Sequence, Structure, and Expression of Opsins in the Monochromatic Stomatopod Squilla empusa

Sequence, Structure, and Expression of Opsins in the Monochromatic Stomatopod Squilla empusa Abstract Most stomatopod crustaceans have complex retinas in their compound eyes, with up to 16 spectral types of photoreceptors, but members of the superfamily Squilloidea have much simpler retinas, thought to contain a single photoreceptor spectral class. In the Atlantic stomatopod Squilla empusa, microspectrophotometry shows that all photoreceptors absorb light maximally at 517 nm, indicating that a single visual pigment is present in all photoreceptors in the retina. However, six distinct, but partial, long wavelength sensitive (LWS) opsin transcripts, which encode the protein component of the visual pigment, have been previously isolated through RT-PCR. In order to investigate the spectral and functional differences among S. empusa’s opsins, we used RT-PCR to complete the 3′ end of sequences for five of the six expressed opsins. The extended sequences spanned from the first transmembrane (TM1) helix to the 3′ end of the coding region. Using homology-based modeling, we predicted the three-dimensional structure of the amino acid translation of the S. empusa opsins. Based on these analyses, S. empusa LWS opsins share a high sequence identity in TM regions and in amino acids within 15 Å of the chromophore-binding lysine on TM helix 7 (TM7), suggesting that these opsins produce spectrally similar visual pigments in agreement with previous results. However, we propose that these spectrally similar opsins differ functionally, as there are non-conservative amino acid substitutions found in intracellular loop 2 (ICL2) and TM5/ICL3, which are critical regions for G-protein binding, and substitutions in extracellular regions suggest different chromophore attachment affinities. In situ hybridization of two of the opsins (Se5 and Se6) revealed strong co-expression in all photoreceptors in both midband and peripheral regions of the retina as well as in selected ocular and cerebral ganglion neuropils. These data suggest the expression of multiple opsins—likely spectrally identical, but functionally different—in multiple types of neuronal cells in S. empusa. This suggests that the multiple opsins characteristic of other stomatopod species may have similar functional specialization. Introduction Stomatopod crustaceans, commonly referred to as mantis shrimps, make up a group of marine crustaceans that has been shown to have complex visual physiology, with up to 16 spectrally distinct photoreceptor classes observed in some species (Cronin et al. 1994; Porter et al. 2009; Cronin et al. 2010). Stomatopods have apposition compound eyes which are composed of many visual units called ommatidia (Marshall et al. 2007). Each ommatidium in the stomatopod eye has its own corneal and crystalline cone optical elements positioned above a rhabdom produced by seven or eight retinular photoreceptor cells (Marshall et al. 2007). In Squilla empusa, each rhabdom is formed by microvilli laden with visual pigments projected from seven photoreceptors, forming a single photoreceptive unit (Schönenberger 1977). Visual pigments are composed of an opsin G-protein-coupled receptor and a light sensitive chromophore. Upon photon absorption, the chromophore undergoes isomerization, typically from 11-cis retinal into all-trans retinal, and starts the phototransduction cascade. The spectral absorbance properties of visual pigments are typically tuned by alterations to the opsin residues that interact with and stabilize the chromophore in its binding pocket. Usually, one spectral class of photoreceptor expresses only one type of visual pigment (and thus a single opsin), although there is evidence for the expression of multiple distinct opsins within a single photoreceptor class from a number of species (e.g., African cichlid fish, Dalton et al. 2015; Limulus polyphemus, Battelle et al. 2016). At the structural level, stomatopod compound eyes are characterized by having two peripheral regions (dorsal and ventral) bisected horizontally by an equatorial midband region of specialized ommatidia (Marshall et al. 2007). While the peripheral regions contain the typical crustacean set of two photoreceptors spectral types, one sensitive to violet or ultraviolet (UV) light and the second sensitive to blue–green wavelengths, photoreceptors within the midband row are typically specialized for polychromatic and polarization vision. Most stomatopod species (superfamilies Gonodactyloidea, Lysiosquilloidea, Pseudosquilloidea, and Hemisquilloidea) have six ommatidial rows in the midband region, but species in the Squillioidea, including S. empusa in the present study, have only two ommatidial rows in the midband and are monochromatic (Cronin 1985; Schiff et al. 1986). Phylogenetic studies of the stomatopods suggest that the common ancestor of the Squilloidea most likely had six midband rows (Ahyong 1997; Porter et al. 2010). Thus, the two-row midband in Squilloidea is likely an evolved loss of photoreceptor diversity and spectral sensitivities. Squilla empusa are found near the coast of the Western Atlantic Ocean, from Maine to the Gulf of Mexico (Schiff et al. 1986). As is common in stomatopods, they make their homes by creating burrows on the ocean floor. Unlike stomatopods found in shallow coral reef habitats, S. empusa tends to burrow in muddy sea floors in dark and murky waters (Schiff et al. 1986). The limited light availability and their nocturnal hunting lifestyle (Schiff et al. 1986) may have contributed to the evolution of reduced visual complexity in S. empusa. Microspectrophotometric (MSP) studies of S. empusa eyes showed that all retinal photoreceptors absorb light maximally at 517 nm (Cronin 1985). The reduced complexity of the S. empusa retinal structure and the presence of a single spectral type of photoreceptor imply that there is also a single expressed opsin in the retina that initiates a conserved visual phototransduction cascade. However, recent studies have suggested that S. empusa visual physiology could be more complex than previously thought. Porter et al. (2009) isolated six unique opsin sequences from S. empusa retinas that cluster with other crustacean long wavelength sensitive (LWS) opsins. This raises an interesting question—why would a species with a monochromatic visual system possesses multiple opsins? The first possibility could be that the opsins differ spectrally and when expressed together, they tune the photoreceptors to their maximal absorbance value. However, this typically leads to a broadened photoreceptor curve, and there is no evidence of multiple visual pigments with different absorbance peaks from past MSP studies (Cronin 1985). Alternatively, the opsins could be identical, or highly similar, in spectral absorbance and yet differ functionally in how they initiate the phototransduction cascade due to structural differences leading to differences in membrane localization or chromophore coupling. There also exists the possibility that the opsins could be evolutionary vestiges, and are not translated into protein. In this study, we extended sequences of five opsin transcripts from Porter et al. (2009) to span from TM1 to the end of the coding region in order to predict the opsins’ functional and spectral differences. We also analyzed the expression of two of these opsins in S. empusa retinal and neural tissues. The data we present here suggest that S. empusa has multiple, spectrally-similar, but functionally distinct opsins expressed in the retina, optic lobes, and cerebral ganglion (CG). We propose that this monochromatic stomatopod possesses a complex molecular toolkit of opsins, perhaps capable of complex visual system modulation and downstream processing. Materials and Methods RT-PCR (3′-RACE) of S. empusa opsins mRNA and sequence analysis Squilla empusa eyes were homogenized in TRIzol (Invitrogen) and RNA was extracted as per the TRIzol Reagent protocol (Invitrogen). Single strand cDNA was synthesized from isolated total RNA using the SuperScript RT III protocol (Invitrogen) and primers designed from published S. empusa opsin partial sequences (Porter et al. 2009; Supplementary Table S1). After first strand synthesis, PCR was performed using Taq DNA polymerase (ThermoFisher Scientific) and specific primers for each of the six opsins identified in Porter et al. (2009) (Supplementary Table S1) to amplify opsin transcripts from the cDNA as per manufacturer’s protocol (ThermoFisher Scientific). PCR amplicons were ligated into the pGEM-T Easy plasmid (Promega) via TA cloning using the manufacturer’s protocol. Opsin sequences ligated into the plasmid were then sequenced (Genewiz). Partial opsin mRNA sequences obtained in Porter et al (2009) (GenBank accession numbers are the following: Se1-GQ221751.1, Se2-GQ221753.1, Se3-GQ221754.1, Se4-GQ221755.1, Se5-GQ221756.1, and Se6-GQ221752.1) were aligned with sequences obtained through RT-PCR (3′RACE) using Geneious software, version R10 (Biomatters Limited) (Kearse et al. 2012) to complete the opsin’s sequence. The mRNA sequences were then translated and aligned using Geneious software to facilitate the identification of non-conservative amino acid substitutions and other analyses. Structural modeling and analysis of S. empusa opsins The amino acid sequence for S. empusa opsin Se5 was used for homology-based three-dimensional structural modeling using LOMETS software (Wu and Zhang 2007). The S. empusa opsin model was generated using squid (Todarodes pacificus) rhodopsin as a template (PDB ID 2ZIY) (Shimamura et al. 2008). In combination with an amino acid alignment of the five analyzed opsins (Fig. 1), the model was used to identify amino acids proximal to the chromophore and potentially able to alter visual pigment spectral tuning. While it is possible to spectrally tune an opsin without a non-conservative amino acid substitution (Fasick and Robinson 1998; Fasick and Robinson 2000), charged amino acids can alter spectral properties of the chromophore (Wang et al. 2014) and are identifiable through bioinformatics. For our analysis, we considered non-conservative amino acid replacements, i.e., positions in the amino acid alignment where the charged/non-charged property of the amino acid has changed between opsins, within a 15 Å (1.5 nm) distance capable of altering chromophore binding chemistry. To generate the models of S. empusa opsin in complex with G-protein and arrestin, the S. empusa opsin structural model was aligned with the crystal structure of human rhodopsin in complex with mouse visual arrestin (PDB 4ZWJ) (Kang et al. 2015) and the crystal structure of human beta-2 adrenergic receptor in complex with bovine Gαs, rat Gβ, and bovine Gγ (PDB ID 3SN6) (Rasmussen et al. 2011) using the cealign tool using Pymol software (Schrodinger). This was done to position S. empusa opsin in complex with the signaling molecules. All amino acid numbering in this article is based on the S. empusa opsin alignments (Fig. 1). Fig. 1 View largeDownload slide Amino acid sequence alignment of five S. empusa opsins. Opsin amino acid sequences were inferred from mRNA nucleotide sequences from Porter et al (2009) and RT-PCR performed in this study. Amino acid residues are colored according to their property—yellow, non-polar; green, polar and uncharged; and red and blue, charged (negatively and positively charged, respectively). High levels of sequence identity are observed throughout, particularly in the TM regions (indicated by red annotations above the alignment) and in residues predicted to be in close proximity (≤15 Å) to the chromophore attachment site, K272 (indicated by blue annotations above the alignment). Sites of non-conservative amino acid substitutions among the opsins are denoted by the yellow annotations above the alignment. The green annotation above the alignment corresponds to residues predicted to be sites of phosphorylation and subsequent arrestin interaction. Fig. 1 View largeDownload slide Amino acid sequence alignment of five S. empusa opsins. Opsin amino acid sequences were inferred from mRNA nucleotide sequences from Porter et al (2009) and RT-PCR performed in this study. Amino acid residues are colored according to their property—yellow, non-polar; green, polar and uncharged; and red and blue, charged (negatively and positively charged, respectively). High levels of sequence identity are observed throughout, particularly in the TM regions (indicated by red annotations above the alignment) and in residues predicted to be in close proximity (≤15 Å) to the chromophore attachment site, K272 (indicated by blue annotations above the alignment). Sites of non-conservative amino acid substitutions among the opsins are denoted by the yellow annotations above the alignment. The green annotation above the alignment corresponds to residues predicted to be sites of phosphorylation and subsequent arrestin interaction. Synthesis of riboprobes for in situ hybridization Riboprobes were synthesized for visual opsins Se1, Se5, and Se6, which represent representative opsins from all three of the identified S. empusa opsin evolutionary clades identified in Porter et al. (2009). To synthesize both sense and antisense probes, pGEM-T Easy plasmid DNA containing the 3′UTR of the visual opsin transcripts were digested with one restriction enzyme (SalI or NotI) to create linear plasmids. Next, the following in vitro transcription reaction was prepared: linear plasmid DNA, DIG-RNA Labeling Mix (Roche), Polymerase buffer (Roche), RNase OUT (Invitrogen), and either T7 RNA polymerase or SP6 RNA polymerase (Roche). The transcription reaction was carried out as per the RNA polymerase protocol (Roche). Following transcription, reaction buffer with MgCl2 (ThermoFisher Scientific) and 0.1 u/μL RNase-free DNase I, (ThermoFisher Scientific) was added to the mixture. The reaction was carried out as described in the RNase-free DNase I protocol (ThermoFisher Scientific). Riboprobes were purified using the RNeasy Minelute Cleanup Kit (QIAGEN). Preparation of S. empusa tissue for in situ hybridization Squilla empusa mantis shrimp were sedated on ice upon arrival. Specimens were decapitated by making a transverse cut to sever the nerve cord between the CG and subesophageal ganglion. Once the anterior portion of the cephalothorax was separated from the body, appendages were removed from the ophthalmic and antennular somites. The eyestalks (which include the optic lobes within) were cut away from the cephalothorax. The eyestalks and cephalothorax were fixed in 4% paraformaldehyde with 12% sucrose in 0.1% diethyl pyrocarbonate (DEPC) 1× phosphate-buffered saline (PBS) overnight at 4°C. For retinal only in situ hybridization (ISH) studies, eyes were frozen and sectioned at 12–14 μm using a cryostat. Because of potentially lower signals expected from opsins expressed in neural tissues, isolated optic lobe and CG tissues were dehydrated using an ethanol gradient and propylene oxide, and then rehydrated, before being embedded in albumin gelatin. Then, the gelatin blocks were fixed overnight in 4% PFA in 0.1% DEPC 1× PBS, transferred to 0.1% DEPC 1× PBS, and sectioned at 60 μm using a vibratome. ISH of S. empusa tissue sections Our protocol is based originally from Ishii et al. (2003), and was also used in Bok et al. (2014) and Cronin et al. (2010) for stomatopod retinas. For all probes, no probe and sense probe controls were run alongside antisense probes (Supplementary Figs. S3 and S4). Squilla empusa sections on microscope slides were fixed in 4% PFA in PBS for 10 min. Next, the slides were washed three times in 0.1% (v/v) DEPC-1× PBS, 3 min per wash. The slides were then acetylated for 10 min in a solution containing 0.1% (v/v) DEPC-H2O, triethanolamine, 0.02 N HCl, and acetic anhydride, followed by three washes in 1× PBS, 5 min per wash. Hybridization solution (50% [v/v] formamide, 5× saline-sodium citrate [SSC] buffer, 5× Denhardt’s solution, and 250 μg/mL herring sperm DNA) was then added to the retina sections and the slides were incubated in a humidified chamber for 1 h. Riboprobes were added to hybridization solution (150–200 ng riboprobe per 100 μL of hybridization solution for retinal tissue, and 100 ng riboprobe per 100 μL of hybridization solution for extraocular tissue) and were incubated at 70°C for 10 min. The hybridization solution on the tissue was poured off and hybridization solution with riboprobe was added to the tissue sections. The slides were incubated at 75°C overnight. The next day, slides were incubated in 0.2× SSC at 65°C three times for 20 min to remove unbound probes. The slides were then incubated in Buffer B1 (0.1 M Tris pH 7.5 and 0.15 M NaCl) for 5 min, and then in Buffer B2 (Buffer B1 and 10% normal goat serum) for 1 h. Anti-digoxigenin-alkaline phosphatase (AP) (Roche) was diluted 1:5000 in Buffer B2 and was placed on the tissue sections, and the slides were incubated for 1–2 days at 4°C. Slides were next washed with Buffer B1 four times for 3 min each. Buffer B3 (0.1 M Tris pH 9.5, 0.1 M NaCl, and 50 mM MgCl2) was added to the slides and incubated for 5 min. Buffer B4 (NBT/BCIP tablet [Roche], 24 mg/mL levamisole) was then applied to the slides and left to incubate for several hours (retina sections) to overnight (extraocular tissue sections). Slides were then mounted and photographed via light microscopy. Results Amino acid sequence analysis of S. empusa opsins Using RT-PCR of S. empusa eyes, we completed the 3′ end of five of the six S. empusa opsin transcript sequences initially described by Porter et al (2009) (Fig. 1). We were unable to amplify the 3′ end of one of the six opsins (Se1), which is missing sequence data for part of TM6 and all of TM7, and so was excluded from further analysis. Based on amino acid translations, these transcript sequences encode for seven-transmembrane (TM) opsins with a mean predicted molecular weight of 37.5 kDa. Also, the opsins contain the critical chromophore attachment site at K272 (numbering based on Fig. 1 alignment), extended TM5 and TM6 helices (compared with bovine rhodopsin, Palczewski et al. 2000), a C-terminus region containing 9 or 10 putative sites of phosphorylation (Table 2), and important rhodopsin-class GPCR domains such as the (E)DRY motif on TM3 and NPXXY motif on TM7 (Fig. 1). Table 2. Comparison and summary of C-terminus amino acids predicted to influence signaling deactivation in S. empusa opsins Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Table 2. Comparison and summary of C-terminus amino acids predicted to influence signaling deactivation in S. empusa opsins Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Among the opsins analyzed there was high amino acid sequence similarity, with the sequence identity between opsins ranging from 76.6% to 93.7% (Supplementary Fig. S1). The percent identity across all opsins is 71.2%, with an average pairwise identity of 83.4%. TM domains also have a high degree of similarity, with percent identity of 76.3% and an average pairwise identity of 86.0%. Despite the high level of sequence identity we identified several sites of non-conservative amino acid substitution. Specifically, non-conservative substitutions exist at functionally relevant locations, including positions 74 on TM3; 112 on intracellular loop (ICL) 2; 189, 192, and 199 on TM5; and 258 on TM7 (Table 1 and Figs. 1 and 2A, B). Table 1. Summary of non-conservative amino acid substitution among S. empusa opsins Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Notes: Number, location, and identities of the amino acids are depicted, along with a hypothesized function of each amino acid of interest. Amino acids marked with (/) in a white cell indicate non-charged residues (includes polar and non-polar). Amino acids marked with (+) in a gray cell indicate positively-charged residues; and those marked with (−) in a black cell indicate negatively-charged residues. TM, transmembrane region; ICL, intraellular loop; ECL, extracellular loop. Amino acid of interest numbering based on alignment consensus. Table 1. Summary of non-conservative amino acid substitution among S. empusa opsins Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Notes: Number, location, and identities of the amino acids are depicted, along with a hypothesized function of each amino acid of interest. Amino acids marked with (/) in a white cell indicate non-charged residues (includes polar and non-polar). Amino acids marked with (+) in a gray cell indicate positively-charged residues; and those marked with (−) in a black cell indicate negatively-charged residues. TM, transmembrane region; ICL, intraellular loop; ECL, extracellular loop. Amino acid of interest numbering based on alignment consensus. Fig. 2 View largeDownload slide Structural modeling of S. empusa opsins suggests amino acids sites of non-conservative substitution function as modulators of G-protein binding and chromophore attachment stability. Front (A) and rear (B) view of structural model of S. empusa opsin Se5, labeling the predicted position of the sites of non-conservative substitution on the opsin’s tertiary structure (refer to Fig. 1 for position of these sites on the opsin amino acid sequences). (C) TM helices form a compact binding pocket around the chromophore, 11-cis retinal (depicted in space-filling format). No non-conservative amino acid substitutions are found within this binding pocket. (D) Model of active-state opsin bound to heterotrimeric G-protein (Gαs used in this model). (E) Model of active-state opsin in complex with arrestin (β-arrestin-1 used in this model). Surface charges plotted on arrestin—the legend below G indicates the charges. (F) Four non-conservative amino acids substitutions are predicted to be proximal to the G-protein binding pocket, particularly amino acids 112 and 199, found on intracellular loops 2 and TM5, respectively. (G) The opsin’s C-terminus is in close proximity to the positively-charged phosphate-sensing domains on arrestin. Acidic/negatively charged residues, serines, and threonines are concentrated in this region of the opsin’s C-terminus. Fig. 2 View largeDownload slide Structural modeling of S. empusa opsins suggests amino acids sites of non-conservative substitution function as modulators of G-protein binding and chromophore attachment stability. Front (A) and rear (B) view of structural model of S. empusa opsin Se5, labeling the predicted position of the sites of non-conservative substitution on the opsin’s tertiary structure (refer to Fig. 1 for position of these sites on the opsin amino acid sequences). (C) TM helices form a compact binding pocket around the chromophore, 11-cis retinal (depicted in space-filling format). No non-conservative amino acid substitutions are found within this binding pocket. (D) Model of active-state opsin bound to heterotrimeric G-protein (Gαs used in this model). (E) Model of active-state opsin in complex with arrestin (β-arrestin-1 used in this model). Surface charges plotted on arrestin—the legend below G indicates the charges. (F) Four non-conservative amino acids substitutions are predicted to be proximal to the G-protein binding pocket, particularly amino acids 112 and 199, found on intracellular loops 2 and TM5, respectively. (G) The opsin’s C-terminus is in close proximity to the positively-charged phosphate-sensing domains on arrestin. Acidic/negatively charged residues, serines, and threonines are concentrated in this region of the opsin’s C-terminus. Structural modeling and analysis of a S. empusa opsin A three-dimensional structural model was constructed for the amino acid sequence of opsin Se5, using homology to T. pacificus rhodopsin, a rhabdomeric visual opsin, to predict the likely molecular conformation of opsins in S. empusa. The predicted structure of Se5 (Fig. 2A, B) reveals a 7-TM opsin with structured cytoplasmic protrusions. Specifically, the cytoplasmic protrusions of the extended TM5 and TM6 helices likely form a structural determinant for G-protein binding specificity, namely to Gαq (Porter et al. 2013; Donohue et al. 2017). The model also predicts a compact chromophore binding pocket (Fig. 2C) comprised of all TM helices, and a chromophore binding site at K272 on TM7. The portions of the TM helices proximal to the extracellular space, and the ECLs (particularly ECL2) form the opsin chromophore “plug,” stabilized by a disulfide bond formed between C76 and C153. To address the possibility that S. empusa opsins are spectrally distinct, we analyzed the identities of the amino acids proximal to the site of chromophore attachment on TM7, K272. Specifically, we considered non-conservative amino acid substitutions between opsins as possible sites of spectral tuning. For this analysis, we considered amino acids within a 15 Å (1.5 nm) distance to be proximal, and potentially able to alter the chromophore binding chemistry. Our structural analysis suggests that the opsins are spectrally identical or similar: no non-conservative amino acid substitutions were found in within 15 Å of K272. Only one residue, site 74, has non-conservative amino acid substitutions within the 15 Å distance of K272 (Figs. 1 and 2A, B). However, this site is unlikely to cause spectral shifts between opsins, given its position on TM3 where it is close to the extracellular space, and its R-group is almost completely out of the 15 Å window (Fig. 2A, B). Interestingly, this site is placed close to the extracellular chromophore “plug,” and while it isn’t likely to affect the opsins spectrally, this site might serve as a tuning site for chromophore binding stability, a mechanism used in the mammalian rhabdomeric-type opsin, melanopsin (Tsukamoto et al. 2015). Additional analysis (Supplementary Fig. S5) reveals 11 amino acids surrounding the chromophore that are identical in all opsins analyzed in this study, and two are predicted to make contact with it (Y171 and W242). This analysis suggests a neutrally charged binding pocket similar to rhodopsin (Sakmar et al. 1989; Zhukovsky and Oprian 1989), however, amino acids containing R-groups with hydroxl moieties are present in the pocket (Supplementary Fig. S5), such as Y171 (which is predicted to contact the chromophore), Y79, and Y245, which support a green-shifted visual pigment (Chan et al. 1992; Asenjo et al. 1994). We also considered whether or not the multiple opsins might differ functionally, even while sharing high sequence and structural similarity. Our structural and sequence analyses identified four sites of non-conservative amino acid substitutions: residues 112 on ICL2, residues 189 and 192 on TM5, and 199 on TM5/ICL3 (Figs. 1 and 2A–F), located on important regions involved in coupling to signaling molecules. Specifically, these regions (ICL2 and ICL3) recognize and bind the opsin’s cognate G-protein, as shown extensively in bovine rhodopsin coupling to transducin (König et al. 1989; Franke et al. 1992; Yamashita et al. 2000; Natochin et al. 2003). We modeled the protein complex consisting of active-state S. empusa opsin and heterotrimeric G-protein (Gs was used in this model) (Fig. 2D, F). We observed the expected helical movement of TM5 and TM6 on the opsin and subsequent insertion of the C-terminus helix of Gα into the newly formed binding pocket in the opsin. Two of our sites, 112 on ICL2 and 199 on TM5 were particularly close to the binding pocket (Fig. 2F). Residue 112 is of particular interest for two reasons: it is on an unstructured coil, which does not sterically hinder its R-group from potentially interacting with several residues on the G-protein. Second, the non-conserved amino acid changes range from a complete switch of charge at that site (Se2 and Se6 are negatively charged, and Se3 is positively charged) to a loss of charge at that site (Se4 and Se5). Residue 199, while very close to the binding pocket, is hindered from movement due to its location on the cytoplasmic end of TM5. However, its proximity to the binding pocket might make it an important site that influences G-protein binding by impacting the overall charge of this region. Sites 189 and 192 are not likely to affect the G-protein binding pocket, but might affect the flexibility of TM5, and thus the formation of the binding pocket in the active state (Rasmussen et al. 2011). We analyzed the C-terminus, specifically for the number of phosphorylation sites and how they might activate arrestin (Fig. 2E, G). All opsins had a similar number of possible phosphorylation sites and negatively charged residues, which work in a synergistic manner to activate arrestin (Zhou et al. 2017). More important than the total number of possible phosphorylation sites is their proximity to the positively-charged phosphorylation-sensing domain on arrestins (Table 2 and Fig. 2G). To model opsin C-terminus–arrestin interaction and predict critical opsin C-terminus phosphorylation sites, we coupled active-state opsin to visual arrestin (crystal structure PDB 4ZWJ) (Figs. 1 and 2E, G) and determined that opsin residues 308–322 were proximal to the positively-charged region on arrestin. These data suggest that possible phosphorylation sites within this region on the opsin C-terminus are likely to be critical for signaling deactivation. Most opsins have a similar amount of possible phosphorylation sites in this region, except for Se3, which has most of its serines and threonines concentrated in this predicted critical region of arrestin interaction (Fig. 1 and Table 2). Expression of opsins in S. empusa retina and extraretinal neural tissue Using the newly generated sequence data (Supplementary Fig. S3), 3′UTR riboprobes were designed to hybridize to visual opsin mRNA in tissue sections in situ. Although riboprobes were synthesized for visual opsins Se1, Se5, and Se6, only Se5 and Se6 showed evidence of hybridization in our preparations. Expression patterns of S. empusa opsin transcripts Se5 (Fig. 3A–D) and Se6 (Fig. 3E–H) reveal that both opsins are robustly expressed in all regions of the retina—in both peripheral/hemispheric regions and in the midband. Expression of opsins Se5 and Se6 is also observed in transverse retinal sections (Fig. 3B, F), where riboprobe labeling is observed in all photoreceptors surrounding the rhabdoms in both hemispheres and in the midband (Fig. 3C, D, G, H). The intensity of Se5 labeling is even and robust in all regions of the retina (Fig. 3C,D), and a similar expression pattern is observed for Se6 (Fig. 3H). These data indicate that there is no preferential expression of either Se5 or Se6 in certain photoreceptors around the rhabdom in any region. Rather, S. empusa opsins Se5 and Se6 are co-expressed at high levels in all photoreceptors in all regions of the retina. Fig. 3 View largeDownload slide Robust transcript co-expression of M/LWS opsins Se5 and Se6 throughout the entire S. empusa retina. Sagittal (A and E) and transverse (B and F) retina sections labeled with Se5 (A–D) and Se6 (E–H) antisense riboprobes. Robust expression is observed in retina sections incubated with Se5 antisense riboprobes (A and B) including strong expression in all photoreceptors surrounding the rhabdom in the midband region (C) and periphery (D). Labeling with Se6 antisense riboprobes (E and F) also suggests robust expression of this opsin throughout the retina, with robust expression in all photoreceptors surrounding the retina in the midband region (G), and to a lesser degree in the periphery (H). DH, dorsal hemisphere; MB: midband; VH, ventral hemisphere. Scale bars: A, B, E, F: 500 μm; C, D, G, H: 100 μm. Fig. 3 View largeDownload slide Robust transcript co-expression of M/LWS opsins Se5 and Se6 throughout the entire S. empusa retina. Sagittal (A and E) and transverse (B and F) retina sections labeled with Se5 (A–D) and Se6 (E–H) antisense riboprobes. Robust expression is observed in retina sections incubated with Se5 antisense riboprobes (A and B) including strong expression in all photoreceptors surrounding the rhabdom in the midband region (C) and periphery (D). Labeling with Se6 antisense riboprobes (E and F) also suggests robust expression of this opsin throughout the retina, with robust expression in all photoreceptors surrounding the retina in the midband region (G), and to a lesser degree in the periphery (H). DH, dorsal hemisphere; MB: midband; VH, ventral hemisphere. Scale bars: A, B, E, F: 500 μm; C, D, G, H: 100 μm. Given such robust co-expression of opsins in the retina, we then tested if the Se5 and Se6 opsins are expressed in extraretinal tissue, which is common in marine crustaceans (Kingston et al. 2015; Kingston and Cronin 2016; Donohue et al. 2017) and terrestrial invertebrates such as Papilio xuthus (Arikawa et al. 2003). Through ISH of thicker (60 μm) tissue sections, we observed expression of retinal opsins Se5 and Se6 in other neural tissues (Fig. 4). Specifically, expression of both opsin transcripts was observed in optic neuropils including the optic lobe lamina, medulla, and lobula, as well as the hemiellipsoid body in the lateral protocerebrum. Se6 was more broadly expressed than Se5 in all neuropils, especially in the lamina and lobula neuropils (Fig. 4). Neither Se5 nor Se6 opsin transcript expression were observed in the ventral eye, but it is possible that other opsins (not probed for in this study, such as Se2–Se4) are present. Se5 and Se6 opsin expression was also observed in the CG, specifically cell bodies that make up the olfactory neuropil (Fig. 4). Thus, given all these results, co-expression of multiple opsins in this stomatopod is not only in photoreceptors, but surprisingly, also in downstream neurons involved in sensory processing. Fig. 4 View largeDownload slide Se5 and Se6 opsin transcripts co-expressed the optic lobes and cerebral ganglion (CG) of S. empusa. Sagittal eyestalk sections (top row) suggest that Se5 and Se6 transcripts appear to trace the lamina (La), medulla (Me), lobula (Lo), and hemiellipsoid body (HB) neuropils. As in Fig. 3, both transcripts are also co-expressed in retinal photoreceptors throughout the retina. Additionally, transverse CG sections show that Se5 and Se6 are co-expressed in the periphery of the olfactory lobes (OL). Antennal neuropil, AnN; lateral antennal neuropil (LAN); olfactory-glomeruli tract (OGT); dorsal hemisphere (DH); two equatorial midband rows (MB); and ventral hemisphere (VH). Fig. 4 View largeDownload slide Se5 and Se6 opsin transcripts co-expressed the optic lobes and cerebral ganglion (CG) of S. empusa. Sagittal eyestalk sections (top row) suggest that Se5 and Se6 transcripts appear to trace the lamina (La), medulla (Me), lobula (Lo), and hemiellipsoid body (HB) neuropils. As in Fig. 3, both transcripts are also co-expressed in retinal photoreceptors throughout the retina. Additionally, transverse CG sections show that Se5 and Se6 are co-expressed in the periphery of the olfactory lobes (OL). Antennal neuropil, AnN; lateral antennal neuropil (LAN); olfactory-glomeruli tract (OGT); dorsal hemisphere (DH); two equatorial midband rows (MB); and ventral hemisphere (VH). Discussion Past MSP analyses suggested that S. empusa, despite having two midband rows, has only a single photoreceptor spectral class (Cronin 1985), in contrast to the large number of spectrally-distinct photoreceptor classes described in other stomatopod species (Porter et al. 2009; Cronin et al. 2010). These physiological data imply that a simple molecular composition exists in its photoreceptors (e.g., fewer expressed opsins), and in combination with past evolutionary studies (Porter et al. 2010) also suggest a reduction in eye complexity compared with stomatopods with many photoreceptor classes. Our data suggest quite the contrary, that the monochromatic S. empusa expresses multiple opsins in both retinal photoreceptor cells and downstream visual processing neurons (Figs. 3 and 4). Homology modeling suggests that these opsins do not differ spectrally, but may differ functionally in phototransduction cascade interactions. The exact function(s) of these opsins in non-retinal tissue and the function of multiple opsins in a monochromatic retina remain unclear. It is also unknown whether or not the opsins expressed in non-retinal neurons bind chromophore and become functional visual pigments. Additionally, it is also unclear if these non-retinal neurons have the required signaling molecules to initiate canonical G-protein signaling. Transcripts putatively encoding the components of a Gq-mediated phototransduction pathway have been identified in other stomatopod species (Porter et al. 2013; Donohue et al. 2017). However, it is conceivable that opsins expressed in these non-retinal neuropils can initiate G-protein independent signal transduction, a well described and common mechanism (Heuss and Gerber 2000; Rajagopal et al. 2005; Shenoy et al. 2006). Thus, these findings of opsin expression in non-retinal neuropils, particularly in visual ones, might implicate these opsins in the inclusion of non-visual photoreception in visual pathways. Co-expression of opsins in the retina, particularly spectrally similar ones, while an interesting finding, is a seemingly redundant mechanism of light detection in a monochromatic organism. However, our molecular modeling results also suggest that functional differences likely exist among the opsins, specifically, “tuning” of chromophore, G-protein binding, and arrestin interactions via non-conservative differences in regions which form the respective binding pockets for each structure. Should the opsins differ functionally, as our analysis suggests, this could be an interesting mechanism to maintain stable visual function in different levels of irradiance. Specifically, our analysis suggests that non-conservative amino acid substitutions in extracellular residues of S. empusa opsins (74 and 258) might tune the stability of the “chromophore plug” by affecting the binding affinity of the retinaldehyde chromophore (Janz and Farrens 2004; Tsukamoto et al. 2015). This would alter the duration of the chromophore’s attachment to the opsin, and thus make some opsins more sensitive to light than others (Tsukamoto et al. 2015). Thus, we propose that co-expression of spectrally identical opsins of varying sensitivity to light, or varying levels and times of activation, might be a mechanism S. empusa employs to maintain a stable visual representation of its environment at different times of day or in variable water depths. For G-protein binding, comprehensive and comparative structural analysis (Flock et al. 2017) of GPCR-Gα binding suggests that residues in ICL2, ICL3, and TM5 are at the interface between these two proteins. Our analysis has identified four residues of non-conservative amino acid substitution precisely at these structures in S. empusa opsins, residues 112 on ECL 2 and 189, 192, and 199 on TM5, which suggests they contribute either to G-protein docking and binding interactions, albeit through different mechanisms (Rasmussen et al. 2011). Therefore, we hypothesize that these sites serve as modulators of G-protein affinity and binding, causing differences in the electrical response of the photoreceptor, either in changes in strength or duration of light-induced depolarization. Additionally, the prolonged depolarizing afterpotential—typical of invertebrate photoreceptors and induced when an extensive population of visual pigments is photo-converted into the active state (Johnson and Pak 1986)—could be altered in opsins with a more transient or low affinity interaction with its cognate G-protein. Thus, opsin expressed in light-sensitive cells of the S. empusa retina and neuropils could have different capabilities to re-sensitize to high intensity light stimuli or have different onset kinetics of phototransduction. Finally, based on the analysis of the number and position of serine and threonine residues in the C-terminus, deactivation or desensitization kinetics are likely to be similar among the opsins, with the exception of opsin Se3 where serines and threonines were concentrated in the predicted region of arrestin interaction. Therefore, we don’t propose this as a common molecular mechanism of modulating phototransduction. Summarizing our unexpected findings, we propose that the monochromatic Atlantic stomatopod S. empusa has a more complex visual system than predicted, based on its single retinal photoreceptor class. While we report co-expression of two opsins in the retina, the possibility of additional opsins should not be discounted. We also cannot discount the possibility of multiple opsins being evolutionary vestiges from ancestral stomatopods, where the complex eye conformation (i.e., six midband rows between dorsal and ventral hemispheres) was likely the structure. This would represent a loss of molecular complexity in S. empusa, specifically in the array of opsins, that would mirror its structural eye loss. Thus, more work is required to ascertain if these multiple opsin transcripts are translated. Additionally, molecular analysis is needed to verify and substantiate our functional and spectral predictions. While stomatopod physiology proves difficult to study, electrophysiological studies of opsin expressing cells would shed light on the larger implications of opsin molecular adaptations. We propose that the expression of opsins in S. empusa is a flexible and versatile tool of not only mediating image formation in the retina, but of also adding nonvisual photoreceptive signals to downstream neurons. 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Sequence, Structure, and Expression of Opsins in the Monochromatic Stomatopod Squilla empusa

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Abstract

Abstract Most stomatopod crustaceans have complex retinas in their compound eyes, with up to 16 spectral types of photoreceptors, but members of the superfamily Squilloidea have much simpler retinas, thought to contain a single photoreceptor spectral class. In the Atlantic stomatopod Squilla empusa, microspectrophotometry shows that all photoreceptors absorb light maximally at 517 nm, indicating that a single visual pigment is present in all photoreceptors in the retina. However, six distinct, but partial, long wavelength sensitive (LWS) opsin transcripts, which encode the protein component of the visual pigment, have been previously isolated through RT-PCR. In order to investigate the spectral and functional differences among S. empusa’s opsins, we used RT-PCR to complete the 3′ end of sequences for five of the six expressed opsins. The extended sequences spanned from the first transmembrane (TM1) helix to the 3′ end of the coding region. Using homology-based modeling, we predicted the three-dimensional structure of the amino acid translation of the S. empusa opsins. Based on these analyses, S. empusa LWS opsins share a high sequence identity in TM regions and in amino acids within 15 Å of the chromophore-binding lysine on TM helix 7 (TM7), suggesting that these opsins produce spectrally similar visual pigments in agreement with previous results. However, we propose that these spectrally similar opsins differ functionally, as there are non-conservative amino acid substitutions found in intracellular loop 2 (ICL2) and TM5/ICL3, which are critical regions for G-protein binding, and substitutions in extracellular regions suggest different chromophore attachment affinities. In situ hybridization of two of the opsins (Se5 and Se6) revealed strong co-expression in all photoreceptors in both midband and peripheral regions of the retina as well as in selected ocular and cerebral ganglion neuropils. These data suggest the expression of multiple opsins—likely spectrally identical, but functionally different—in multiple types of neuronal cells in S. empusa. This suggests that the multiple opsins characteristic of other stomatopod species may have similar functional specialization. Introduction Stomatopod crustaceans, commonly referred to as mantis shrimps, make up a group of marine crustaceans that has been shown to have complex visual physiology, with up to 16 spectrally distinct photoreceptor classes observed in some species (Cronin et al. 1994; Porter et al. 2009; Cronin et al. 2010). Stomatopods have apposition compound eyes which are composed of many visual units called ommatidia (Marshall et al. 2007). Each ommatidium in the stomatopod eye has its own corneal and crystalline cone optical elements positioned above a rhabdom produced by seven or eight retinular photoreceptor cells (Marshall et al. 2007). In Squilla empusa, each rhabdom is formed by microvilli laden with visual pigments projected from seven photoreceptors, forming a single photoreceptive unit (Schönenberger 1977). Visual pigments are composed of an opsin G-protein-coupled receptor and a light sensitive chromophore. Upon photon absorption, the chromophore undergoes isomerization, typically from 11-cis retinal into all-trans retinal, and starts the phototransduction cascade. The spectral absorbance properties of visual pigments are typically tuned by alterations to the opsin residues that interact with and stabilize the chromophore in its binding pocket. Usually, one spectral class of photoreceptor expresses only one type of visual pigment (and thus a single opsin), although there is evidence for the expression of multiple distinct opsins within a single photoreceptor class from a number of species (e.g., African cichlid fish, Dalton et al. 2015; Limulus polyphemus, Battelle et al. 2016). At the structural level, stomatopod compound eyes are characterized by having two peripheral regions (dorsal and ventral) bisected horizontally by an equatorial midband region of specialized ommatidia (Marshall et al. 2007). While the peripheral regions contain the typical crustacean set of two photoreceptors spectral types, one sensitive to violet or ultraviolet (UV) light and the second sensitive to blue–green wavelengths, photoreceptors within the midband row are typically specialized for polychromatic and polarization vision. Most stomatopod species (superfamilies Gonodactyloidea, Lysiosquilloidea, Pseudosquilloidea, and Hemisquilloidea) have six ommatidial rows in the midband region, but species in the Squillioidea, including S. empusa in the present study, have only two ommatidial rows in the midband and are monochromatic (Cronin 1985; Schiff et al. 1986). Phylogenetic studies of the stomatopods suggest that the common ancestor of the Squilloidea most likely had six midband rows (Ahyong 1997; Porter et al. 2010). Thus, the two-row midband in Squilloidea is likely an evolved loss of photoreceptor diversity and spectral sensitivities. Squilla empusa are found near the coast of the Western Atlantic Ocean, from Maine to the Gulf of Mexico (Schiff et al. 1986). As is common in stomatopods, they make their homes by creating burrows on the ocean floor. Unlike stomatopods found in shallow coral reef habitats, S. empusa tends to burrow in muddy sea floors in dark and murky waters (Schiff et al. 1986). The limited light availability and their nocturnal hunting lifestyle (Schiff et al. 1986) may have contributed to the evolution of reduced visual complexity in S. empusa. Microspectrophotometric (MSP) studies of S. empusa eyes showed that all retinal photoreceptors absorb light maximally at 517 nm (Cronin 1985). The reduced complexity of the S. empusa retinal structure and the presence of a single spectral type of photoreceptor imply that there is also a single expressed opsin in the retina that initiates a conserved visual phototransduction cascade. However, recent studies have suggested that S. empusa visual physiology could be more complex than previously thought. Porter et al. (2009) isolated six unique opsin sequences from S. empusa retinas that cluster with other crustacean long wavelength sensitive (LWS) opsins. This raises an interesting question—why would a species with a monochromatic visual system possesses multiple opsins? The first possibility could be that the opsins differ spectrally and when expressed together, they tune the photoreceptors to their maximal absorbance value. However, this typically leads to a broadened photoreceptor curve, and there is no evidence of multiple visual pigments with different absorbance peaks from past MSP studies (Cronin 1985). Alternatively, the opsins could be identical, or highly similar, in spectral absorbance and yet differ functionally in how they initiate the phototransduction cascade due to structural differences leading to differences in membrane localization or chromophore coupling. There also exists the possibility that the opsins could be evolutionary vestiges, and are not translated into protein. In this study, we extended sequences of five opsin transcripts from Porter et al. (2009) to span from TM1 to the end of the coding region in order to predict the opsins’ functional and spectral differences. We also analyzed the expression of two of these opsins in S. empusa retinal and neural tissues. The data we present here suggest that S. empusa has multiple, spectrally-similar, but functionally distinct opsins expressed in the retina, optic lobes, and cerebral ganglion (CG). We propose that this monochromatic stomatopod possesses a complex molecular toolkit of opsins, perhaps capable of complex visual system modulation and downstream processing. Materials and Methods RT-PCR (3′-RACE) of S. empusa opsins mRNA and sequence analysis Squilla empusa eyes were homogenized in TRIzol (Invitrogen) and RNA was extracted as per the TRIzol Reagent protocol (Invitrogen). Single strand cDNA was synthesized from isolated total RNA using the SuperScript RT III protocol (Invitrogen) and primers designed from published S. empusa opsin partial sequences (Porter et al. 2009; Supplementary Table S1). After first strand synthesis, PCR was performed using Taq DNA polymerase (ThermoFisher Scientific) and specific primers for each of the six opsins identified in Porter et al. (2009) (Supplementary Table S1) to amplify opsin transcripts from the cDNA as per manufacturer’s protocol (ThermoFisher Scientific). PCR amplicons were ligated into the pGEM-T Easy plasmid (Promega) via TA cloning using the manufacturer’s protocol. Opsin sequences ligated into the plasmid were then sequenced (Genewiz). Partial opsin mRNA sequences obtained in Porter et al (2009) (GenBank accession numbers are the following: Se1-GQ221751.1, Se2-GQ221753.1, Se3-GQ221754.1, Se4-GQ221755.1, Se5-GQ221756.1, and Se6-GQ221752.1) were aligned with sequences obtained through RT-PCR (3′RACE) using Geneious software, version R10 (Biomatters Limited) (Kearse et al. 2012) to complete the opsin’s sequence. The mRNA sequences were then translated and aligned using Geneious software to facilitate the identification of non-conservative amino acid substitutions and other analyses. Structural modeling and analysis of S. empusa opsins The amino acid sequence for S. empusa opsin Se5 was used for homology-based three-dimensional structural modeling using LOMETS software (Wu and Zhang 2007). The S. empusa opsin model was generated using squid (Todarodes pacificus) rhodopsin as a template (PDB ID 2ZIY) (Shimamura et al. 2008). In combination with an amino acid alignment of the five analyzed opsins (Fig. 1), the model was used to identify amino acids proximal to the chromophore and potentially able to alter visual pigment spectral tuning. While it is possible to spectrally tune an opsin without a non-conservative amino acid substitution (Fasick and Robinson 1998; Fasick and Robinson 2000), charged amino acids can alter spectral properties of the chromophore (Wang et al. 2014) and are identifiable through bioinformatics. For our analysis, we considered non-conservative amino acid replacements, i.e., positions in the amino acid alignment where the charged/non-charged property of the amino acid has changed between opsins, within a 15 Å (1.5 nm) distance capable of altering chromophore binding chemistry. To generate the models of S. empusa opsin in complex with G-protein and arrestin, the S. empusa opsin structural model was aligned with the crystal structure of human rhodopsin in complex with mouse visual arrestin (PDB 4ZWJ) (Kang et al. 2015) and the crystal structure of human beta-2 adrenergic receptor in complex with bovine Gαs, rat Gβ, and bovine Gγ (PDB ID 3SN6) (Rasmussen et al. 2011) using the cealign tool using Pymol software (Schrodinger). This was done to position S. empusa opsin in complex with the signaling molecules. All amino acid numbering in this article is based on the S. empusa opsin alignments (Fig. 1). Fig. 1 View largeDownload slide Amino acid sequence alignment of five S. empusa opsins. Opsin amino acid sequences were inferred from mRNA nucleotide sequences from Porter et al (2009) and RT-PCR performed in this study. Amino acid residues are colored according to their property—yellow, non-polar; green, polar and uncharged; and red and blue, charged (negatively and positively charged, respectively). High levels of sequence identity are observed throughout, particularly in the TM regions (indicated by red annotations above the alignment) and in residues predicted to be in close proximity (≤15 Å) to the chromophore attachment site, K272 (indicated by blue annotations above the alignment). Sites of non-conservative amino acid substitutions among the opsins are denoted by the yellow annotations above the alignment. The green annotation above the alignment corresponds to residues predicted to be sites of phosphorylation and subsequent arrestin interaction. Fig. 1 View largeDownload slide Amino acid sequence alignment of five S. empusa opsins. Opsin amino acid sequences were inferred from mRNA nucleotide sequences from Porter et al (2009) and RT-PCR performed in this study. Amino acid residues are colored according to their property—yellow, non-polar; green, polar and uncharged; and red and blue, charged (negatively and positively charged, respectively). High levels of sequence identity are observed throughout, particularly in the TM regions (indicated by red annotations above the alignment) and in residues predicted to be in close proximity (≤15 Å) to the chromophore attachment site, K272 (indicated by blue annotations above the alignment). Sites of non-conservative amino acid substitutions among the opsins are denoted by the yellow annotations above the alignment. The green annotation above the alignment corresponds to residues predicted to be sites of phosphorylation and subsequent arrestin interaction. Synthesis of riboprobes for in situ hybridization Riboprobes were synthesized for visual opsins Se1, Se5, and Se6, which represent representative opsins from all three of the identified S. empusa opsin evolutionary clades identified in Porter et al. (2009). To synthesize both sense and antisense probes, pGEM-T Easy plasmid DNA containing the 3′UTR of the visual opsin transcripts were digested with one restriction enzyme (SalI or NotI) to create linear plasmids. Next, the following in vitro transcription reaction was prepared: linear plasmid DNA, DIG-RNA Labeling Mix (Roche), Polymerase buffer (Roche), RNase OUT (Invitrogen), and either T7 RNA polymerase or SP6 RNA polymerase (Roche). The transcription reaction was carried out as per the RNA polymerase protocol (Roche). Following transcription, reaction buffer with MgCl2 (ThermoFisher Scientific) and 0.1 u/μL RNase-free DNase I, (ThermoFisher Scientific) was added to the mixture. The reaction was carried out as described in the RNase-free DNase I protocol (ThermoFisher Scientific). Riboprobes were purified using the RNeasy Minelute Cleanup Kit (QIAGEN). Preparation of S. empusa tissue for in situ hybridization Squilla empusa mantis shrimp were sedated on ice upon arrival. Specimens were decapitated by making a transverse cut to sever the nerve cord between the CG and subesophageal ganglion. Once the anterior portion of the cephalothorax was separated from the body, appendages were removed from the ophthalmic and antennular somites. The eyestalks (which include the optic lobes within) were cut away from the cephalothorax. The eyestalks and cephalothorax were fixed in 4% paraformaldehyde with 12% sucrose in 0.1% diethyl pyrocarbonate (DEPC) 1× phosphate-buffered saline (PBS) overnight at 4°C. For retinal only in situ hybridization (ISH) studies, eyes were frozen and sectioned at 12–14 μm using a cryostat. Because of potentially lower signals expected from opsins expressed in neural tissues, isolated optic lobe and CG tissues were dehydrated using an ethanol gradient and propylene oxide, and then rehydrated, before being embedded in albumin gelatin. Then, the gelatin blocks were fixed overnight in 4% PFA in 0.1% DEPC 1× PBS, transferred to 0.1% DEPC 1× PBS, and sectioned at 60 μm using a vibratome. ISH of S. empusa tissue sections Our protocol is based originally from Ishii et al. (2003), and was also used in Bok et al. (2014) and Cronin et al. (2010) for stomatopod retinas. For all probes, no probe and sense probe controls were run alongside antisense probes (Supplementary Figs. S3 and S4). Squilla empusa sections on microscope slides were fixed in 4% PFA in PBS for 10 min. Next, the slides were washed three times in 0.1% (v/v) DEPC-1× PBS, 3 min per wash. The slides were then acetylated for 10 min in a solution containing 0.1% (v/v) DEPC-H2O, triethanolamine, 0.02 N HCl, and acetic anhydride, followed by three washes in 1× PBS, 5 min per wash. Hybridization solution (50% [v/v] formamide, 5× saline-sodium citrate [SSC] buffer, 5× Denhardt’s solution, and 250 μg/mL herring sperm DNA) was then added to the retina sections and the slides were incubated in a humidified chamber for 1 h. Riboprobes were added to hybridization solution (150–200 ng riboprobe per 100 μL of hybridization solution for retinal tissue, and 100 ng riboprobe per 100 μL of hybridization solution for extraocular tissue) and were incubated at 70°C for 10 min. The hybridization solution on the tissue was poured off and hybridization solution with riboprobe was added to the tissue sections. The slides were incubated at 75°C overnight. The next day, slides were incubated in 0.2× SSC at 65°C three times for 20 min to remove unbound probes. The slides were then incubated in Buffer B1 (0.1 M Tris pH 7.5 and 0.15 M NaCl) for 5 min, and then in Buffer B2 (Buffer B1 and 10% normal goat serum) for 1 h. Anti-digoxigenin-alkaline phosphatase (AP) (Roche) was diluted 1:5000 in Buffer B2 and was placed on the tissue sections, and the slides were incubated for 1–2 days at 4°C. Slides were next washed with Buffer B1 four times for 3 min each. Buffer B3 (0.1 M Tris pH 9.5, 0.1 M NaCl, and 50 mM MgCl2) was added to the slides and incubated for 5 min. Buffer B4 (NBT/BCIP tablet [Roche], 24 mg/mL levamisole) was then applied to the slides and left to incubate for several hours (retina sections) to overnight (extraocular tissue sections). Slides were then mounted and photographed via light microscopy. Results Amino acid sequence analysis of S. empusa opsins Using RT-PCR of S. empusa eyes, we completed the 3′ end of five of the six S. empusa opsin transcript sequences initially described by Porter et al (2009) (Fig. 1). We were unable to amplify the 3′ end of one of the six opsins (Se1), which is missing sequence data for part of TM6 and all of TM7, and so was excluded from further analysis. Based on amino acid translations, these transcript sequences encode for seven-transmembrane (TM) opsins with a mean predicted molecular weight of 37.5 kDa. Also, the opsins contain the critical chromophore attachment site at K272 (numbering based on Fig. 1 alignment), extended TM5 and TM6 helices (compared with bovine rhodopsin, Palczewski et al. 2000), a C-terminus region containing 9 or 10 putative sites of phosphorylation (Table 2), and important rhodopsin-class GPCR domains such as the (E)DRY motif on TM3 and NPXXY motif on TM7 (Fig. 1). Table 2. Comparison and summary of C-terminus amino acids predicted to influence signaling deactivation in S. empusa opsins Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Table 2. Comparison and summary of C-terminus amino acids predicted to influence signaling deactivation in S. empusa opsins Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Squilla empusa opsin  Possible phosphorylation sites (Ser and Thr)  Possible phosphorylation sites in arrestin interacting region  Negatively charged amino acids (Asp and Glu)  Se2  9  5  8  Se3  10  8  6  Se4  10  5  8  Se5  10  5  8  Se6  9  6  7  Among the opsins analyzed there was high amino acid sequence similarity, with the sequence identity between opsins ranging from 76.6% to 93.7% (Supplementary Fig. S1). The percent identity across all opsins is 71.2%, with an average pairwise identity of 83.4%. TM domains also have a high degree of similarity, with percent identity of 76.3% and an average pairwise identity of 86.0%. Despite the high level of sequence identity we identified several sites of non-conservative amino acid substitution. Specifically, non-conservative substitutions exist at functionally relevant locations, including positions 74 on TM3; 112 on intracellular loop (ICL) 2; 189, 192, and 199 on TM5; and 258 on TM7 (Table 1 and Figs. 1 and 2A, B). Table 1. Summary of non-conservative amino acid substitution among S. empusa opsins Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Notes: Number, location, and identities of the amino acids are depicted, along with a hypothesized function of each amino acid of interest. Amino acids marked with (/) in a white cell indicate non-charged residues (includes polar and non-polar). Amino acids marked with (+) in a gray cell indicate positively-charged residues; and those marked with (−) in a black cell indicate negatively-charged residues. TM, transmembrane region; ICL, intraellular loop; ECL, extracellular loop. Amino acid of interest numbering based on alignment consensus. Table 1. Summary of non-conservative amino acid substitution among S. empusa opsins Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Amino acid of interest  Location in opsin  Se2  Se3  Se4  Se5  Se6  Hypothesized function  74  TM3  Thr (/)  Arg (+)  Arg (+)  Thr (/)  Thr (/)  Chromophore binding stability  112  ICL2  Glu (−)  Lys (+)  Thr (/)  Thr (/)  Glu (−)  Modulation of Gα binding  189  TM5  His (+)  Phe (/)  Phe (/)  Tyr (/)  His (+)  Helical flexibility  192  TM5  Ser (/)  Lys (+)  Lys (+)  Gln (/)  Ser (/)  Helical flexibility  199  TM5  Lys (+)  Gln (/)  Lys (+)  Arg (+)  Lys (+)  Modulation of Gα binding  258  ECL3  Lys (+)  Lys (+)  Val (/)  Val (/)  Lys (+)  Chromophore binding stability  Notes: Number, location, and identities of the amino acids are depicted, along with a hypothesized function of each amino acid of interest. Amino acids marked with (/) in a white cell indicate non-charged residues (includes polar and non-polar). Amino acids marked with (+) in a gray cell indicate positively-charged residues; and those marked with (−) in a black cell indicate negatively-charged residues. TM, transmembrane region; ICL, intraellular loop; ECL, extracellular loop. Amino acid of interest numbering based on alignment consensus. Fig. 2 View largeDownload slide Structural modeling of S. empusa opsins suggests amino acids sites of non-conservative substitution function as modulators of G-protein binding and chromophore attachment stability. Front (A) and rear (B) view of structural model of S. empusa opsin Se5, labeling the predicted position of the sites of non-conservative substitution on the opsin’s tertiary structure (refer to Fig. 1 for position of these sites on the opsin amino acid sequences). (C) TM helices form a compact binding pocket around the chromophore, 11-cis retinal (depicted in space-filling format). No non-conservative amino acid substitutions are found within this binding pocket. (D) Model of active-state opsin bound to heterotrimeric G-protein (Gαs used in this model). (E) Model of active-state opsin in complex with arrestin (β-arrestin-1 used in this model). Surface charges plotted on arrestin—the legend below G indicates the charges. (F) Four non-conservative amino acids substitutions are predicted to be proximal to the G-protein binding pocket, particularly amino acids 112 and 199, found on intracellular loops 2 and TM5, respectively. (G) The opsin’s C-terminus is in close proximity to the positively-charged phosphate-sensing domains on arrestin. Acidic/negatively charged residues, serines, and threonines are concentrated in this region of the opsin’s C-terminus. Fig. 2 View largeDownload slide Structural modeling of S. empusa opsins suggests amino acids sites of non-conservative substitution function as modulators of G-protein binding and chromophore attachment stability. Front (A) and rear (B) view of structural model of S. empusa opsin Se5, labeling the predicted position of the sites of non-conservative substitution on the opsin’s tertiary structure (refer to Fig. 1 for position of these sites on the opsin amino acid sequences). (C) TM helices form a compact binding pocket around the chromophore, 11-cis retinal (depicted in space-filling format). No non-conservative amino acid substitutions are found within this binding pocket. (D) Model of active-state opsin bound to heterotrimeric G-protein (Gαs used in this model). (E) Model of active-state opsin in complex with arrestin (β-arrestin-1 used in this model). Surface charges plotted on arrestin—the legend below G indicates the charges. (F) Four non-conservative amino acids substitutions are predicted to be proximal to the G-protein binding pocket, particularly amino acids 112 and 199, found on intracellular loops 2 and TM5, respectively. (G) The opsin’s C-terminus is in close proximity to the positively-charged phosphate-sensing domains on arrestin. Acidic/negatively charged residues, serines, and threonines are concentrated in this region of the opsin’s C-terminus. Structural modeling and analysis of a S. empusa opsin A three-dimensional structural model was constructed for the amino acid sequence of opsin Se5, using homology to T. pacificus rhodopsin, a rhabdomeric visual opsin, to predict the likely molecular conformation of opsins in S. empusa. The predicted structure of Se5 (Fig. 2A, B) reveals a 7-TM opsin with structured cytoplasmic protrusions. Specifically, the cytoplasmic protrusions of the extended TM5 and TM6 helices likely form a structural determinant for G-protein binding specificity, namely to Gαq (Porter et al. 2013; Donohue et al. 2017). The model also predicts a compact chromophore binding pocket (Fig. 2C) comprised of all TM helices, and a chromophore binding site at K272 on TM7. The portions of the TM helices proximal to the extracellular space, and the ECLs (particularly ECL2) form the opsin chromophore “plug,” stabilized by a disulfide bond formed between C76 and C153. To address the possibility that S. empusa opsins are spectrally distinct, we analyzed the identities of the amino acids proximal to the site of chromophore attachment on TM7, K272. Specifically, we considered non-conservative amino acid substitutions between opsins as possible sites of spectral tuning. For this analysis, we considered amino acids within a 15 Å (1.5 nm) distance to be proximal, and potentially able to alter the chromophore binding chemistry. Our structural analysis suggests that the opsins are spectrally identical or similar: no non-conservative amino acid substitutions were found in within 15 Å of K272. Only one residue, site 74, has non-conservative amino acid substitutions within the 15 Å distance of K272 (Figs. 1 and 2A, B). However, this site is unlikely to cause spectral shifts between opsins, given its position on TM3 where it is close to the extracellular space, and its R-group is almost completely out of the 15 Å window (Fig. 2A, B). Interestingly, this site is placed close to the extracellular chromophore “plug,” and while it isn’t likely to affect the opsins spectrally, this site might serve as a tuning site for chromophore binding stability, a mechanism used in the mammalian rhabdomeric-type opsin, melanopsin (Tsukamoto et al. 2015). Additional analysis (Supplementary Fig. S5) reveals 11 amino acids surrounding the chromophore that are identical in all opsins analyzed in this study, and two are predicted to make contact with it (Y171 and W242). This analysis suggests a neutrally charged binding pocket similar to rhodopsin (Sakmar et al. 1989; Zhukovsky and Oprian 1989), however, amino acids containing R-groups with hydroxl moieties are present in the pocket (Supplementary Fig. S5), such as Y171 (which is predicted to contact the chromophore), Y79, and Y245, which support a green-shifted visual pigment (Chan et al. 1992; Asenjo et al. 1994). We also considered whether or not the multiple opsins might differ functionally, even while sharing high sequence and structural similarity. Our structural and sequence analyses identified four sites of non-conservative amino acid substitutions: residues 112 on ICL2, residues 189 and 192 on TM5, and 199 on TM5/ICL3 (Figs. 1 and 2A–F), located on important regions involved in coupling to signaling molecules. Specifically, these regions (ICL2 and ICL3) recognize and bind the opsin’s cognate G-protein, as shown extensively in bovine rhodopsin coupling to transducin (König et al. 1989; Franke et al. 1992; Yamashita et al. 2000; Natochin et al. 2003). We modeled the protein complex consisting of active-state S. empusa opsin and heterotrimeric G-protein (Gs was used in this model) (Fig. 2D, F). We observed the expected helical movement of TM5 and TM6 on the opsin and subsequent insertion of the C-terminus helix of Gα into the newly formed binding pocket in the opsin. Two of our sites, 112 on ICL2 and 199 on TM5 were particularly close to the binding pocket (Fig. 2F). Residue 112 is of particular interest for two reasons: it is on an unstructured coil, which does not sterically hinder its R-group from potentially interacting with several residues on the G-protein. Second, the non-conserved amino acid changes range from a complete switch of charge at that site (Se2 and Se6 are negatively charged, and Se3 is positively charged) to a loss of charge at that site (Se4 and Se5). Residue 199, while very close to the binding pocket, is hindered from movement due to its location on the cytoplasmic end of TM5. However, its proximity to the binding pocket might make it an important site that influences G-protein binding by impacting the overall charge of this region. Sites 189 and 192 are not likely to affect the G-protein binding pocket, but might affect the flexibility of TM5, and thus the formation of the binding pocket in the active state (Rasmussen et al. 2011). We analyzed the C-terminus, specifically for the number of phosphorylation sites and how they might activate arrestin (Fig. 2E, G). All opsins had a similar number of possible phosphorylation sites and negatively charged residues, which work in a synergistic manner to activate arrestin (Zhou et al. 2017). More important than the total number of possible phosphorylation sites is their proximity to the positively-charged phosphorylation-sensing domain on arrestins (Table 2 and Fig. 2G). To model opsin C-terminus–arrestin interaction and predict critical opsin C-terminus phosphorylation sites, we coupled active-state opsin to visual arrestin (crystal structure PDB 4ZWJ) (Figs. 1 and 2E, G) and determined that opsin residues 308–322 were proximal to the positively-charged region on arrestin. These data suggest that possible phosphorylation sites within this region on the opsin C-terminus are likely to be critical for signaling deactivation. Most opsins have a similar amount of possible phosphorylation sites in this region, except for Se3, which has most of its serines and threonines concentrated in this predicted critical region of arrestin interaction (Fig. 1 and Table 2). Expression of opsins in S. empusa retina and extraretinal neural tissue Using the newly generated sequence data (Supplementary Fig. S3), 3′UTR riboprobes were designed to hybridize to visual opsin mRNA in tissue sections in situ. Although riboprobes were synthesized for visual opsins Se1, Se5, and Se6, only Se5 and Se6 showed evidence of hybridization in our preparations. Expression patterns of S. empusa opsin transcripts Se5 (Fig. 3A–D) and Se6 (Fig. 3E–H) reveal that both opsins are robustly expressed in all regions of the retina—in both peripheral/hemispheric regions and in the midband. Expression of opsins Se5 and Se6 is also observed in transverse retinal sections (Fig. 3B, F), where riboprobe labeling is observed in all photoreceptors surrounding the rhabdoms in both hemispheres and in the midband (Fig. 3C, D, G, H). The intensity of Se5 labeling is even and robust in all regions of the retina (Fig. 3C,D), and a similar expression pattern is observed for Se6 (Fig. 3H). These data indicate that there is no preferential expression of either Se5 or Se6 in certain photoreceptors around the rhabdom in any region. Rather, S. empusa opsins Se5 and Se6 are co-expressed at high levels in all photoreceptors in all regions of the retina. Fig. 3 View largeDownload slide Robust transcript co-expression of M/LWS opsins Se5 and Se6 throughout the entire S. empusa retina. Sagittal (A and E) and transverse (B and F) retina sections labeled with Se5 (A–D) and Se6 (E–H) antisense riboprobes. Robust expression is observed in retina sections incubated with Se5 antisense riboprobes (A and B) including strong expression in all photoreceptors surrounding the rhabdom in the midband region (C) and periphery (D). Labeling with Se6 antisense riboprobes (E and F) also suggests robust expression of this opsin throughout the retina, with robust expression in all photoreceptors surrounding the retina in the midband region (G), and to a lesser degree in the periphery (H). DH, dorsal hemisphere; MB: midband; VH, ventral hemisphere. Scale bars: A, B, E, F: 500 μm; C, D, G, H: 100 μm. Fig. 3 View largeDownload slide Robust transcript co-expression of M/LWS opsins Se5 and Se6 throughout the entire S. empusa retina. Sagittal (A and E) and transverse (B and F) retina sections labeled with Se5 (A–D) and Se6 (E–H) antisense riboprobes. Robust expression is observed in retina sections incubated with Se5 antisense riboprobes (A and B) including strong expression in all photoreceptors surrounding the rhabdom in the midband region (C) and periphery (D). Labeling with Se6 antisense riboprobes (E and F) also suggests robust expression of this opsin throughout the retina, with robust expression in all photoreceptors surrounding the retina in the midband region (G), and to a lesser degree in the periphery (H). DH, dorsal hemisphere; MB: midband; VH, ventral hemisphere. Scale bars: A, B, E, F: 500 μm; C, D, G, H: 100 μm. Given such robust co-expression of opsins in the retina, we then tested if the Se5 and Se6 opsins are expressed in extraretinal tissue, which is common in marine crustaceans (Kingston et al. 2015; Kingston and Cronin 2016; Donohue et al. 2017) and terrestrial invertebrates such as Papilio xuthus (Arikawa et al. 2003). Through ISH of thicker (60 μm) tissue sections, we observed expression of retinal opsins Se5 and Se6 in other neural tissues (Fig. 4). Specifically, expression of both opsin transcripts was observed in optic neuropils including the optic lobe lamina, medulla, and lobula, as well as the hemiellipsoid body in the lateral protocerebrum. Se6 was more broadly expressed than Se5 in all neuropils, especially in the lamina and lobula neuropils (Fig. 4). Neither Se5 nor Se6 opsin transcript expression were observed in the ventral eye, but it is possible that other opsins (not probed for in this study, such as Se2–Se4) are present. Se5 and Se6 opsin expression was also observed in the CG, specifically cell bodies that make up the olfactory neuropil (Fig. 4). Thus, given all these results, co-expression of multiple opsins in this stomatopod is not only in photoreceptors, but surprisingly, also in downstream neurons involved in sensory processing. Fig. 4 View largeDownload slide Se5 and Se6 opsin transcripts co-expressed the optic lobes and cerebral ganglion (CG) of S. empusa. Sagittal eyestalk sections (top row) suggest that Se5 and Se6 transcripts appear to trace the lamina (La), medulla (Me), lobula (Lo), and hemiellipsoid body (HB) neuropils. As in Fig. 3, both transcripts are also co-expressed in retinal photoreceptors throughout the retina. Additionally, transverse CG sections show that Se5 and Se6 are co-expressed in the periphery of the olfactory lobes (OL). Antennal neuropil, AnN; lateral antennal neuropil (LAN); olfactory-glomeruli tract (OGT); dorsal hemisphere (DH); two equatorial midband rows (MB); and ventral hemisphere (VH). Fig. 4 View largeDownload slide Se5 and Se6 opsin transcripts co-expressed the optic lobes and cerebral ganglion (CG) of S. empusa. Sagittal eyestalk sections (top row) suggest that Se5 and Se6 transcripts appear to trace the lamina (La), medulla (Me), lobula (Lo), and hemiellipsoid body (HB) neuropils. As in Fig. 3, both transcripts are also co-expressed in retinal photoreceptors throughout the retina. Additionally, transverse CG sections show that Se5 and Se6 are co-expressed in the periphery of the olfactory lobes (OL). Antennal neuropil, AnN; lateral antennal neuropil (LAN); olfactory-glomeruli tract (OGT); dorsal hemisphere (DH); two equatorial midband rows (MB); and ventral hemisphere (VH). Discussion Past MSP analyses suggested that S. empusa, despite having two midband rows, has only a single photoreceptor spectral class (Cronin 1985), in contrast to the large number of spectrally-distinct photoreceptor classes described in other stomatopod species (Porter et al. 2009; Cronin et al. 2010). These physiological data imply that a simple molecular composition exists in its photoreceptors (e.g., fewer expressed opsins), and in combination with past evolutionary studies (Porter et al. 2010) also suggest a reduction in eye complexity compared with stomatopods with many photoreceptor classes. Our data suggest quite the contrary, that the monochromatic S. empusa expresses multiple opsins in both retinal photoreceptor cells and downstream visual processing neurons (Figs. 3 and 4). Homology modeling suggests that these opsins do not differ spectrally, but may differ functionally in phototransduction cascade interactions. The exact function(s) of these opsins in non-retinal tissue and the function of multiple opsins in a monochromatic retina remain unclear. It is also unknown whether or not the opsins expressed in non-retinal neurons bind chromophore and become functional visual pigments. Additionally, it is also unclear if these non-retinal neurons have the required signaling molecules to initiate canonical G-protein signaling. Transcripts putatively encoding the components of a Gq-mediated phototransduction pathway have been identified in other stomatopod species (Porter et al. 2013; Donohue et al. 2017). However, it is conceivable that opsins expressed in these non-retinal neuropils can initiate G-protein independent signal transduction, a well described and common mechanism (Heuss and Gerber 2000; Rajagopal et al. 2005; Shenoy et al. 2006). Thus, these findings of opsin expression in non-retinal neuropils, particularly in visual ones, might implicate these opsins in the inclusion of non-visual photoreception in visual pathways. Co-expression of opsins in the retina, particularly spectrally similar ones, while an interesting finding, is a seemingly redundant mechanism of light detection in a monochromatic organism. However, our molecular modeling results also suggest that functional differences likely exist among the opsins, specifically, “tuning” of chromophore, G-protein binding, and arrestin interactions via non-conservative differences in regions which form the respective binding pockets for each structure. Should the opsins differ functionally, as our analysis suggests, this could be an interesting mechanism to maintain stable visual function in different levels of irradiance. Specifically, our analysis suggests that non-conservative amino acid substitutions in extracellular residues of S. empusa opsins (74 and 258) might tune the stability of the “chromophore plug” by affecting the binding affinity of the retinaldehyde chromophore (Janz and Farrens 2004; Tsukamoto et al. 2015). This would alter the duration of the chromophore’s attachment to the opsin, and thus make some opsins more sensitive to light than others (Tsukamoto et al. 2015). Thus, we propose that co-expression of spectrally identical opsins of varying sensitivity to light, or varying levels and times of activation, might be a mechanism S. empusa employs to maintain a stable visual representation of its environment at different times of day or in variable water depths. For G-protein binding, comprehensive and comparative structural analysis (Flock et al. 2017) of GPCR-Gα binding suggests that residues in ICL2, ICL3, and TM5 are at the interface between these two proteins. Our analysis has identified four residues of non-conservative amino acid substitution precisely at these structures in S. empusa opsins, residues 112 on ECL 2 and 189, 192, and 199 on TM5, which suggests they contribute either to G-protein docking and binding interactions, albeit through different mechanisms (Rasmussen et al. 2011). Therefore, we hypothesize that these sites serve as modulators of G-protein affinity and binding, causing differences in the electrical response of the photoreceptor, either in changes in strength or duration of light-induced depolarization. Additionally, the prolonged depolarizing afterpotential—typical of invertebrate photoreceptors and induced when an extensive population of visual pigments is photo-converted into the active state (Johnson and Pak 1986)—could be altered in opsins with a more transient or low affinity interaction with its cognate G-protein. Thus, opsin expressed in light-sensitive cells of the S. empusa retina and neuropils could have different capabilities to re-sensitize to high intensity light stimuli or have different onset kinetics of phototransduction. Finally, based on the analysis of the number and position of serine and threonine residues in the C-terminus, deactivation or desensitization kinetics are likely to be similar among the opsins, with the exception of opsin Se3 where serines and threonines were concentrated in the predicted region of arrestin interaction. Therefore, we don’t propose this as a common molecular mechanism of modulating phototransduction. Summarizing our unexpected findings, we propose that the monochromatic Atlantic stomatopod S. empusa has a more complex visual system than predicted, based on its single retinal photoreceptor class. While we report co-expression of two opsins in the retina, the possibility of additional opsins should not be discounted. We also cannot discount the possibility of multiple opsins being evolutionary vestiges from ancestral stomatopods, where the complex eye conformation (i.e., six midband rows between dorsal and ventral hemispheres) was likely the structure. This would represent a loss of molecular complexity in S. empusa, specifically in the array of opsins, that would mirror its structural eye loss. Thus, more work is required to ascertain if these multiple opsin transcripts are translated. Additionally, molecular analysis is needed to verify and substantiate our functional and spectral predictions. While stomatopod physiology proves difficult to study, electrophysiological studies of opsin expressing cells would shed light on the larger implications of opsin molecular adaptations. We propose that the expression of opsins in S. empusa is a flexible and versatile tool of not only mediating image formation in the retina, but of also adding nonvisual photoreceptive signals to downstream neurons. 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Integrative and Comparative BiologyOxford University Press

Published: Apr 25, 2018

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