Photoreceptor Current and Photoorientation in Chlamydomonas Mediated by 9-Demethylchlamyrhodopsin (original) (raw)
2001
efficient reduction of a specific gene product in a green alga. In opsin-deprived transformants, flash-induced photoreceptor currents (PC) are left unchanged. Moreover, photophobic responses as studied by motion analysis and phototaxis tested in a light-scattering assay were indistinguishable from the responses of untransformed wild-type cells. We conclude that phototaxis and photophobic responses in C. reinhardtii are triggered by an as yet unidentified rhodopsin species.
Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas
Biophysical Journal, 1997
Both phototactic and photophobic responses of Chlamydomonas are mediated by a visual system comprising a rhodopsin photoreceptor. Suction pipette recordings have revealed that flash stimulation causes calcium currents into the eyespot and the flagella. These photocurrents have been suggested to be the trigger for all behavioral light responses of the cell. But this has never been shown experimentally. Here we describe a detection technique that combines electrical and optical measurements from individual algae held in a suction pipette. Thus it is possible to record photocurrents and flagellar beating simultaneously and establish a direct link between the two. We demonstrate that in Chlamydomonas only the photoreceptor current in conjuction with a fast flagellar current constitutes the trigger for photophobic responses. Within the time of the action-potential-like flagellar current, the flagella switch from forward to backward swimming, which constitutes the beginning of the photoshock reaction. The switch is accompanied by a complex frequency change and beating pattern modulation. The results are interpreted in terms of a general model for phototransduction in green algae (Chlorophyceae).
Biophysical Journal, 1996
In the green alga Chlamydomonas chlamyrhodopsin fulfills its role as a light sensor by absorbing light and activating photoreceptor channels within the eyespot area. At intense light stimuli, the photoreceptor (P) current triggers a fast and a slow flagellar current that finally leads to backward swimming (stop response). Here we report about probing the photoreceptor current directly at the eyespot. This allows the detection of the whole P current with a size of above 50 pA. The P current appears with a delay of less than 50 ,us, suggesting that rhodopsin and the P channel are closely coupled or form one ion channel complex. The Ca2+ dependence of the P current has been demonstrated with the established suction technique in a capacitive mode. The P current shows the maximum amplitude at only 300 nM Ca2+, and it gradually declines at higher Ca2 . In addition to Ca2 , the photoreceptor and the fast flagellar current can be carried by Sr2+ and Ba2 . Mg2+ is conducted less efficiently and at high concentrations blocks the photoreceptor channel. A motion analysis of the cells shows that only Ca2+ and Sr2' can induce physiological stop responses, whereas the large Ba2+ currents cause abnormal long-lasting cell spiraling.
Desensitization and Dark Recovery of the Photoreceptor Current in Chlamydomonas reinhardtii
Plant physiology
Photoexcitation of rhodopsin in Chlamydomonas reinhardtii triggers a complex of rapid bioelectric processes in the cell membrane. Photoreceptor and flagellar currents are the major components of this cascade and are the basis for the phototaxis and photoshock response, respectively. Desensitization and dark recovery of the extracellularly recorded photoreceptor current were investigated in double-flash excitation experiments. l h e data obtained show that the desensitization is determined by membrane depolarization rather than by rhodopsin bleaching. At external K+ concentrations less than 0.6 mM, generation of the flagellar current triggers a transient, depolarization-activated K+ efflux that contributes to membrane repolarization after light excitation. Acceleration of the dark recovery at 5 to 10 mM Ca2+ can be partially attributed to a blockade of K+ influx, which is triggered at higher external K+ concentrations. K+ currents constitute a nove1 component of the rhodopsin-mediated signaling system in C. reinhardtii that is involved in the process of dark adaptation of the system.
Chlamydomonas Sensory Rhodopsins A and B: Cellular Content and Role in Photophobic Responses
Biophysical Journal, 2004
Two retinylidene proteins, CSRA and CSRB, have recently been shown by photoelectrophysiological analysis of RNAi-transformants to mediate phototaxis signaling in Chlamydomonas reinhardtii. Here we report immunoblot detection of CSRA and CSRB apoproteins in C. reinhardtii cells enabling assessment of the cellular content of the receptors. We obtain 9 3 10 4 CSRA and 1.5 3 10 4 CSRB apoprotein molecules per cell in vegetative cells of the wild-type strain 495, a higher value than that for functional receptor cellular content estimated previously from photosensitivity measurements and retinal extraction yields. Exploiting our ability to control the CSRA/CSRB ratio by transformation with receptor gene-directed RNAi, we report analysis of the CSRA and CSRB roles in the photophobic response of the organism by action spectroscopy with automated cell tracking/motion analysis. The results show that CSRA and CSRB each mediate the photophobic swimming response, a second known retinal-dependent photomotility behavior in C. reinhardtii. Due to the different light saturation and spectral properties of the two receptors, CSRA is dominantly responsible for photophobic responses, which appear at high light intensity.
Integration of photo- and chemosensory signaling pathways in Chlamydomonas
Planta, 2003
The behavior of Chlamydomonas reinhardtii Dangeard is regulated by both light and chemical stimuli. Generation of a transmembrane photoreceptor current is the earliest so far resolved event in phototaxis of green flagellates. Tryptone rapidly inhibits the photoreceptor current in gametes of C. reinhardtii and induces their accumulation. The time-course, concentration dependence and induction during gametogenesis of these two processes coincide. On the other hand, tryptone causes a weak stimulation of the photoreceptor current in the absence of any behavioral responses in vegetative cells. This shows that the tryptone-induced inhibition of the photoreceptor current in C. reinhardtii is due to activation of a gamete-specific chemosensory system, and that integration of the photo- and chemosensory signals already occurs at the initial steps of the signaling pathways.
PLANT PHYSIOLOGY, 2007
Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca 21 -binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.
THE PLANT CELL ONLINE, 2008
Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H þ -channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H þ , CHR1 also conducts Na þ , K þ , and Ca 2þ . The kinetic selectivity analysis demonstrates that H þ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1 505 and CHR1 463 , that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.
The Photoreceptor for Phototaxis in the Photosynthetic Flagellate Euglena gracilis
Photochemistry and Photobiology, 1998
The unicellular flagellate Euglena gracilis shows positive phototaxis at low fluence rates (510 W m-2) and negative phototaxis at high fluence rates (2100 W m-2). Currently, retinal or flavidpterins are discussed as chromophores of the photoreceptor. When grown in the presence of 4 mM nicotine, a retinal inhibitor, for several generations, the cells still showed both responses, indicating that retinal is unlikely to be the chromophoric group of the photoreceptor responsible for phototaxis. The native flavin(s) can be substituted by growing the cells in roseoflavin dissolved in the medium. The absorption spectrum of roseoflavin extends well beyond the action spectrum for phototaxis (up to 600 nm). Excitation at wavelengths >550 nm does not cause phototactic orientation in control cells but causes both positive and negative phototaxis in roseoflavin-grown cells, indicating an uptake and assembly of the chromophore in the photoreceptor complex. The white mutant strain 122&5/1f, induced by streptomycin treatment, lacks flavins as indicated by fluorescence spectroscopy. The phototaxis-deficient phenotype cannot be complemented by the addition of external riboflavin. Fluorescence spectra of intact paraxonemal bodies (PAB) indicate that both pterins and flavins are involved in photoperception and that the excitation energy is efficiently funneled from the pterins to the flavins. This energy transfer is disrupted by solubilization of the PAB. In intact PAB flavins are not accessible to reducing or oxidizing substances, indicating that they are located inside the structure, while pterins are accessible, so that their localization can be assumed to be on the surface. The results described above are discussed with regard to the potential involvement of flavins and pterins as well as retinal in photoperception.
Plant Physiology, 2007
Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii. Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction, we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a Ca 21-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively. Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly light-gated ion channels relevant for the photoresponses of C. reinhardtii.