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Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

ABSTRACT: Arrestin was identified in ciliary photoreceptors of Pecten irradians, and its role in terminating the light response was established electrophysiologically. Downstream effectors in these unusual visual cells diverge from both microvillar photoreceptors and rods and cones; the finding that key regulatory mechanisms of the early steps of visual excitation are conserved across such distant lineages of photoreceptors underscores that a common blueprint for phototransduction exists across metazoa. Arrestin was detected by Western blot analysis of retinal lysates, and localized in ciliary photoreceptors by immunostaining of whole-eye cryosections and dissociated cells. Two arrestin isoforms were molecularly identified by PCR; these present the canonical N- and C-arrestin domains, and are identical at the nucleotide level over much of their sequence. A high degree of homology to various ?-arrestins (up to 70% amino acid identity) was found. In situ hybridization localized the two transcripts within the retina, but failed to reveal finer spatial segregation, possibly because of insufficient differences between the riboprobes. Intracellular dialysis of anti arrestin antibodies into voltage-clamped ciliary photoreceptors produced a gradual slow-down of the photocurrent falling phase, leaving a tail that decayed over many seconds after light termination. The antibodies also caused spectrally neutral flashes to elicit prolonged aftercurrents in the absence of large metarhodopsin accumulation; such aftercurrents could be quenched by chromatic illumination that photoconverts metarhodopsin back to rhodopsin. These observations indicate that the antibodies depleted functionally available arrestin, and implicate this molecule in the deactivation of the photoresponse at the rhodopsin level.

SUBMITTER: Gomez Mdel P

PROVIDER: S-EPMC6623731 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

Gomez Maria Del Pilar Mdel P Espinosa Lady L Ramirez Nelson N Nasi Enrico E

The Journal of neuroscience : the official journal of the Society for Neuroscience 20110201 5

Arrestin was identified in ciliary photoreceptors of Pecten irradians, and its role in terminating the light response was established electrophysiologically. Downstream effectors in these unusual visual cells diverge from both microvillar photoreceptors and rods and cones; the finding that key regulatory mechanisms of the early steps of visual excitation are conserved across such distant lineages of photoreceptors underscores that a common blueprint for phototransduction exists across metazoa. A ...[more]

PMID: 21289191

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Project description:The two basic animal photoreceptor types, ciliary and microvillar, use different light-transduction schemes: their photopigments couple to Gt versus Gq proteins, respectively, to either mobilize cyclic nucleotides or trigger a lipid signaling cascade. A third class of photoreceptors has been described in the dual retina of some marine invertebrates; these present a ciliary morphology but operate via radically divergent mechanisms, prompting the suggestion that they comprise a novel lineage of light sensors. In one of these organisms, an uncommon putative opsin was uncovered that was proposed to signal through Go Orthologues subsequently emerged in diverse phyla, including mollusks, echinoderms, and chordates, but the cells in which they express have not been identified, and no studies corroborated their function as visual pigments or their suggested signaling mode. Conversely, in only one invertebrate species, Pecten irradians, have the ciliary photoreceptors been physiologically characterized, but their photopigment has not been identified molecularly. We used the transcriptome of Pecten retina to guide the cloning by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) extensions of a new member of this group of putative opsins. In situ hybridization shows selective transcription in the distal retina, and specific antibodies identify a single band of the expected molecular mass in Western blots and distinctly label ciliary photoreceptors in retina sections. RNA interference knockdown resulted in a reduction in the early receptor current-the first manifestation of light transduction-and prevented the prolonged aftercurrent, which requires a large buildup of activated rhodopsin. We also obtained a full-length clone of the ?-subunit of a Go from Pecten retina complementary DNA and localized it by in situ hybridization to the distal photoreceptors. Small interfering RNA targeting this Go caused a specific depression of the photocurrent. These results establish this novel putative opsin as a bona fide visual pigment that couples to Go to convey the light signal.

Project description:Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trispora IMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.

Project description:BackgroundEyes in bilaterian metazoans have been described as being composed of either ciliary or rhabdomeric photoreceptors. Phylogenetic distribution, as well as distinct morphologies and characteristic deployment of different photopigments (ciliary vs. rhabdomeric opsins) and transduction pathways argue for the co-existence of both of these two photoreceptor types in the last common bilaterian ancestor. Both receptor types exist throughout the Bilateria, but only vertebrates are thought to use ciliary photoreceptors for directional light detection in cerebral eyes, while all other invertebrate bilaterians studied utilize rhabdomeric photoreceptors for this purpose. In protostomes, ciliary photoreceptors that express c-opsin have been described only from a non-visual deep-brain photoreceptor. Their homology with vertebrate rods and cones of the human eye has been hypothesized to represent a unique functional transition from non-visual to visual roles in the vertebrate lineage.ResultsTo test the hypothesis that protostome cerebral eyes employ exclusively rhabdomeric photoreceptors, we investigated the ultrastructure of the larval eyes in the brachiopod Terebratalia transversa. We show that these pigment-cup eyes consist of a lens cell and a shading pigment cell, both of which are putative photoreceptors, deploying a modified, enlarged cilium for light perception, and have axonal connections to the larval brain. Our investigation of the gene expression patterns of c-opsin, Pax6 and otx in these eyes confirms that the larval eye spots of brachiopods are cerebral eyes that deploy ciliary type photoreceptors for directional light detection. Interestingly, c-opsin is also expressed during early embryogenesis in all potential apical neural cells, becoming restricted to the anterior neuroectoderm, before expression is initiated in the photoreceptor cells of the eyes. Coincident with the expression of c-opsin in the presumptive neuroectoderm, we found that middle gastrula stage embryos display a positive photoresponse behavior, in the absence of a discrete shading pigment or axonal connections between cells.ConclusionsOur results indicate that the dichotomy in the deployment of ciliary and rhabdomeric photoreceptors for directional light detection is not as clear-cut as previously thought. Analyses of brachiopod larval eyes demonstrate that the utilization of c-opsin expressing ciliary photoreceptors in cerebral eyes is not limited to vertebrates. The presence of ciliary photoreceptor-based eyes in protostomes suggests that the transition between non-visual and visual functions of photoreceptors has been more evolutionarily labile than previously recognized, and that co-option of ciliary and rhabdomeric photoreceptor cell types for directional light detection has occurred multiple times during animal evolution. In addition, positive photoresponse behavior in gastrula stage embryos suggests that a discrete shading pigment is not requisite for directional photoreception in metazoans. Scanning photoreception of light intensities mediating cell-autonomous changes of ciliary movement may represent an ancient mechanism for regulating locomotory behavior, and is likely to have existed prior to the evolution of eye-mediated directional light detection employing axonal connections to effector cells and a discreet shading pigment.

Dataset Information

Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

Publications

Arrestin in ciliary invertebrate photoreceptors: molecular identification and functional analysis in vivo.

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