR signaling itself.

Materials and Methods

Generating Tg C. elegans.

Based on the information from WormBase (66), the 2.4-kbp promoter sequence of sra-6 (42) was obtained from the C. elegans genomic DNA by PCR. The 3.3-kbp promoter sequence of unc-17 was a generous gift from Prof. Robert Lucas (56). The vector backbone containing SL1 and GFP was obtained by digestion of the plasmid [pEM1 = flp-21::LoxPStopLoxP::npr-1 SL2 GFP] (Addgene plasmid # 24033) (67) with NotI and KpnI. The sra-6 promoter linked with MosOpn3 (AB753162), bovine rhodopsin (AB062417), mouse SWS1 opsin (U49720), and human LWS opsin (NM_020061) cDNA or the unc-17 promoter linked with LamPP cDNA was introduced into the vector. Each plasmid was coinjected into the wild-type C. elegans strain N2 obtained from the Caenorhabditis Genetics Center with pmyo-2::mCherry as a selection marker. The wild and Tg strains (psra-6::MosOpn3::GFP, psra-6::BovRh::GFP, psra-6::MouSWS1::GFP, psra-6::HumLWS::GFP, and punc-17::LamPP::GFP) were cultured according to standard methods (68). The plasmids and strains are available on request.

Behavioral Experiments.

Adult worms were used for behavioral experiments. Tg worms were fed E. coli strain OP50, which are mixed with or without certain amounts of retinal at 24 h before experiments. The standard concentration of 11-cis retinal or all-trans retinal in OP50 for our experiment was 0.38 mM or 0.67 mM, respectively, and diluted to 1/100 to 1/10,000. Worms were monitored under infrared light illumination. White lights (I0 = ~0.8 mW/mm2) supplied by a 200-W metal-halide lamp (PhotoFluor II, 89 North) with or without ND filter(s) were applied as light stimuli for MosOpn3-worms. Narrow-band violet (410 nm) and green (510 nm) LED lights were applied as light stimuli for LamPP. Red (600 nm) LED light was used as a background light, while LamPP-worms were fed all-trans retinal. Avoidance response probabilities were calculated by the ratio or the number of responded worms to five worms examined.

Generation of Chimeric Mutants of Opsins.

Chimeric mutants of MosOpn3 and LamPP were generated by replacing cytoplasmic regions of hamster ß2-adrenergic receptor (ß2AR) and human α1-adrenergic receptor (α1AR) by combining DNA fragments of each region with PCR. The cytoplasmic regions were determined according to previous reports (5, 50) and applied to MosOpn3 and LamPP based on the alignment including bovine rhodopsin. We deposited a set of plasmids for the chimeric mutants in Addgene.

Bioluminescent Reporter Assays for Ca2+ and cAMP.

The intracellular cAMP and Ca2+ levels in opsin-expressing HEK293S cells were measured using the GloSensor cAMP assay and the aequorin assay, respectively, as described previously (22, 26, 69, 70). The pGloSensor-22F cAMP plasmid (Promega) was used for the GloSensor cAMP assay. The wild-type aequorin obtained by introducing two reverse mutations into the plasmid [pcDNA3.1+/mit-2mutAEQ] (Addgene #45539) (71) was used for the aequorin assay. A broadband green LED light and narrow-band violet (410 nm) and blue (430 nm) LED lights were applied for 5 s in the GloSensor cAMP assay and for 1 s in the aequorin assay as light stimuli.

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix. Some study data available (The plasmids and strains are available on request).

Acknowledgments

We thank Robert J. Lucas (The University of Manchester) for providing the unc-17 promoter and his helpful comments on this manuscript. We also thank Takahiro Yamashita (Kyoto University) for providing the mouse SWS1 opsin and human LWS opsin clones. This work was supported by the JSPS KAKENHI grant numbers JP18H02482, JP20K21433, and JP21H00435 (M.K.), JP15H05777 and JP20K21434 (A.T.), Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) grant JPMJPR13A2 (M.K.), and JST Core Research for Evolutional Science and Technology (CREST) grant JPMJCR1753 (A.T.).

Author contributions

M.K. and A.T. designed research; M.K., B.S., T.N., L.S., and S.K. performed research; M.K., B.S., T.N., L.S., S.W., S.K., E.K.-N., and A.T. contributed new reagents/analytic tools; M.K., B.S., T.N., S.W., S.K., E.K.-N., and A.T. analyzed data; and M.K. and A.T. wrote the paper.

Competing interests

The author declares no competing interest.

Supporting Information

Appendix 01 (PDF)
Movie S1
Movies showing typical light-induced avoidance behaviors of Tg C. elegans expressing MosOpn3 or BovRh in ASH neurons. (A) MosOpn3/11-worm. (B) MosOpn3/NoRet-worm. (C) MosOpn3/AT-worm. (D) BovRh/11-worm. (E) BovRh/NoRet-worm. (F) BovRh/AT-worm. Timings of light illumination were indicated by emergence of white frames.
Movie S2
The movie showing color-dependent control of behaviors of Tg C. elegans expressing LamPP in cholinergic motor neurons. A LamPP-worm was moving before illumination, stopped moving and coiled upon violet light illumination, and restarted movement upon green light illumination, which occurred repeatedly. Timings of violet and green light illumination were indicated by emergence of violet and green frames, respectively. Signals derived from mCherry were also detected by green light illumination.

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 119 | No. 48
November 29, 2022
PubMed: 36417444

Classifications

Data, Materials, and Software Availability

All study data are included in the article and/or SI Appendix. Some study data available (The plasmids and strains are available on request).

Submission history

Received: March 11, 2022
Accepted: October 5, 2022
Published online: November 23, 2022
Published in issue: November 29, 2022

Keywords

  1. rhodopsin
  2. second messenger
  3. cAMP
  4. Ca2+
  5. signal transduction

Acknowledgments

We thank Robert J. Lucas (The University of Manchester) for providing the unc-17 promoter and his helpful comments on this manuscript. We also thank Takahiro Yamashita (Kyoto University) for providing the mouse SWS1 opsin and human LWS opsin clones. This work was supported by the JSPS KAKENHI grant numbers JP18H02482, JP20K21433, and JP21H00435 (M.K.), JP15H05777 and JP20K21434 (A.T.), Japan Science and Technology Agency (JST) Precursory Research for Embryonic Science and Technology (PRESTO) grant JPMJPR13A2 (M.K.), and JST Core Research for Evolutional Science and Technology (CREST) grant JPMJCR1753 (A.T.).
Author Contributions
M.K. and A.T. designed research; M.K., B.S., T.N., L.S., and S.K. performed research; M.K., B.S., T.N., L.S., S.W., S.K., E.K.-N., and A.T. contributed new reagents/analytic tools; M.K., B.S., T.N., S.W., S.K., E.K.-N., and A.T. analyzed data; and M.K. and A.T. wrote the paper.
Competing Interests
The author declares no competing interest.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Department of Biology, Graduate School of Science, Osaka Metropolitan University, Sumiyoshi-ku, Osaka 558-8585, Japan
The OMU Advanced Research Institute for Natural Science and Technology, Osaka Metropolitan University, Sumiyoshi-ku, Osaka 558-8585, Japan
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan
Lanfang Sun
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan
Seiji Wada
Department of Biology, Graduate School of Science, Osaka Metropolitan University, Sumiyoshi-ku, Osaka 558-8585, Japan
Satomi Kamimura
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan
The OMU Advanced Research Institute for Natural Science and Technology, Osaka Metropolitan University, Sumiyoshi-ku, Osaka 558-8585, Japan
Department of Nutrition, Graduate School of Human Life and Ecology, Osaka Metropolitan University, Sumiyoshi-ku, Osaka Osaka 558-8585, Japan
Department of Biology, Graduate School of Science, Osaka Metropolitan University, Sumiyoshi-ku, Osaka 558-8585, Japan
The OMU Advanced Research Institute for Natural Science and Technology, Osaka Metropolitan University, Sumiyoshi-ku, Osaka 558-8585, Japan
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan

Notes

2
To whom correspondence may be addressed. Email: [email protected] or [email protected].
1
M.K. and B.S. contributed equally to this work.

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