Laura J. Robles

Autoradiographic and biochemical analysis of photoreceptor membrane renewal inOctopus retina

SummaryUsing autoradiographic and biochemical methods, we have demonstrated the renewal of light-sensitive membranes and photopigments inOctopus visual cells. After the injection ofOctopus 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 inOctopus 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 ofOctopus 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]retrnol 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.

Immunocytochemical localization of a rhodopsin-like protein in the lipochondria in photosensitive neurons of Aplysia californica

SummaryPolyclonal antibodies directed against squid opsin were used in immunocytochemical and immunoblot experiments to identify a rhodopsin-like protein in photosensitive neurons of Aplysia. Aldehyde-fixed abdominal and cerebral ganglia were embedded in paraffin for peroxidase anti-peroxidase analysis or used whole for immunofluorescence studies. Ganglia were embedded in Lowicryl K4M for electron-microscope immunocytochemistry. In both the cerebral and abdominal ganglia, light-microscope immunocytochemical results showed reaction product deposited around the neuronal cell periphery corresponding in position to the lipochondria. In the abdominal ganglion, the giant cell R2, located in the right rostral quarter, and neurons in the right caudal quarter were consistently labeled with anti-opsin. Electron-microscopic studies demonstrated ferritin-labeling of the lipochondria in R2 and other immunoreactive neurons. Immunoblot analysis of R2 and cerebral neuron extracts was used to identify two prominent immunoreactive protein bands at 85000 and 67500 molecular weight.

Immunocytochemical localization of retinal binding protein in the octopus retina: A shuttle protein for 11-cis retinal

The cephalopod retina contains two photopigments that are spatially separated within the photoreceptors; rhodopsin, localized in the light-sensitive rhabdoms, and retinochrome, present in the myeloid bodies of the photoreceptor inner segments. In the light, the chromophore of retinochrome, all-trans retinal, is photoisomerized to 11-cis to form metaretinochrome. Metaretinochrome is believed to serve as a store for 11-cis retinal used in the regeneration or biosynthesis of rhodopsin. Previous studies suggest that a soluble retinal binding protein (RALBP) serves as a shuttle between retinochrome and rhodopsin, and, in the dark, may transport chromophore from the myeloid bodies to the rhabdoms. Our study supports this hypothesis and demonstrates that RALBP is in the correct cellular locations to function as a shuttle. Dark- and light-adapted octopus retinas were labeled with anti-RALBP using immunofluorescence and immunogold techniques. Our results showed that RALBP was distributed differently in the dark- and light-adapted retinas. Our most significant observation was that myeloid bodies from light-adapted retinas were more heavily labeled by anti-RALBP than myeloid bodies in dark-adapted retinas. The rhabdomeres, interphotoreceptor matrix, and inner limiting membrane were also labeled in both light and dark conditions. Based on these results and evidence from previous biochemical studies, we conclude that in the dark RALBP leaves the myeloid bodies and transports 11-cis retinal to the rhabdoms where chromophore exchange with metarhodopsin may occur.

Light-activated retinoid transport in cephalopod photoreceptors

Retinoid transport and chromophore exchange have been investigated in cephalopods using autoradiographic and radiobiochemical techniques. In dark adapted retinas, [3H]-retinoid is concentrated in myeloid bodies present in the photoreceptor inner segments and is bound to the photopigment retinochrome. In retinas exposed to light, there is a shift in the distribution of [3H]-retinoid. The rhabdomes become more heavily labeled than the inner segments, and rhodopsin labeling exceeds that of retinochrome. In animals returned to the dark, another shift in retinoid distribution occurs and the inner segments are again more labeled than the rhabdomes. In these animals [3H]-retinoid is bound primarily to retinochrome. Exposure to light seems to activate a transport mechanism that results in the redistribution of retinoid between the inner segments and rhabdomes and chromophore exchange among the photopigments.

Light-/dark-induced changes in rhabdom structure in the retina of Octopus bimaculoides

Abstract. 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.

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.

Rho GTPases regulate rhabdom morphology in octopus photoreceptors

In the cephalopod retina, light/dark adaptation is accompanied by a decrease/increase in rhabdom size and redistribution of rhodopsin and retinochrome. Rearrangements in the actin cytoskeleton probably govern changes in rhabdom size by regulating the degradation/formation of rhabdomere microvilli. Photopigment movements may be directed by microtubules present in the outer segment core cytoplasm. We believe that rhodopsin activation by light stimulates Rho and Rac signaling pathways, affecting these cytoskeletal systems and their possible functions in controlling rhabdom morphology and protein movements. In this study, we localized cytoskeletal and signaling proteins in octopus photoreceptors to determine their concurrence between the lighting conditions. We used toxin B from Clostridium difficile to inhibit the activity of Rho/Rac and observed its effect on the location of signaling proteins and actin and tubulin. In both lighting conditions, we found Rho in specific sets of juxtaposed rhabdomeres in embryonic and adult retinas. In the light, Rho and actin were localized along the length of the rhabdomere, but, in the dark, both proteins were absent from a space beneath the inner limiting membrane. Rac colocalized with tubulin in the outer segment core cytoplasm and, like Rho, the two proteins were also absent beneath the inner limiting membrane in the dark. The distribution of actin and Rho was affected by toxin B and, in dark-adapted retinas, actin and Rho distribution was similar to that observed in the light. Our results suggest that the Rho/Rac GTPases are candidates for the regulation of rhabdomere size and protein movements in light-dark-adapted octopus photoreceptors.