The lateral eyes of the horseshoe crab Limulus polyphemus undergo dramatic daily changes in structure and function that lead to enhanced retinal sensitivity and responsiveness to light at night. These changes are controlled by a circadian neural input that alters photoreceptor and pigment cell shape, pigment migration, and phototransduction. Clock input to the eyes also regulates photomechanical movements within photoreceptors, including membrane shedding. The biochemical mechanisms underlying these diverse effects of the clock on the retina are unknown, but a major biochemical consequence of activating clock input to the eyes is a rise in the concentration of cAMP in photoreceptors and the phosphorylation of a 122 kDa visual system-specific protein. We have cloned and sequenced cDNA encoding the clock-regulated 122 kDa phosphoprotein and show here that it is a new member of the myosin III family. We report that Limulus myosin III is similar to other unconventional myosins in that it binds to calmodulin in the absence of Ca 2ϩ ; it is novel in that it is phosphorylated within its myosin globular head, probably by cAMP-dependent protein kinase. The protein is present throughout the photoreceptor, including the region occupied by the photosensitive rhabdom. We propose that the phosphorylation of Limulus myosin III is involved in one or more of the structural and functional changes that occur in Limulus eyes in response to clock input.
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.
Histamine has been proposed as a photoreceptor neurotransmitter in two major groups of arthropods, the insects and the crustacea. In this study biochemical and immunocytochemical approaches were used to examine the synthesis, endogenous content, and cellular distribution of histamine in the visual system of the horseshoe crab Limulus polyphemus, an ancient chelicerate arthropod. Studies with this animal have been critical to our understanding of the basic processes of vision. High-voltage paper electrophoresis was used to assay for histamine synthesis in Limulus tissues incubated with radiolabeled histidine; histamine synthesis was detected in the lateral, median, and ventral eyes and optic nerves and in the visual centers in the brain. Endogenous histamine, assayed as its orthophthalaldehyde derivative by high-performance liquid chromatography and electrochemical detection, was also detected in these tissues. Immunocytochemical analyses, with an antiserum directed against a protein conjugate of histamine, revealed histamine-like immunoreactivity in the somata of photoreceptors in each of the eyes and in the regions of the brain where the photoreceptors terminate. Histamine-like immunoreactivity was also intense in the cell bodies and axon collaterals of eccentric cells in the lateral eye and in eccentric cell projections in the brain. These results show that histamine is a major biogenic amine in the Limulus visual system, and they suggest that this amine is involved in transmitting visual information from the eyes to the brain and in lateral inhibition, a fundamental mechanism for processing visual information in the lateral eye.
Studies of lateral, median, and ventral eyes of the chelicerate arthropod Limulus polyphemus (the common American horseshoe crab) are providing important basic information about mechanisms for information processing in the peripheral visual system and for the modulation of visual responses by light and circadian rhythms. The processing of visual information in Limulus brain is less well understood in part because the specific central projections of the various classes of visual neurons are not known. This study describes a mouse monoclonal antibody, 3C6A3, which binds to Limulus photoreceptor cell bodies, their axons, and terminals, but not to any other cell type in the central nervous system. This antibody, and intracellular injection of biocytin, are used to demonstrate the central projections of each type of photoreceptor. Our main conclusions are that: 1) the photoreceptors (retinular cells) of the lateral eye project only to the lamina; 2) the photoreceptors of the lateral rudimentary eye project to both the lamina and medulla; 3) the photoreceptors of the median ocellus project only to the ocellar ganglion; and 4) the photoreceptors of the rudimentary median (endoparietal) eye project to the ocellar ganglion and also into the optic tract. These results, along with previous studies, allow us to infer the projections of the secondary cells. The eccentric cells of the lateral eye project to the lamina, medulla, optic tract, and ocellar ganglion. The arhabdomeral cells of the median ocellus project through the ocellar ganglion and to optic tract to the medulla.
Efferent fibers innervate all of the eyes of the horseshoe crab, Limulus polyphemus. Driven by a circadian clock located in the central nervous system, the activity of the fibers at night is responsible for anatomical, biochemical, and physiological changes in the eyes, which increase their ability to detect and respond to light. We showed previously that octopamine, a putative efferent neurotransmitter, stimulates the phosphorylation of a 122 kD protein in in vitro preparations of both ventral and lateral eyes by means of a cAMP-dependent mechanism. We now report that phosphorylation of the 122 kD protein in the lateral eye is enhanced in vivo: (1) at night, in correlation with efferent nerve input activated by the circadian clock; and (2) during the day, in response to electrical stimulation of efferent axons. We show further that the 122 kD protein is enriched in, and may be restricted to, tissues that contain photoreceptors. We postulate that this protein is involved in the efferent-stimulated increase in retinal sensitivity.
We performed genome-wide mutagenesis of C57BL/6J mice using the mutagen N-ethyl-N-nitrosourea (ENU) and screened the third generation (G3) offspring for visual system alterations using electroretinography and fundus photography. Several mice in one pedigree showed characteristics of retinal degeneration when tested at 12-14 weeks of age: no recordable electroretinogram (ERG), attenuation of retinal vessels, and speckled pigmentation of the fundus. Histological studies showed that the retinas undergo a photoreceptor degeneration with apoptotic loss of outer nuclear layer nuclei but visual acuity measured using the optomotor response under photopic conditions persists in spite of considerable photoreceptor loss. The Noerg-1 mutation showed an autosomal dominant pattern of inheritance in progeny. Studies in early postnatal mice showed degeneration to occur after formation of partially functional rods. The Noerg-1 mutation was mapped genetically to chromosome 6 by crossing C57BL/6J mutants with DBA/2J or BALB/cJ mice to produce an N2 generation and then determining the ERG phenotypes and the genotypes of the N2 offspring at multiple loci using SSLP and SNP markers. Fine mapping was accomplished with a set of closely spaced markers. A non-recombinant region from 112.8 Mb to 115.1 Mb was identified, encompassing the rhodopsin (Rho) coding region. A single nucleotide transition from G to A was found in the Rho gene that is predicted to result in a substitution of Tyr for Cys at position 110, in an intradiscal loop. This mutation has been found in patients with autosomal dominant retinitis pigmentosa (RP) and results in misfolding of rhodopsin expressed in vitro. Thus, ENU mutagenesis is capable of replicating mutations that occur in human patients and is useful for generating de novo models of human inherited eye disease. Furthermore, the availability of the mouse genomic sequence and extensive DNA polymorphisms made the rapid identification of this gene possible, demonstrating that the use of ENU-induced mutations for functional gene identification is now practical for individual laboratories.
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