Pawns are behavioural mutants which show impairment in membrane excitability and are, therefore, devoid of normal avoiding reactions. We obtained 103 lines of Pawns through mutagenesis and screening. Among those that yielded to breeding studies, 59 lines belonged to the pwA complementation group and 38 lines belonged to the pwB group. No other genie loci were found in this extensive analysis. Mutants of the pwA locus showed various degrees of phenotypic leakiness. Based on the pheno-and genotypic differences, we estimated that at least 45 independent mutational events were represented in these 103 Pawn lines.

In cephalopods, the complex rhodopsin-retinochrome system serves to regenerate metarhodopsin and metaretinochrome after illumination. In the dark, a soluble protein, retinal-binding protein (RALBP), shuttles 11-cis retinal released from metaretinochrome located in the photoreceptor inner segments to metarhodopsin present in the rhabdoms. While in the rhabdoms, RALBP delivers 11-cis retinal to regenerate rhodopsin and in turn binds the all-trans isomer released by metarhodopsin. RALBP then returns all-trans retinal to the inner segments to restore retinochrome. The conventional interpretation of retinoid cycling is contradicted by immunocytochemical studies showing that, in addition to rhodopsin, retinochrome is present in the rhabdomal compartment, making possible the direct exchange of chromophores between the metapigments with the potential exclusion of RALBP. By using immunofluorescence and laser scanning confocal microscopy, we have precisely located opsin, aporetinochrome, and RALBP in light-/dark-adapted octopus retinas. We found differences in the distribution of all three proteins throughout the retina. Most significantly, comparison of cross sections though light- and dark-adapted rhabdoms showed a dramatic shift in position of the proteins. In the dark, opsin and retinochrome colocalized at the base of the rhabdomal microvilli. In the light, opsin redistributed along the length of the microvillar membranes, and retinochrome retreated to a location that is perhaps extracellular. RALBP was present in the core cytoplasm of the photoreceptor outer segments in the dark, and RALBP moved to the periphery in the light. Because of the colocalization of opsin and retinochrome in the dark, we believe that the two metapigments participate directly in chromophore exchange. RALBP may serve to transport additional chromophore from the inner segments to the rhabdoms and may not be immediately involved in the exchange process.

The photopigments, rhodopsin and retinochrome, have been localized in cephalopod retinae using light and electron microscopic immunocytochemical methods. Polyclonal antibodies prepared against squid opsin demonstrated the presence of this protein in the photoreceptor rhabdomes, Golgi zone, Golgi-associated vesicles, plasma membrane, large cytoplasmic vesicles, and axonal membranes of octopus retinae. Monoclonal anti-opsin immunostained the rhabdomes and multivesicular bodies in the photoreceptor inner segments of squid. We believe the multivesicular bodies are involved in rhabdome turnover. Polyclonal anti-retinochrome localized this photopigment to the myeloid bodies of the photoreceptor inner segments, the rhabdomes, and to the extracellular space between opposing rhabdomeres in octopus retina. The results suggest some interesting functional relationships between rhodopsin and retinochrome with regard to chromophore exchange between illuminated forms of these photopigments and chromophore addition to newly synthesized opsin.

We examined rhabdom structure and the distribution of filamentous actin in the photoreceptor outer segments of the retina of Octopus bimaculoides. Animals were dark- or light-adapted, fixed, embedded and sectioned for light and electron microscopy. Statistical analyses were used to compare relative cross-sectional areas of rhabdom microvilli and core cytoplasm within and between the two lighting conditions. Dark-/light-adapted rhabdoms were also prepared for confocal laser scanning microscopy and labeled with fluorescence-tagged phalloidin. Results show differences in the morphology of dark- and light-adapted octopus rhabdoms with the cross-sectional areas of the rhabdoms increasing in dark-adapted retinas and diminishing in the light. Comparisons between the lighting conditions show that an avillar portion of the photoreceptor outer segment membrane, prominent in the light-adapted retina, is recruited to form new rhabdomere microvilli in dark-adapted eyes. Filamentous actin was associated with the avillar membrane in light-adapted retinas, which may indicate that actin and other microvillus core proteins remain linked to the avillar membrane to support rapid microvillus formation in the dark. Photopigment redistributions also occur in light- and dark-adapted retinas, and this study suggests that these changes must be coordinated with the simultaneous breakdown and reformation of the rhabdomere microvilli.

Using autoradiographic and biochemical methods, we have demonstrated the renewal of light-sensitive membranes and photopigments in Octopus visual cells. After the injection of Octopus with [3H]leucine, electron microscope autoradiography revealed an intracellular pathway similar to that in vertebrates for the synthesis and transport of nascent protein from the inner segments to the rhabdomes. However, migration of labelled protein from synthetic sites to the light-sensitive rhabdomes took longer in Octopus than the equivalent process in vertebrates. Biochemical analysis of [3H]leucine-labelled retinas identified some of the labelled protein observed in autoradiographs of the rhabdomes as the visual pigment, rhodopsin. We have shown that retinochrome, a second photopigment in cephalopod retinas, is also renewed. Biochemical analysis 8 h after injection of [3H]leucine revealed heavy labelling of this photoprotein. Light microscope autoradiography of Octopus retina 8 h after injection of [3H]retinol showed labelling of both the rhabdomes and the myeloid bodies of the inner segments. Biochemical data gathered 8 h after injection of [3H]retinol indicated chromophore addition to both rhodopsin and retinochrome with retinochrome being more heavily labelled than rhodopsin. Thus, silver grains observed over the rhabdomes and inner segments could arise from one or both photopigments. These data suggest that retinal is stored in the myeloid bodies of the photoreceptor inner segments. Retinal could then be transferred, perhaps via retinochrome, to newly synthesized opsin before the visual pigment is assembled into new rhabdomeric membranes. Alternatively, retinochrome may serve to transport retinal from the myeloid bodies to the rhabdomes to regenerate rhodopsin as previously proposed.