Reference: Bid. BUN. 193: 97-105. (August. 1997) Identification and Localization of a Possible Rhodopsin in the Echinoderms Asterias forbesi (Asteroidea) and Ophioderma brevispinum (Ophiuroidea) SijNKE JOHNSEN Department of Biology, University ofNorth Carolina, Chapel Hill, North Carolina 27599 Abstract. Protein extracts of optic cushion tissue from intra-ossicular, etc.) and ultrastructure (ciliary, rhab- the asteroid Asterias forbesi and arm tissue from the domeric, unspecialized, etc.) of extraocular photorecep- ophiuroid Ophioderma brevispinum were subjected to tors remain controversial (Cobb and Hendler, 1990; Western blot analysis. Both tissues contain a membrane- Cobb and Moore, 1986; Millott and Yoshida, 1960; associated protein that reacts with two monoclonal anti- Stubbs, 198 1; Yoshida et al., 1983). Among extraocular bodies raised against bovine rhodopsin. This protein mi- photoreceptors, those of ophiuroids are of particular in- grates slightly behind bovine rhodopsin during sodium- terest because portions of the calcitic endoskeleton in dodecyl-sulfate polyacrylamide gel electrophoresis, sug- ophiuroids transmit, polarize, and possibly focus light gesting that its molecular weight is slightly larger. Immu- (Hendler and Byrne, 1987; Johnsen, 1994). In particular, nohistochemical examination of the optic cushions of A. the crystalline structure of the brachial spines of the firbesi revealed a substance that reacts with both mono- ophiuroid Ophioderma brevispinum is ideal for analysis clonal antibodies; moreover, this substance is more of the polarization of downwelling skylight (Johnsen, abundant in dark-adapted animals than in light-adapted 1994). It has been shown that 0. brevispinum is sensitive animals. The arms and central disk of 0. brevispinum to polarized light (Johnsen, 1994) and it is hypothesized were also examined immunohistochemically. The tips of that this sensitivity is mediated by photoreceptors within the arm spines contain a substance that reacts with both the brachial spines. monoclonal antibodies, and at higher magnification this If the visual pigment in echinoderms were identified, immunoreactive material is localized to small regions the photoreceptors could be localized using immunohis- within the stroma of the ossicles. Taken together, the bio- tochemical techniques. The pigment is virtually un- chemical and immunochemical evidence suggests that known (reviewed in Yoshida et al., 1983), but several as- the cross-reacting protein is homologous to other known sumptions can be made about it. First, the visual pig- rhodopsins and is serving as a visual pigment in echino- ment is likely to be a rhodopsin, because all described derms. visual pigments are members of this protein family. Sec- ond, it is assumed that essentially the same visual pig- Introduction ment occurs in the photoreceptors of all echinoderms. Third, it is assumed that the optic cushions of asteroids Extraocular photosensitivity in echinoderms is wide- are functionally similar to other metazoan eyes, because spread, well documented, and ecologically important (reviewed in Yoshida, 1979; Yoshida et al., 1983), but a large body of chemical, morphological, and behavioral the functional characteristics, morphology, and distribu- results suggests that optic cushions are visual organs (Ta- tion of the receptors mediating this sense are poorly un- kasu and Yoshida, 1983; reviewed in Yoshida et al., derstood. At present, the location (subepithelial, dermal, 1983). Given these assumptions, the asteroid optic cush- ion can be used as a model structure for determining the visual pigment in echinoderms. The optic cushion of As- Received 6 December 1996: accepted 6 May 1997. terias forbesi is particularly suitable as a model because 97 98 S. JOHNSEN it is known that the microvilli of the putative sensory irreversibly aggregate, the samples were not boiled be- cells within the optic cup of a congener (A. amurensis) fore electrophoresis. The supernatant was electropho- become more numerous and increase in size and retinal resed on an 8% SDS polyacrylamide gel and transferred protein content after long exposure to darkness (Takasu to a polyvinyl membrane (Immobilon-P, Millipore and Yoshida, 1983). Inc.) using the Western transfer process (Harlow and This study describes an immunochemical search for Lane, 1988). The gel lanes were loaded with the maxi- homologues of rhodopsin, first in the optic cushion of mum amount of protein that could run correctly on the the starfish Asterias forbesi and then in the tissues of the gel apparatus. Nonspecific binding was blocked by in- brittlestar Ophioderma brevispinum. In both animals, cubating the membrane in 1 pg/ml polyvinyl alcohol the search revealed a membrane-associated protein, for 5 min. The membrane was then incubated in a slightly heavier than bovine rhodopsin, that reacts with monoclonal antibody raised against bovine rhodopsin two monoclonal antibodies raised against bovine rho- (diluted 1:50 in phosphate buffered saline (140 mM dopsin. The same antibodies also react with a substance NaCl, 3 mM KCl, 8 mM Na2HP04, 1.5 mM KH2P04, in the optic cups of the optic cushion of A. forbesi. In pH 7.4, referred to hereafter as PBS) containing 0.1% addition, the cross-reaction is stronger in dark-adapted Tween-20 detergent and 3% bovine serum albumin animals than in light-adapted animals. The two mono- (BSA)) for about 20 h at 20°C. Fourteen monoclonal clonal antibodies also cross-reacted with a substance antibodies raised against bovine rhodopsin were tested. within the tips of the brachial spines of 0. brevispinum, Their epitope was then investigated using competitive suggesting that they contain concentrated amounts of binding assays with various peptide sequences from bo- echinoderm rhodopsin. These data all suggest that the vine rhodopsin (Adamus et al., 1991). Figure 1 shows purified protein in both animals is homologous to the names and putative epitopes of the antibodies. All known rhodopsins and that this protein is the visual pig- 14 antibodies are known to bind with bovine rhodopsin ment in echinoderms. on a Western blot (Hargrave, pers. comm.). Antibodies raised against bovine rhodopsin were chosen because Materials and Methods echinoderms are more closely related to chordates than to any other phylum for which rhodopsin antibodies are Source and maintenance qf animals available. After reaction with the primary antibody, the Specimens of A. .forbesi were obtained from the Ma- membrane was washed three times for 5 min each in rine Biological Laboratory, Woods Hole, Massachusetts. PBS, and then incubated for 1 h at room temperature Specimens of 0. brevispinum were collected from Old in goat anti-mouse IgG + IgM conjugated with alkaline Dan Bank, about 2 km north of the Keys Marine Lab, phosphatase (Sigma Immunochemicals; diluted 1:5000 Layton, Long Key, Florida. Animals were maintained in in same buffer as primary antibody). The membrane aquaria in artificial seawater (ASW), salinity 35 ppt, on was then washed three times for 20 min each in PBS a 12/ 12 hour light/dark cycle. 0. brevispinum and A. and developed according to standard procedures for al- forbesi were kept at 24°C and 10°C respectively. kaline phosphatase detection (Harlow and Lane, 1988). The procedure for 0. brevispinum was identical except that tissue from the arms was used. As a negative con- Extraction and Western blot analysis trol, tissue from the gut of A. forbesi was prepared and An adult specimen ofA.,forbesi was dark-adapted for analyzed in the above manner. about 20 h. The adaptation was performed because pre- vious studies on several asteroids have shown that the Determination of cellular location ofproteins from microvilli of the putative sensory cells in the optic cush- A. forbesi and 0. brevispinum ions increase in number, size, and retinal protein content after long exposure to darkness (Takasu and Tissue from A. forbesi or 0. brevispinum was frozen Yoshida, 1983). Under dim red light, the tips of four and pulverized as described above. The powder was sus- arms (containing optic cushions) were removed and im- pended in 200 ~1 of PBS with 1 mM phenylmethysulfo- mediately frozen in liquid nitrogen. The frozen tissue nyl fluoride and 20 pg/ml leupeptin (referred to hereafter was pulverized with a mortar and pestle and the powder as PBS/PI). The solution was centrifuged for 20 min at suspended in 100 ~1 sodium-dodecyl-sulfate (SDS) 16,000 X g. The supernatant was then centrifuged at polyacrylamide gel electrophoresis (PAGE) sample 125,000 X g for 1 h to remove all insoluble material. The buffer. The solution was incubated at 20°C for 5 min to supernatant from the high-speed spin was kept and re- dissolve all proteins and centrifuged at 16,000 X g for ferred to as “C.” The pellet from the low-speed spin was 10 min to precipitate insoluble material. Because rho- resuspended in 200 ~1 of PBS/PI with 2% Triton X- 100 dopsins that have been boiled in the presence of SDS and incubated for 30 min. The suspension was then spun RHODOPSIN IN ASTEROIDS AND ECHINOIDS 99 B6-30N b!NG TEG PNF WP FSN KTG VVR SPF EYP QYY LAE PWQ YSA LAA YMF LLI LLG 33'1 R2-15N 1 K62-12N NFM TLY WR TPL NY1 LLN LVF AX3 FMV E’GG FTV TI’Q TSJ, HGY FVF GPT BlOcL + B3uL Al-55M GCY m FFA TLG GEG ALW SLV VLA IER YIV VCK PMA NFR FGE NHA IMG VAF T.W% MAL ACA APP LAG WSR YIP EGM OCS CGV DYY TTlK PEI NNB SFV IYN FVV HF?' IP5 Tl3-34L + Tl3-35L III F'FC YGQ LVF Tw ATT OKA EKE VTR MVI z;Kv IAF LIC WVP YAS K42 -4lL VA?? YIF THQ GSN FGP VFM TIP AFF AKS SAI YNP VIY II*??4 MO FRN CMV TTT, CCG All-275C K16-5OC m-K- K57-91C K16-155C + k55-75C Figure1. Amino acid sequence of bovine rhodopsin showing the epitopes of the various antibodies used in the study. Light type denotes transmembrane regions that are inaccessible to antibodies in immu- nohistochemistry.