Immunocytochemical localization of opsin, visual arrestin, myosin III, and calmodulin in Limulus lateral eye retinular cells and ventral photoreceptors

Abstract

The photoreceptors of the horseshoe crab Limulus polyphemus are classical preparations for studies of the photoresponse and its modulation by circadian clocks. An extensive literature details their physiology and ultrastructure, but relatively little is known about their biochemical organization largely because of a lack of antibodies specific for Limulus photoreceptor proteins. We developed antibodies directed against Limulus opsin, visual arrestin, and myosin III, and we have used them to examine the distributions of these proteins in the Limulus visual system. We also used a commercial antibody to examine the distribution of calmodulin in Limulus photoreceptors. Fixed frozen sections of lateral eye were examined with conventional fluorescence microscopy; ventral photoreceptors were studied with confocal microscopy. Opsin, visual arrestin, myosin III, and calmodulin are all concentrated at the photosensitive rhabdomeral membrane, which is consistent with their participation in the photoresponse. Opsin and visual arrestin, but not myosin III or calmodulin, are also concentrated in extra-rhabdomeral vesicles thought to contain internalized rhabdomeral membrane. In addition, visual arrestin and myosin III were found widely distributed in the cytosol of photoreceptors, suggesting that they have functions in addition to their roles in phototransduction. Our results both clarify and raise new questions about the functions of opsin, visual arrestin, myosin III, and calmodulin in photoreceptors and set the stage for future studies of the impact of light and clock signals on the structure and function of photoreceptors.

Evolution of arthropod visual systems: Development of the eyes and central visual pathways in the horseshoe crabLimulus polyphemusLinnaeus, 1758 (Chelicerata, Xiphosura)

“…2) and sections of adult L. polyphemus brains (Battelle, unpublished observations) demonstrated that the antibody directed against Lp myosin III not only stained photoreceptor cell bodies but also their axonal projections and terminals. Studies with adult animals had also shown that the arhabdomeral cells of the lateral eye do not stain with this antibody, suggesting the protein to be photoreceptor-specific (Battelle et al, 2001). We used this antibody to explore the architecture of the developing central visual pathway in more detail.…”

“…The production and characterization of the antibodies against L. polyphemus (Lp) opsin and Lp myosin III were described in Battelle et al (2001); the antibody against Lp visual arrestin in Battelle et al (2000). Opsin is the protein moiety of the visual pigment rhodopsin, which is responsible for absorbing light and initiating the phototransduction cascade (Smith et al, 1993).…”

“…Opsin is the protein moiety of the visual pigment rhodopsin, which is responsible for absorbing light and initiating the phototransduction cascade (Smith et al, 1993). Lp opsin localizes to the rhabdom of photoreceptors and is associated with cytosplasmic vesicles containing internalized rhabdomeral membranes (Battelle et al, 2001;Sacunas et al, 2002). Lp myosin III is present throughout the photoreceptors, including the region occupied by the photosensitive rhabdom, the cytosol of their cell bodies (Battelle et al, 1998(Battelle et al, , 2001, and their axons and terminals (Edwards et al, 1990;and Battelle, unpublished observations).…”

“…Aspects that have been explored in the past include adult ommatidial morphology of the lateral eyes (review Fahrenbach, 1975), organization of the retinal projections into the brain as well as efferent projections Barlow, 1980, 1982;Batra and Chamberlain, 1985;, and distribution of the visual pathway neurotransmitters (reviewed in Battelle et al, 1999) octopamine (Evans et al, 1983), serotonin , FMRFamide and substance P (Chamberlain and Engbretson, 1982;Lewandoswski et al, 1989), histamine , and acetylcholine (Hornstein et al, 1994). Additionally, circadian modulation of the photoresponse (e.g., Battelle et al, 1998Battelle et al, , 2000Battelle et al, , 2001Sacunas et al, 2002;Waren and Chamberlain, 2002;Ankrom and Chamberlain, 2002;Dalal et al, 2003;Pieprzyk et al, 2003;Runyon et al, 2004;reviews, Barlow, 2001;Battelle, 2002), and both computational and behavioral aspects of vision (reviewed in Barlow et al, 2001) are active areas of current research.…”

“…Thus, we applied the proliferation marker bromodeoxyuridine (BrdU; Harzsch et al, 1999;Harzsch and Walossek, 2001;Wildt and Harzsch, 2002) and immunohistochemistry with photoreceptor-specific markers that have been characterized in adult animals (opsin, visual arrestin, myosin III; Battelle et al, 1998Battelle et al, , 2001). Our analyses focused on embryonic stages shortly before hatching, trilobite larvae, and young juveniles, because the study by French (1980) indicated that this time period includes rapid morphogenetic events.…”