Photobehavioral Responses Research Articles

Characterization of Chlamydomonas voltage-gated calcium channel and its interaction with photoreceptor support VGCC modulated photobehavioral response in the green alga

Calcium (Ca2+) signaling plays a major role in regulating multiple processes in living cells. The photoreceptor potential in Chlamydomonas triggers the generation of all or no flagellar Ca2+ currents that cause membrane depolarization across the eyespot and flagella. Modulation in membrane potential causes changes in the flagellar waveform, and hence, alters the beating patterns of Chlamydomonas flagella. The rhodopsin-mediated eyespot membrane potential is generated by the photoreceptor Ca2+ current or P-current however, the flagellar Ca2+ currents are mediated by unidentified voltage-gated calcium (VGCC or CaV) and potassium channels (VGKC). The voltage-gated ion channel that associates with ChRs to generate Ca2+ influx across the flagella and its cellular distribution has not yet been identified. Here, we identified putative VGCCs from algae and predicted their novel properties through insilico analysis. We further present experimental evidence on Chlamydomonas reinhardtii VGCCs to predict their novel physiological roles. Our experimental evidences showed that CrVGCC4 localizes to the eyespot and flagella of Chlamydomonas and associates with channelrhodopsins (ChRs). Further in silico interactome analysis of CrVGCCs suggested that they putatively interact with photoreceptor proteins, calcium signaling, and intraflagellar transport components. Expression analysis indicated that these VGCCs and their putative interactors can be perturbed by light stimuli. Collectively, our data suggest that VGCCs in general, and VGCC4 in particular, might be involved in the regulation of the photobehavioral response of Chlamydomonas.

Channelrhodopsin-Dependent Photo-Behavioral Responses in the Unicellular Green Alga Chlamydomonas reinhardtii.

Channelrhodopsins (ChRs) are the light-gated ion channels that have opened the research field of optogenetics. They were originally identified in the green alga Chlamydomonas reinhardtii, a biciliated unicellular alga that inhabits in freshwater, swims with the cilia, and undergoes photosynthesis. It has various advantages as an experimental organism and is used in a wide range of research fields including photosynthesis, cilia, and sexual reproduction. ChRs function as the primary photoreceptor for the cell's photo-behavioral responses, seen as changes in the manner of swimming after photoreception. In this chapter, we will introduce C. reinhardtii as an experimental organism and explain our current understanding of how the cell senses light and shows photo-behavioral responses.

Characterization of Novel BBS Mutants in Chlamydomonas reinhardtii

Defects of cilia and flagella contribute to a multitude of human diseases, including Bardet‐Biedl Syndrome (BBS), a rare genetically inherited disorder characterized by obesity, retinal degeneration, polydactyly, and mental retardation. Thus far, defects in 12 genes have been linked to BBS, classified as BBS 1–12. Chlamydomonas reinhardtii, a biflagellate unicellular alga, has eight well conserved BBS genes (BBS 1, 2, 3, 4, 5, 7, 8, and 9) suggesting it could serve as unicellular model for BBS. Seven of the eight (excluding BBS3) the gene products form a complex, the BBSome, whose functions are currently unknown. Here, we attempt to characterize 13 BBS mutants, seven containing normal amounts of BBS4 in the flagella but sharing a nonphototactic phenotype with established BBS mutants of Chlamydomonas, five lacking BBS4 in the flagella, and one harboring reduced amounts of BBS4 in the flagella. The goal was to identify the genetic defects in these uncharacterized mutants, and to assay their photo‐behavioral responses. The results suggest that assembly, stability, and/or transport of the BBSome can be impaired despite the presence of the genes encoding all seven BBSome proteins. These findings indicate that other genes are involved in the functionality of the BBSome pathway and that the identification of these genes could further studies of BBS in humans. Supported by NIH T34GM008411

Spectral sensitivity of vertically migrating marine copepods.

Light is a critical factor in the proximate basis of diel vertical migration (DVM) in zooplankton. A photobehavioral approach was used to examine the spectral sensitivity of four coastal species of calanoid copepod, representing a diversity of DVM patterns, to test whether species that migrate (nocturnal or reverse DVM) have response spectra that differ from non-migratory surface dwellers. The following species were given light stimuli at wavelengths from 350 to 740 nm, and their photoresponses were measured: Centropages typicus (nocturnal migrator), Calanopia americana (nocturnal migrator), Anomalocera ornata (reverse migrator), and Labidocera aestiva (non-migrator). Centropages typicus and A. ornata had peak responses at 500 and 520 nm, respectively, while Calanopia americana had maximum responses at 480 and 520 nm. Thus, the species that undergo DVM have peak photobehavioral responses at wavelengths corresponding to those available during twilight in coastal water, although the range of wavelengths to which they respond is variable. Non-migratory surface-dwelling L. aestiva had numerous response peaks over a broad spectral range, which may serve to maximize photon capture for vision in their broad-spectrum shallow-water habitat.

Evidence for the archaebacterial-type conformation about the bond between the β-ionone ring and the polyene chain of the chromophore retinal in chlamyrhodopsin

Previous studies using retinal analogs (Wada, A., Sakai, M., Kinumi, T., Tsujimoto, K., Yamauchi, M. and Ito, M. (1994) J. Org. Chem. 59, 6992–6997; Wada, A., Sakai, M., Imamoto, Y., Shichida., Y., Yoshizawa, T. and Ito, M. (1993) Chem. Pharm. Bull. 41, 793–795) revealed that both retinochrome and visual pigments share a common chromophoric conformation in which the ring region of retinal is twisted ca. 50° with respect to the plane of the polyene chain, suggesting a highly conserved 6-s- cis conformation throughout rhodopsin-like pigments in animals. By contrast, 6-s- trans conformation was commonly observed or suggested in archaebacterial rhodopsins examined thus far. Here we have reconstituted in vivo both the photoreceptor for photobehavioral responses of the unicellular alga Chlamydomonas reinhardtii and the second archaeal sensory photoreceptor phoborhodopsin from a series of retinal analogs. Results exclusively point to the conclusion that in both photoreceptors retinal has the coplanar 6-s- trans conformation, though recent molecular cloning studies revealed no homology between Chlamydomonas photoreceptor (chlamyrhodopsin) and archaeal rhodopsins.

Mutational analysis of the phototransduction pathway of Chlamydomonas reinhardtii.

Chlamydomonas has two photobehavioral responses, phototaxis and photoshock. Rhodopsin is the photoreceptor for these responses and the signal transduction process involves transmembrane Ca2+ fluxes. This causes transient changes in flagellar beating, ultimately resulting in phototaxis or photoshock. To identify components that make up this signal transduction pathway, we generated nonphototactic strains by insertional mutagenesis. Seven new phototaxis genes were identified (ptx2-ptx8); alleles of six of these are tagged by the transforming DNA and therefore should be easily cloned. To order the mutants in the pathway, we characterized them electrophysiologically, behaviorally, and structurally, ptx5, ptx6, and ptx7 have normal light-induced photoreceptor currents (PRC) and flagellar currents (FC) but their pattern of swimming does not change in the normal manner when the intraflagellar Ca2+ concentration is decreased, suggesting that they have defects in the ability of their axonemes to respond to changes in Ca2+ concentration. ptx2 and ptx8 lack the FC but have normal PRCs, suggesting that they are defective in the flagellar Ca2+ channel or some factor that regulates it. ptx4 mutants have multiple eye-spots. ptx3 mutants are defective in a component essential for phototaxis but bypassed during photoshock; this component appears to be located downstream of the PRC but upstream of the axoneme.

Sensitivity increase in the photophobic response of Halobacterium halobium reconstituted with retinal analogs: a novel interpretation for the fluence-response relationship and a kinetic modeling.

Phoborhodopsin (also called sensory rhodopsin II) is a photoreceptor protein which mediates photophobic responses of Halobacterium halobium to blue-green light. Under conditions where the synthesis of the chromophore retinal is inhibited, the photophobic system is reconstituted in vivo by incorporation of all-trans retinal or retinal analogs into the apoprotein of phoborhodopsin. Retinal analogs which retard the cyclic photoreaction kinetics of phoborhodopsin increase significantly the sensitivity of the photophobic response. This supports the previously reported hypothesis that signal amplification occurs during the lifetime of intermediate states of the photocycle. The sensitivity increase caused by the chromophore substitution is observed in cells at several different growth stages, i.e. the naturally occurring chromophore (all-trans retinal) does not produce maximal sensitivity at any stage of the culture growth. These results are difficult to interpret in terms of the proposal by Marwan et al. (J. Mol. Biol. 199, 663-664, 1988) that only a single photon is sufficient to cause the photobehavioral response in cells containing native phoborhodopsin. A new interpretation for the fluence-response curves is described based in part on their Poisson statistical analysis. Further, a kinetic model which relates the receptor photochemical reaction cycle to the behavioral response is developed, which accounts for both the sensitivity increase and the shape of the fluence-response curves.

Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: Simultaneous measurements of phototactic and photophobic responses

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all- trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All- trans retinal was more effective than 11- cis retinal to regenerate both photobehavioral responses. Analogs having locked 11- cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all- trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13–14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.

Thresholds of retinal and extraretinal photoreceptors measured by photobehavioral response in catfish,Silurus asotus

Light thresholds of retinal and extraretinal photoreceptors in catfish were examined by the photobehavioral response using a method in which reflex body movements are recorded. Thresholds were determined in six groups, (A) intact, (B) ophthalmectomized, (C) pinealectomized, (D) ophthalmectomized + pinealectomized, (E) ophthalmectomized, pinealectomized and skinless over the brain (skinless fish) and (F) ophthalmectomized, pinealectomized and dorsally covered with aluminum foil over the brain (covered fish). All these fishes displayed short term activity to white light stimulation after being dark adapted for more than 5 h. The lowest threshold was obtained in the intact group (2.0×10−4 μW/cm2). The thresholds of lateral eyes and the pineal organ were 3.4×10−3 and 1.5×10−2 μW/cm2, respectively. Without lateral eyes and pineal organ, catfish still responded to light, indicating the possible existence of extraretinal nonpineal photoreceptors (ENPs). The threshold of ENPs was 3.3 μW/cm2. The localization of ENPs was assumed to be in the brain from the experiment with the combination of skinless and covered fish.

Photobehavior of marine invertebrates: extraocular photoreception.

How do animals sense light? What are their responses? The researches into photobehavior of animals has had a long and exciting history. In the 1930s John H. Welsh began a series of researches on the photobehavior of invertebrate animals that he continued to pursue into the 1960s. Welsh’s studies, to cite a few, included the effects of light on locomotor muscles, on the reactions of their tentacles, on rhythmic behavior, and on neurohormones (Welsh, 1932, 1933a.b, 1934a,b, 1961). These photobehavioral studies bear directly on our understanding of the mechanisms of neurosensory and visual systems of animals, but how these behavioral responses are carried out via their receptors is not completely understood. Invertebrates exhibit a variety of photobehaviors; that is, they move, orient or swim to or away from the light source. The movement may be in the bending or contraction of part or all of the body. Other photobehavioral responses are migration and circadian rhythms. These photobehavioral responses, or a set of responses that are not initiated via the eye but through photoreceptors closely bound to dermal and neural cells, including the brain, are defined as extraocular or are referred to as the extraretinal photoreceptor system. The search into extraocular photoreception was pioneered by the investigations of Steven (1963) and Millott (1968). Their studies and more recent investigations are reviewed by Menaker (1976, 1977), Millott (1978) Yoshida (1979) Wolken (1986) and Wolken and Mogus (1979, 1981). In attempts to understand the mechanisms of extraocular photoreception and behavior, questions arise as to where are the photoreceptors located, and what are the photoreceptor molecules? How are the photoreceptors structured, and are they similar to or do they differ from the visual photoreceptors? Extraocular photoreceptors are widely distributed throughout the animal, and their function is to inform the animal of the presence or absence of light, monitor the light intensity, measure the duration of light, and select specific wavelengths of light. In some animals, the extraocular photoreceptor analyzes the plane of vibration of polarized light for orientation and navigation. A brief review of some behavioral responses of marine invertebrates will be illustrative of extraocular photosensitivity. For example, many invertebrates respond to a sudden change in light intensity by a withdrawal reaction, which is observed for numerous marine animals and is referred to as the shadow response. Steven (1963) attributed such photobehavior to a dermal light sense, but Millott (1968) preferred the all-inclusive term extraocular for all types of photobehavior that was not initiated through the eye. The shadow response of the sea urchin Diadema exhibits a withdrawal from the light and is accompanied by a complex spine waving reaction (Millott, 1968). In annelids, there is withdrawal of the tail. In Nereis diuersicolor, the photosensitive area is located on the parapodia and proand peristromium (Gwilliam, 1969). In nematodes, there is a similar phototactic response to light (Croll et al., 1975). The marine worm GolJingia gouldii reacts to light by retraction of the proboscis (O’Benar and Matsumoto, 1976). The burrowing sea anemone Calamactis praelongus bends toward the light (Marks, 1976). The adult sea squirt Ciona intestinalis orients in the direction of the light, accompanied by the opening and closing of its siphons (Dilly and Wolken, 1973). The Ciona body surface is also sensitive to changes in light intensity, and the most light-sensitive area is found in the region of the ganglion cells. The response to changes in light intensity are localized contractions or a total contraction of the body. These contractions and elongations continue in Ciona even when the siphons are removed. Migration is a behavioral response for many marine animals that is associated with extraocular photosensitivity. Migrating rhythms have been studied in the squid Todurodes sagittatus and in the tubellarian flatworm Convoiuta roscoffensis. Convoluta lives in the sand at night and during high tides, and emerges onto the surface at low tides during the daytime. If the animals are brought into the laboratory and exposed to a constant light source, vertical migration continues for up to seven days in synchronization with the tides. The migratory rhythm persists in continuous light but ceases in continuous darkness (Palmer, 1974; Wolken, 1975; Naylor, 1985). However, if they are subjected to a flash of light in their dark world, they will resynchronize their rhythm. The extraocular system is also involved in the entrainment of circadian rhythms (Bennett, 1979; Hisano et al., 1972a). The daily rhythmic behavior can directly mediate the response, or entrainment, of the circadian oscillator, via the sixth abdominal

Photodynamically induced chemoresponses of the colorless flagellate, Astasia longa, in the presence of riboflavin

In the presence of the photosensitizer riboflavin, Astasia accumulates in illuminated fields at high fluence rates. The quenchers of riboflavin excited states, NaN3 and KI, abolish the photodynamic effect of riboflavin. Crocetin, a 1O2 quencher, does not influence the photodynamic action of riboflavin while 1,4-benzoquinone very strongly depresses its effect. This indicates a type I pathway forming H2O2 as a photoproduct. The photodynamic effect is abolished by the addition of 10-5 M catalase which breaks down H2O2. Astasia shows chemoaccumulations around the opening of a capillary filled either with riboflavin (under high intensity irradiation) or H2O2 which proves the hypothesis that the photobehavioral response in the presence of riboflavin is based on a chemoresponse toward H2O2, produced when the dye is irradiated. The accumulation around the capillary opening is not due to a direct chemotactic movement of cells but rather to a chemophobic response which prevents the cells from swimming away from the chemoattractant.

Light-induced chemotactic responses of the colorless flagellate,Polytomella magna, in the presence of photodynamic dyes

The colorless flagellate,Polytomella magna, does not show natural reactions toward light like phototaxis, photophobic response or photokinesis. However, if photosensitizers (e.g. riboflavin) are added to the culture an avoidance response toward a light field is induced. The photodynamic reaction depends on the presence of O2. The action spectra are in good agreement with the absorption spectra of the tested dyes. The photobehavioral response in the presence of riboflavin seems to be based on a negative chemotaxis toward H2O2 which is produced when the dye is irradiated.

Photoperiodic activity changes in juvenile sockeye salmon (Oncorhynchus nerka)

Spontaneous locomotor activity was studied in juvenile sockeye salmon under controlled environmental conditions (LD 9.5:14.5 or 12:12; 5 °C; 0.1–34.4 lux). Siblings were hatched in activity chambers and swimming movements were monitored with an ultrasonic system for 11 months. The experiments gave evidence of a bimodal activity rhythm in sockeye fry immediately after hatching. The bimodal, dark-active pattern persisted until 9 days after the fish emerged from the gravel. The photobehavioral response was reversed and the fish expressed a unimodal, light-active pattern 10–14 days after first emergence. This light-active response was then maintained for 11 months.The possible interrelationships between age, photobehavioral response, and activity rhythms underlying the sockeye fry migrations to nursery lakes are discussed.