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How do animals use visual systems to extract specific features of a visual scene and respond appropriately? The medicinal leech, Hirudo verbana, is a predatory, quasi-amphibious annelid with a rich sensorium that is an excellent system in which to study how sensory cues are encoded, and how key features of visual images are mapped into the CNS. The leech visual system is broadly distributed over its entire body, consisting of five pairs of cephalic eyecups and seven segmentally iterated pairs of dermal sensilla in each mid-body segment. Leeches have been shown to respond behaviorally to both green and near ultraviolet light (UV, 365-375 nm). Here, we used electrophysiological techniques to show that spectral responses by dermal sensilla are mapped across the dorsal-ventral axis, such that the ventral sensilla respond strongly to UV light, while dorsal sensilla respond strongly to visible light, broadly tuned around green. These results establish how key features of visual information are initially encoded by spatial mapping of photo-response profiles of primary photoreceptors and provide insight into how these streams of information are presented to the CNS to inform behavioral responses., (© 2021. The Author(s).)

Photoreceptor cells in the eyes of Bilateria are often classified into microvillar cells with rhabdomeric opsin and ciliary cells with ciliary opsin, each type having specialized molecular components and physiology. First data on the recently discovered xenopsin point towards a more complex situation in protostomes. In this study, we provide clear evidence that xenopsin enters cilia in the eye of the larval bryozoan Tricellaria inopinata and triggers phototaxis. As reported from a mollusc, we find xenopsin coexpressed with rhabdomeric-opsin in eye photoreceptor cells bearing both microvilli and cilia in larva of the annelid Malacoceros fuliginosus . This is the first organism known to have both xenopsin and ciliary opsin, showing that these opsins are not necessarily mutually exclusive. Compiling existing data, we propose that xenopsin may play an important role in many protostome eyes and provides new insights into the function, evolution, and possible plasticity of animal eye photoreceptor cells., Competing Interests: CD, SK, ST, IK, HH No competing interests declared, (© 2020, Döring et al.)

Visual opsins coupled with G q -type G protein have been considered to be responsible for the vision in mollusks. Recent transcriptomic studies, however, revealed the presence of opsin mRNA belonging to different groups of opsin subfamilies in the eyes of mollusks. In the present study, we found that at least three different opsins, G q -coupled rhodopsin, opsin5A, and xenopsin, are co-expressed in the rhabdomeric photoreceptor cell in the eyes of the terrestrial slug Limax valentianus. These opsins were all localized to the microvilli of the rhabdomere. Co-expression of rhodopsin and opsin5A mRNA was also demonstrated by dual fluorescence in situ hybridization. Co-expression of multiple opsins in the rhabdomeric photoreceptors cells may explain the previously reported shift in the action spectra of the electroretinogram of eyes of Limax flavus between the light- and dark-adapted states, which was also reproduced in the present study in L. valentianus., (© 2019 Wiley Periodicals, Inc.)

Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells.

The coloration of flowers is due to the wavelength-selective absorption by pigments of light backscattered by structures inside the petals. We investigated the optical properties of flowers using (micro)spectrophotometry and anatomical methods. To assess the contribution of different structures to the overall visual signal of flowers, we used an optical model, where a petal is considered as a stack of differently pigmented and structured layers and we interpreted the visual signals of the model petals with insect vision models. We show that the reflectance depends, in addition to the pigmentation, on the petal's thickness and the inhomogeneity of its interior. We find large between-species differences in floral pigments, pigment concentration and localization, as well as floral interior structure. The fractions of reflected and transmitted light are remarkably similar between the studied species, suggesting common selective pressures of pollinator visual systems. Our optical model highlights that pigment localization crucially determines the efficiency of pigmentary filtering and thereby the chromatic contrast and saturation of the visual signal. The strongest visual signal occurs with deposition of pigments only on the side of viewing. Our systematic approach and optical modelling open new perspectives on the virtues of flower colour., (© 2016 The Author(s).)

Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.

Tsetse flies transmit trypanosomes that cause nagana in cattle, and sleeping sickness in humans. Therefore, optimising visual baits to control tsetse is an important priority. Tsetse are intercepted at visual baits due to their initial attraction to the bait, and their subsequent contact with it due to landing or accidental collision. Attraction is proposed to be driven in part by a chromatic mechanism to which a UV-blue photoreceptor contributes positively, and a UV and a green photoreceptor contribute negatively. Landing responses are elicited by stimuli with low luminance, but many studies also find apparently strong landing responses when stimuli have high UV reflectivity, which would imply that UV wavelengths contribute negatively to attraction at a distance, but positively to landing responses at close range. The strength of landing responses is often judged using the number of tsetse sampled at a cloth panel expressed as a proportion of the combined catch of the cloth panel and a flanking net that samples circling flies. I modelled these data from two previously published field studies, using calculated fly photoreceptor excitations as predictors. I found that the proportion of tsetse caught on the cloth panel increased with an index representing the chromatic mechanism driving attraction, as would be expected if the same mechanism underlay both long- and close-range attraction. However, the proportion of tsetse caught on the cloth panel also increased with excitation of the UV-sensitive R7p photoreceptor, in an apparently separate but interacting behavioural mechanism. This R7p-driven effect resembles the fly open-space response which is believed to underlie their dispersal towards areas of open sky. As such, the proportion of tsetse that contact a cloth panel likely reflects a combination of deliberate landings by potentially host-seeking tsetse, and accidental collisions by those seeking to disperse, with a separate visual mechanism underlying each behaviour.

The tropical disease vector mosquito Anopheles gambiae possesses 11 rhodopsin genes. Three of these, GPROP1, GPROP3, and GPROP4, encode rhodopsins with >99% sequence identity. We created antisera against these rhodopsins and used immunohistology to show that one or more of these rhodopsins are expressed in the major R1-6 photoreceptor class of the adult A.gambiae eye. Under dark conditions, rhodopsin accumulates within the light-sensitive rhabdomere of the photoreceptor. Light treatment, however, causes extensive movement of rhodopsin to the cytoplasmic compartment. Protein electrophoresis showed that the rhodopsin is present in two different forms. The larger form is an immature species that is deglycosylated during the posttranslational maturation process to generate the smaller, mature form. The immature form is maintained at a constant level regardless of lighting conditions. These results indicate that rhodopsin biosynthesis and movement into the rhabdomere occurs at a constant rate. In contrast, the mature form increases in abundance when animals are placed in dark conditions. Light-triggered internalization and protein degradation counteracts this rhodopsin increase and keeps rhabdomeric rhodopsin levels low in light conditions. The interplay of the constant maturation rate with light-triggered degradation causes rhodopsin to accumulate within the rhabdomere only in dark conditions. Thus, Anopheles photoreceptors possess a mechanism for adjusting light sensitivity through light-dependent control of rhodopsin levels and cellular location., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The eyes of the horseshoe crab, Limulus polyphemus, are a model for studies of visual function and the visual systems of euarthropods. Much is known about the structure and function of L. polyphemus photoreceptors, much less about their photopigments. Three visible-light-sensitive L. polyphemus opsins were characterized previously (LpOps1, 2 and 5). Here we characterize a UV opsin (LpUVOps1) that is expressed in all three types of L. polyphemus eyes. It is expressed in most photoreceptors in median ocelli, the only L. polyphemus eyes in which UV sensitivity was previously detected, and in the dendrite of eccentric cells in lateral compound eyes. Therefore, eccentric cells, previously thought to be non-photosensitive second-order neurons, may actually be UV-sensitive photoreceptors. LpUVOps1 is also expressed in small photoreceptors in L. polyphemus ventral larval eyes, and intracellular recordings from these photoreceptors confirm that LpUVOps1 is an active, UV-sensitive photopigment. These photoreceptors also express LpOps5, which we demonstrate is an active, long-wavelength-sensitive photopigment. Thus small photoreceptors in ventral larval eyes, and probably those of the other larval eyes, have dual sensitivity to UV and visible light. Interestingly, the spectral tuning of small ventral photoreceptors may change day to night, because the level of LpOps5 in their rhabdoms is lower during the day than during the night, whereas LpUVOps1 levels show no diurnal change. These and previous findings show that opsin co-expression and the differential regulation of co-expressed opsins in rhabdoms is a common feature of L. polyphemus photoreceptors., (© 2014. Published by The Company of Biologists Ltd.)

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

Phototransduction, the mechanism underlying the electrical response to light in photoreceptor cells, has been thoroughly investigated in Drosophila melanogaster, an essential model in signal transduction research. These cells present a highly specialized photosensitive membrane consisting of thousands of microvilli forming a prominent structure termed a rhabdomere. These microvilli encompass the phototransduction proteins, most of which are transmembrane and exclusively rhabdomeric. Rhabdomere membrane lipids play a crucial role in the activation of the transient receptor potential ionic channels (TRP and TRPL) responsible for initiating the photoresponse. Despite its importance, rhabdomere lipid composition has not been established. We developed a novel preparation enriched in rhabdomere membranes to perform a thorough characterization of the lipidomics of Drosophila rhabdomeres. Isolated eyes (500) were homogenized and subjected to a differential centrifugation protocol that generates a fraction enriched in rhabdomere membrane. Lipids extracted from this preparation were identified and quantified by gas chromatography coupled to mass spectrometry. We found an abundance of low sterol esters (C16:0, C18:0), highly abundant and diverse triglycerides, free fatty acids, a moderate variety of mono and diacyglycerols (C:16:0, 18:0, C18:1) and abundant phospholipids (principally C18:2). This preparation opens a new avenue for investigating essential aspects of phototransduction.

Optical insulation plays a critical role in the fine visual acuity of the Drosophila compound eye. Screening pigments expressed by a number of cell types contribute to this phenomenon. They provide optical insulation that prevents extraneous light rays from inappropriately activating the photoreceptors. This optical insulation can be divided into two categories; the insulation of the individual ommatidia, and the insulation of the compound eye as a whole. The whole-eye insulation is provided by two sources. The sides of the eye are optically insulated by the pigment rim, a band of pigment cells that circumscribes the eye. The base of the eye is insulated by the subretinal pigment layer; a thick layer of pigment that lies directly underneath the retina. How this subretinal pigment layer is generated has not been clearly described. Here, experiments that manipulate pigment expression during eye development suggest that the subretinal pigment layer is directly derived from pigment cells in the overlying retina., (Copyright © 2012 Wiley Periodicals, Inc.)

A shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The principal eyes of jumping spiders have a unique retina with four tiered photoreceptor layers, on each of which light of different wavelengths is focused by a lens with appreciable chromatic aberration. We found that all photoreceptors in both the deepest and second-deepest layers contain a green-sensitive visual pigment, although green light is only focused on the deepest layer. This mismatch indicates that the second-deepest layer always receives defocused images, which contain depth information of the scene in optical theory. Behavioral experiments revealed that depth perception in the spider was affected by the wavelength of the illuminating light, which affects the amount of defocus in the images resulting from chromatic aberration. Therefore, we propose a depth perception mechanism based on how much the retinal image is defocused.

The crystalline photoreceptor lattice in the Drosophila eye is a paradigm for pattern formation during development. During eye development, activation of proneural genes at a moving front adds new columns to a regular lattice of R8 photoreceptors. We present a mathematical model of the governing activator-inhibitor system, which indicates that the dynamics of positive induction play a central role in the selection of certain cells as R8s. The "switch and template" patterning mechanism we observe is mathematically very different from the well-known Turing instability. Unlike a standard lateral inhibition model, our picture implies that R8s are defined before the appearance of the complete group of proneural cells. The model reproduces the full time course of proneural gene expression and accounts for specific features of the refinement of proneural groups that had resisted explanation. It moreover predicts that perturbing the normal template can lead to eyes containing stripes of R8 cells. We observed these stripes experimentally after manipulation of the Notch and scabrous genes. Our results suggest an alternative to the generally assumed mode of operation for lateral inhibition during development; more generally, they hint at a broader role for bistable switches in the initial establishment of patterns as well as in their maintenance.

The eyes on the backs of molluscs known as chitons are shadow and motion detectors, the lenses of which are made of birefringent aragonite. These provide a focus both in and out of water., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hundreds of ocelli are embedded in the dorsal shell plates of certain chitons. These ocelli each contain a pigment layer, retina, and lens, but it is unknown whether they provide chitons with spatial vision. It is also unclear whether chiton lenses are made from proteins, like nearly all biological lenses, or from some other material. Electron probe X-ray microanalysis and X-ray diffraction revealed that the chiton Acanthopleura granulata has the first aragonite lenses ever discovered. We found that these lenses allow A. granulata's ocelli to function as small camera eyes with an angular resolution of about 9°-12°. Animals responded to the sudden appearance of black, overhead circles with an angular size of 9°, but not to equivalent, uniform decreases in the downwelling irradiance. Our behavioral estimates of angular resolution were consistent with estimates derived from focal length and receptor spacing within the A. granulata eye. Behavioral trials further indicated that A. granulata's eyes provide the same angular resolution in both air and water. We propose that one of the two refractive indices of the birefringent chiton lens places a focused image on the retina in air, whereas the other does so in water., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Scallop eyes contain two retinas, one proximal and one distal. Molecular evidence suggests that each retina expresses a different visual pigment. To test whether these retinas have different spectral sensitivities, we used microspectrophotometry to measure the absorption spectra of photoreceptors from the eyes of two different scallop species. Photoreceptors from the proximal and distal retinas of the sea scallop Placopecten magellanicus had absorption peak wavelengths (λ(max)) of 488 ± 1 nm (mean ± s.e.m.; N=20) and 513 ± 3 nm (N=26), respectively. Photoreceptors from the corresponding retinas of the bay scallop Argopecten irradians had λ(max) values of 506 ± 1 nm (N=21) and 535 ± 3 nm (N=14). Assuming that the proximal and distal receptors had equal absorption coefficients (k(D)=0.0067 microm(-1)), we found that self-screening within the scallop eye caused the proximal and distal receptors in P. magellanicus to have peak absorption at 490 and 520 nm, respectively, and the corresponding receptors in A. irradians to have peak absorption at 504 and 549 nm. We conclude that environment may influence the λ(max) of scallop visual pigments: P. magellanicus, generally found in blue oceanic water, has visual pigments that are maximally sensitive to shorter wavelengths than those found in A. irradians, which lives in greener inshore water. Scallop distal retinas may be sensitive to longer wavelengths of light than scallop proximal retinas to correct for either self-screening by the retinas or longitudinal chromatic aberration of the lens.

In this work, a new biomaterial resulting from the isolation of octopus rhodopsin (OR) starting from octopus photoreceptor membranes is presented. Mass spectroscopic characterization was employed in order to verify the presence of rhodopsin in the extract. Photoreversibility and photochromic properties were investigated using spectrophotometric measurements and pulsed light. Thin films of OR were realized using the gel-matrix entrapment method in polyvinyl alcohol solution. The results indicate that the photoreversibility and the photostability of the OR in gel-matrices are maintained. Several measurements were performed to test the stability of the resulting biomaterial in time and at room temperature. Preliminary tests demonstrate that the photoreversibility and the photostability are still found after few days from the biomaterial preparation and after the exposure for several hours at room temperature.

In animals, successful production of the visual chromophore (11-cis-retinal or derivatives thereof such as 11-cis-3-hydroxy-retinal) is essential for photoreceptor cell function and survival. These carotenoid-derived compounds must combine with a protein moiety (the opsin) to establish functional visual pigments. Evidence from cell culture systems has implicated that the retinal pigment epithelium protein of 65 kDa (RPE65) is the long-sought all-trans to 11-cis retinoid isomerase. RPE65 is structurally related to nonheme iron oxygenases that catalyze the conversion of carotenoids into retinoids. In vertebrate genomes, two carotenoid oxygenases and RPE65 are encoded, whereas in insect genomes only a single representative of this protein family, named NinaB (denoting neither inactivation nor afterpotential mutant B), is encoded. We here cloned and functionally characterized the ninaB gene from the great wax moth Galleria mellonella. We show that the recombinant purified enzyme combines isomerase and oxygenase (isomerooxygenase) activity in a single polypeptide. From kinetics and isomeric composition of cleavage products of asymmetrical carotenoid substrates, we propose a model for the spatial arrangement between substrate and enzyme. In Drosophila, we show that carotenoid-isomerooxygenase activity of NinaB is more generally found in insects, and we provide physiological evidence that carotenoids such as 11-cis-retinal can promote visual pigment biogenesis in the dark. Our study demonstrates that trans/cis isomerase activity can be intrinsic to this class of proteins and establishes these enzymes as key components for both invertebrate and vertebrate vision.

The vertebrate-type opsin, Ci-opsin1, is localized in the outer segments of the photoreceptor cells of larvae of the ascidian Ciona intestinalis. The absorption spectrum of the photopigment reconstituted from Ci-opsin1 and 11-cis-retinal suggested that the photopigment is responsible for photic behavior of the larvae. The structure and function of Ci-opsin1-positive photoreceptor cells were examined by immunohistochemistry, confocal microscopy, electron microscopy, laser ablation, and behavioral analysis. Ciona larvae have three morphologically distinct groups of photoreceptor cells in the brain vesicle. Group I and group II photoreceptor cells are associated with the ocellus pigment cell on the right side of the brain vesicle. The outer segments of the group I photoreceptor cells are regularly arranged inside the small cavity encircled by the cup-shaped pigment cell. The outer segments of the group II photoreceptor cells are located outside the pigment cavity and exposed to the lumen of the brain vesicle. The outer segments of the group III photoreceptor cells are located near the otolith on the left ventral side of the brain vesicle. Thus, the brain vesicle of the ascidian larva has two ocelli: a 'conventional' pigmented ocellus containing the group I and group II photoreceptor cells and a novel nonpigmented ocellus solely consisting of the group III photoreceptor cells. Laser ablation experiments suggest that the pigmented ocellus is responsible for the photic swimming behavior. The nonpigmented ocellus might relate to later developmental or physiological events, such as metamorphosis, because Ci-opsin1 immunoreactivity appears in the late larval stage and becomes intense just before the onset of metamorphosis., ((c) 2008 Wiley-Liss, Inc.)

PDZ domains are common building blocks of scaffold proteins that enhance specificity and speed in signal transduction cascades. Although PDZ modules are often viewed as passive participants, Mishra et al. (2007) now show that a PDZ domain in INAD, a scaffold protein in photoreceptor cells of the fruit fly, undergoes a light-dependent conformational change, which has important consequences for signaling and animal behavior.

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.

Cryptochrome (CRY) is a photolyase-like flavoprotein with no DNA-repair activity but with known or presumed blue-light receptor function. Animal CRYs have DNA-binding and autokinase activities, and their flavin cofactor is reduced by photoinduced electron transfer. In Drosophila, CRY is a major circadian photoreceptor, and in mammals, the two CRY proteins are core components of the molecular clock and potential circadian photoreceptors. In mammals, CRYs participate in cell cycle regulation and the cellular response to DNA damage by controlling the expression of some cell cycle genes and by directly interacting with checkpoint proteins.

The closely related crickets Dianemobius nigrofasciatus and Allonemobius allardi exhibit similar circadian rhythms and photoperiodic responses, suggesting that they possess similar circadian and seasonal clocks. To verify this assumption, antisera to Period (PER), Doubletime (DBT), and Cryptochrome (CRY) were used to visualize circadian clock neurons in the cephalic ganglia. Immunoreactivities referred to as PER-ir, DBT-ir, and CRY-ir were distributed mainly in the optic lobes (OL), pars intercerebralis (PI), dorsolateral protocerebrum, and the subesophageal ganglion (SOG). A system of immunoreactive cells in the OL dominates in D. nigrofasciatus, while immunoreactivities in the PI and SOG prevail in A. allardi. Each OL of D. nigrofasciatus contains 3 groups of cells that coexpress PER-ir and DBT-ir and send processes over the frontal medulla face to the inner lamina surface, suggesting functional linkage to the compound eye. Only 2 pairs of PER-ir cells (no DBT-ir) were found in the OL of A. allardi. Several groups of PER-ir cells occur in the brain of both species. The PI also contains DBT-ir and CRY-ir cells, but in A. allardi, most of the DBT-ir is confined to the SOG. Most immunoreactive cells in the PI and in the dorsolateral brain send their fibers to the contralateral corpora cardiaca and corpora allata. The proximity and, in some cases, proven identity of the PER-ir, DBT-ir, and CRY-ir perikarya are consistent with presumed interactions between the examined clock components. The antigens were always found in the cytoplasm, and no diurnal oscillations in their amounts were detected. The photoperiod, which controls embryonic diapause, the rate of larval development, and the wing length of crickets, had no discernible effect on either distribution or the intensity of the immunostaining.

The spectral, angular and polarization sensitivities of photoreceptors in the compound eye of the monarch butterfly (Danaus plexippus) are examined using electrophysiological methods. Intracellular recordings reveal a spectrally homogenous population of UV receptors with optical axes directed upwards and >or=10 degrees to the contralateral side. Based on optical considerations and on the opsin expression pattern (Sauman et al. 2005), we conclude that these UV receptors belong to the anatomically specialized dorsal rim area (DRA) of the eye. Photoreceptors in the main retina with optical axes <10 degrees contralateral or ipsilateral have maximal sensitivities in the UV (lambda(max)

The compound eye of the butterfly Papilio xuthus is composed of three spectrally distinct types of ommatidia. We investigated the blue and double-peaked green receptors that are encountered distally in type I and III ommatidia, by means of intracellular recordings, in vivo fluorescence microscopy, and histology. The blue receptors are R1 and/or R2 photoreceptors; they contain the same mRNA encoding the opsin of the blue-absorbing visual pigment. However, here we found that the sensitivity in the UV wavelength region strongly depends on the ommatidial type; the blue receptors in type I ommatidia have a distinctly depressed UV sensitivity, which is attributed to lateral filtering in the fused rhabdom. In the main, fronto-ventral part of the eye, the R3 and R4 photoreceptors of all ommatidia contain the same set of two mRNAs encoding the opsins of green-absorbing visual pigments, PxL1 and PxL2. The spectral sensitivities are double-peaked, but the UV sensitivity of the R3 and R4 photoreceptors in type I ommatidia appears to be reduced, similar to that of the co-localized blue receptors.

Sponges (phylum Porifera) of the classes Hexactinellida and Demospongiae possess a skeleton composed of siliceous spicules, which are synthesized enzymatically. The longest spicules are found among the Hexactinellida, with the stalk spicules (length: 30 cm; diameter: 300 microm) of Hyalonema sieboldi as prominent examples. These spicules are constructed around a central axial filament, which is formed by approximately 40 siliceous layers. The stratified spicules function as optical glass fibers with unique properties. If free-spaced coupled with a white light source (WLS), the entire fiber is illuminated. Special features of the light transmission: (i) only wavelengths between 615 and 1310 nm can pass through the fibers and (ii) light below wavelengths of 615 nm and above 1310 nm is completely cut-off. The transmission efficiency is around 60% (measured at 1080-1100 nm [length of the fiber: 5 cm]). The spicules acts as sharp high- and low-pass filters, suggesting that these silica-based fibers might be involved in a photoreception system. This assumption is supported by the finding that sponges are provided with a bioluminescent system. It is hypothesized that the spicules/siliceous fibers might be involved in a photoreception system in these animals.

Studies using various mutants of Drosophila melanogaster bearing defects in their visual system, including those of the retinal and extraretinal photoreceptor systems, have indicated that the extraretinal photoreceptor known as the Hofbauer-Buchner (H-B) eyelet plays an active, if subsidiary, role in the entrainment of circadian rhythms. In the present study, in the context of unraveling the function of extraretinal photoreception on circadian rhythms and photoperiodic responses, we searched for extraretinal photoreceptors in the blowfly, Protophormia terraenovae, and found that this fly has a homolog of the H-B eyelet. In addition, we show morphologically direct synaptic connections between the eyelet of P. terraenovae (called here Pt-eyelet, after the species' name) and pigment-dispersing factor (PDF)-immunoreactive neurons, which are putative circadian pacemaker neurons, by immunogold electron microscopy combined with intracellular dye injection. The Pt-eyelet was found to reside in the middle of the posterior surface of the optic lobe between the retina and the lamina, as does the H-B eyelet. This extraretinal photoreceptor was composed of at least four photoreceptor cells equipped with well-organized microvillar rhabdomeres. Rhodopsin 6-like immunoreactivity and also the response to light stimuli clearly showed the Pt-eyelet to be functional. The Pt-eyelet terminals in the accessory medulla exhibited synaptic bouton-like appearances and formed divergent multiple-contact output synapses. Synaptic contacts from the Pt-eyelet terminal to the PDF-immunoreactive neurons were identified by the presence of presynaptic ribbons and accumulated synaptic vesicles. Their possible function is discussed in relation to previous studies on circadian rhythm and photoperiodic response of P. terraenovae.

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti-arrestin (anti-Ci-Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro-posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero-posteriorly at the midline of the posterior sensory vesicle (SV). The Ci-arr gene was expressed throughout the SV at the embryonic mid-tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci-Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti-Ci-Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step-down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive.

Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. Here we report the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila melanogaster Sec15. This C-terminal domain was found to bind a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. We also provide evidence that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-A crystal structure of the C-terminal domain revealed a novel fold consisting of ten alpha-helices equally distributed between two subdomains (N and C subdomains). We show that the C subdomain, mainly via a single helix, is sufficient for Rab binding.

Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.

Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis(Iseki et al., Nature, 2002, 415, 1047--1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACalpha and PACbeta). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD"(BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACalpha by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28--0.32, and the half-life was 34--44 s at 25 degrees C for the recombinant PACalpha F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tll0078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACalpha F2 and the F2 domain of the PACalpha homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.

Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores (blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicum's light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell (oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment-protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, free oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640--750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein acceptor residue as it had been suggested in previous studies.

Lepidopterans display biological rhythms associated with egg laying, eclosion and flight activity but the photoreceptors that mediate these behavioural patterns are largely unknown. To further our progress in identifying candidate light-input channels for the lepidopteran circadian system, we have developed polyclonal antibodies against ultraviolet (UV)-, blue- and extraretinal long-wavelength (LW)-sensitive opsins and examined opsin immunoreactivity in the adult optic lobes of four hawk moths, Manduca sexta, Acherontia atropos, Agrius convolvuli and Hippotion celerio. Outside the retina, UV and blue opsin protein expression is restricted to the adult stemmata, with no apparent expression elsewhere in the brain. Melatonin, which is known to have a seasonal influence on reproduction and behaviour, is expressed with opsins in adult stemmata together with visual arrestin and chaoptin. By contrast, the LW opsin protein is not expressed in the retina or stemmata but rather exhibits a distinct and widespread distribution in dorsal and ventral neurons of the optic lobes. The lamina, medulla, lobula and lobula plate, accessory medulla and adjacent neurons innervating this structure also exhibit strong LW opsin immunoreactivity. Together with the adult stemmata, these neurons appear to be functional photoreceptors, as visual arrestin, chaoptin and melatonin are also co-expressed with LW opsin. These findings are the first to suggest a role for three spectrally distinct classes of opsin in the extraretinal detection of changes in ambient light and to show melatonin-mediated neuroendocrine output in the entrainment of sphingid moth circadian and/or photoperiodic rhythms.

Phot proteins are homologs of the blue-light receptor phototropin. We report a comparative study of the photocycles of the isolated, light-sensitive domains LOV1 and LOV2 from Chlamydomonas reinhardtii phot protein, as well as the construct LOV1/2 containing both domains. Transient absorption measurements revealed a short lifetime of the LOV2-wt triplet state (500 ns), but a long lifetime (287 micros) of the triplet in the mutant LOV2-C250S, in which the reactive cysteine is replaced by serine. For LOV1, in comparison, corresponding numbers of 800 ns and 4 micros for the two conformers in LOV1-wt, and 27 micros for LOV1-C57S have been reported. The triplet decay kinetics in the mixed domains LOV1/2-wt, LOV1/2-C57S, and LOV1/2-C250S can be analyzed as the superposition of the behavior of the corresponding single domains. The situation is different for the slow, thermal reaction of the photoadduct back to the dark form. Whereas the individual domains LOV1 and LOV2 show two decay components, the double domains LOV1/2-C57S and LOV1/2-C250S both show only a single component. The interaction of the two domains does therefore not manifest itself during the lifetime of the triplet states, but changes the decay behavior of the adduct states.

Purpose: To take advantage of specialized microscopic methods and transgenic stocks, to understand the properties of each rhodopsin now that Drosophila's six rhodopsins (Rh1-Rh6) have been isolated., Methods: The visual pigment containing organelles, the rhabdomeres, were imaged in live flies with the pseudopupil in standard and confocal microscopes. Five transgenic Drosophila strains in which Rh2-Rh6 replaced the native Rh1 in R1-6 receptors were compared with normal controls (Rh1 in R1-6) for two lines of work: (1) autofluorescence of rhodopsin; and (2) imaging rhodopsin. Other transgenic Drosophila in which the Rh1, Rh3, and Rh4 promoters drive the green fluorescent protein (GFP) reporter were used for other purposes, especially distinguishing the R7/8 types., Results: We show, for the first time, that visual pigment appears pink in white light, especially for Rh1 and Rh6. While showing that rhodopsin-metarhodopsin conversions were understood by their respective wavelengths, we discovered that, for Rh6, rhodopsin and metarhodopsin could not be spectrally separated. Relative fluorescent emission, Rh1=Rh5>Rh6>Rh2>Rh4>Rh3, was of little value in explaining differences between bright and dim autofluorescence in R7. Rather, analysis of GFP driven by Rh3 and Rh4 promoters show that the rhabdomeres with bright autofluorescence are the ones that contain Rh4., Conclusions: Careful imaging provides a useful approach to analyzing Drosophila rhodopsins. Amid a considerable body of microscopic data, we identify the sources of bright and dim R7 rhabdomeres, and we demonstrate the unique properties of Rh6.

For vision, insect and vertebrate eyes use rhabdomeric and ciliary photoreceptor cells, respectively. These cells show distinct architecture and transduce the light signal by different phototransductory cascades. In the marine rag-worm Platynereis, we find both cell types: rhabdomeric photoreceptor cells in the eyes and ciliary photoreceptor cells in the brain. The latter use a photopigment closely related to vertebrate rod and cone opsins. Comparative analysis indicates that both types of photoreceptors, with distinct opsins, coexisted in Urbilateria, the last common ancestor of insects and vertebrates, and sheds new light on vertebrate eye evolution.

CRYPTOCHROME (CRY) is the primary circadian photoreceptor in Drosophila. We show that CRY binding to TIMELESS (TIM) is light-dependent in flies and irreversibly commits TIM to proteasomal degradation. In contrast, CRY degradation is dependent on continuous light exposure, indicating that the CRY-TIM interaction is transient. A novel cry mutation (cry(m)) reveals that CRY's photolyase homology domain is sufficient for light detection and phototransduction, whereas the carboxyl-terminal domain regulates CRY stability, CRY-TIM interaction, and circadian photosensitivity. This contrasts with the function of Arabidopsis CRY domains and demonstrates that insect and plant cryptochromes use different mechanisms.

In Diptera, subsets of small retinotopic neurons provide a discrete channel from achromatic photoreceptors to large motion-sensitive neurons in the lobula complex. This pathway is distinguished by specific affinities of its neurons to antisera raised against glutamate, aspartate, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and a N-methyl-D-aspartate type 1 receptor protein (NMDAR1). Large type 2 monopolar cells (L2) and type 1 amacrine cells, which in the external plexiform layer are postsynaptic to the achromatic photoreceptors R1-R6, express glutamate immunoreactivity as do directionally selective motion-sensitive tangential neurons of the lobula plate. L2 monopolar cells ending in the medulla are accompanied by terminals of a second efferent neuron T1, the dendrites of which match NMDAR1-immunoreactive profiles in the lamina. L2 and T1 endings visit ChAT and GABA-immunoreactive relays (transmedullary neurons) that terminate from the medulla in a special layer of the lobula containing the dendrites of directionally selective retinotopic T5 cells. T5 cells supply directionally selective wide-field neurons in the lobula plate. The present results suggest a circuit in which initial motion detection relies on interactions among amacrines and T1, and the subsequent convergence of T1 and L2 at transmedullary cell dendrites. Convergence of ChAT-immunoreactive and GABA-immunoreactive transmedullary neurons at T5 dendrites in the lobula, and the presence there of local GABA-immunoreactive interneurons, are suggested to provide excitatory and inhibitory elements for the computation of motion direction. A comparable immunocytological organization of aspartate- and glutamate-immunoreactive neurons in honeybees and cockroaches further suggests that neural arrangements providing directional motion vision in flies may have early evolutionary origins., (Copyright 2003 Wiley-Liss, Inc.)

The visual cycle system in a primitive chordate, ascidian Ciona intestinalis, was studied by whole-mount in situ hybridization and by whole-mount immunohistochemistry. Three visual cycle proteins, Ciona homologue of RGR (Ci-opsin3), CRALBP (Ci-CRALBP), and BCO/RPE65 (Ci-BCO/RPE65) were widely distributed in the brain vesicle and visceral ganglion. To identify the visual cycle system in a primitive chordate, we compared the localization of photoreceptor-specific proteins (visual pigment and arrestin) and visual cycle proteins (Ci-opsin3 and Ci-CRALBP). The ascidian visual cycle is composed of two cellular compartments, the photoreceptors and the brain vesicle, but some photoreceptor cells also contain visual cycle proteins.

Phot photoreceptors make up an important protein family regulating biological processes in response to blue light. They contain two light, oxygen, and voltage sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN). Upon absorption of blue light, the LOV domains undergo a photocycle, transiently forming a covalent adduct of a cysteine residue and the FMN (LOV-390). The mechanism of formation of this flavin-thiol adduct is still unclear. We studied a mutant of the LOV1 domain from the green alga Chlamydomonas reinhardtii with a methionine replacing the reactive cysteine 57 (C57M). As in the wild type, irradiation leads to formation of a photoadduct, which, however, is irreversibly converted into a red absorbing species, C57M-675. On the basis of spectroscopic results and the 2.1 A resolution crystal structure, this highly unusual FMN species was assigned to a neutral flavin radical covalently attached to the apoprotein at the N(5) position. In contrast to other flavoprotein neutral radicals, C57M-675 is stable even under aerobic or denaturing conditions. Pathways for the photoinduced formation of the adduct are discussed for the C57M mutant as well as the wild-type LOV1 domain.

In the flavin mononucleotide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.

To provide new information on the series of structural changes that Euglena photoreceptive proteins undergo inside the photoreceptor in response to light, we measured in vivo emission fluorescence spectra in the stable intermediates of its photocycle. Our emission spectra give a certain indication that fluorescent proteins are present in the Euglena photoreceptor and that they undergo a photocycle. On the basis of our data, we suggested that at least two stable intermediates, one of which is fluorescent, can be discriminated at room temperature and with our time resolution.

The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. In spite of much progress, the gating mechanism of TRP channels is still unknown. However, recent studies have shown multi-faceted functions of the Drosophila light-sensitive TRP channel that may shed light on TRP gating. Accordingly, metabolic stress, which leads to depletion of cellular ATP, reversibly activates the Drosophila TRP and TRPL channels in the dark in a constitutive manner. In several Drosophila mutants, constitutive activity of TRP channels lead to a rapid retinal degeneration in the dark, while genetic elimination of TRP protects the cells from degeneration. Additional studies have shown that TRPL translocates in a light-dependent manner between the signaling membranes and the cell body. This light-activated translocation is accompanied by reversible morphological changes leading to partial and reversible collapse of the microvillar signaling membranes into the cytosol, which allows turnover of signaling molecules. These morphological changes are also blocked by genetic elimination of TRP channels. The link of TRP gating to the metabolic state and maintenance of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death upon malfunction.

The compound eye of Pieris rapae crucivora contains ventrally three types of histologically distinct ommatidia. An ommatidium contains nine photoreceptors, four of which (R1-4) construct the distal tier of the rhabdom. We determined the sensitivity spectra of the R1-4 distal photoreceptors in each type of ommatidia by intracellular electrophysiology and identified UV, blue, double-peaked blue, green, and a green receptor with depressed sensitivity in the violet. We localized these receptors in each type of ommatidia by injecting dye after the recording. In type I ommatidia the R1 and R2 cells are UV and blue receptors. When R1 is UV sensitive, R2 is always blue sensitive, or vice versa. R3 and R4 in type I are both green receptors. In type II, R1 and R2 are both double-peaked blue receptors and R3 and R4 are both green receptors with depressed sensitivity in the violet. In type III, R1 and R2 are both UV, and R3 and R4 are green receptors. The double-peaked blue, and green receptors with depressed sensitivity in the violet in type II ommatidia have depressed sensitivity at 420 nm, which is probably due to the filtering effect of a fluorescing material present in the type II ommatidia. Spectral heterogeneity of ommatidia seems to be a common design of insect compound eyes.

The "Phot" protein family comprises blue-light photoreceptors that consist of two flavin mononucleotide (FMN)-binding LOV (light, oxygen, and voltage) domains and a serine/threonine kinase domain. We have investigated the LOV1 domain of Phot1 from Chlamydomonas reinhardtii by time-resolved absorption spectroscopy. Photoexcitation of the dark form, LOV1-447, causes transient bleaching and formation of two spectrally similar red-shifted intermediates that are both assigned to triplet states of the FMN. The triplet states decay with time constants of 800 ns and 4 micro s with an efficiency of >90% into a blue-shifted intermediate, LOV1-390, that is attributed to a thiol adduct of cysteine 57 to FMN C(4a). LOV1-390 reverts to the dark form in hundreds of seconds, the time constant being dependent on pH and salt concentration. In the mutant C57S, where the thiol adduct cannot be formed, the triplet state displays an oxygen-dependent decay directly to the dark form. We present here a spectroscopic characterization of an algal sensory photoreceptor in general and of a LOV1 domain photocycle in particular. The results are discussed with respect to the behavior of the homologous LOV2 domain from oat.

Hermissenda CSP24 (cytoskeletal-related protein 24) is a 24 kDa beta-thymosin-like protein that is associated with intermediate memory. We showed previously that one-trial conditioning resulted in a significant increase in the phosphorylation of CSP24 detected in lysates of the pathway supporting the conditioned stimulus (CS). Here we report the association of the protein with the actin cytoskeleton and the distribution of CSP24-immunoreactive neurons in two sensory structures and the circumesophageal nervous system. Identified photoreceptors, hair cells, and neurons in the cerebropleural and pedal ganglia were immunoreactive for CSP24. Immunoprecipitation experiments with 32PO4-labeled lysates of the circumesophageal nervous system identified a 44 kDa protein band (consistent with actin) that coprecipitates with CSP24. An analysis of immunoprecipitates on Western blots probed with anti-actin antibody also showed that actin coprecipitates with CSP24. Laser confocal microscopy of photoreceptors costained with fluorescently labeled anti-actin antibody and anti-CSP24 antibody, or fluorescent phalloidin and anti-CSP24 antibody showed that CSP24 is localized with actin in the cytosol of photoreceptor cell bodies and colocalized with presumed G-actin, but not F-actin, in regions adjacent to the plasma membrane. Although CSP24 is widely distributed in the Hermissenda nervous system, its regulation by one-trial conditioning was observed only in the CS pathway. Our findings suggest that CSP24 may interact with components of the actin cytoskeleton that contribute to structural changes underlying the formation and maintenance of enduring forms of memory.