Photosensory transduction in unicellular eukaryotes: a comparison between related ciliates Blepharisma japonicum and Stentor coeruleus and photoreceptor cells of higher organisms

Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. I. AN ANALYSIS OF LIGHT-INDUCED ELECTRICAL AND MOTILE RESPONSES IN Stentor coeruleus

Abstract— Light-induced membrane potantial changes and motile responses have been studied in Stentor cells with intracellular microelectrodes and video microscopy, respectively. Intracellulae microelectrode showed that step-up increase in light induced an electrical membrane response which consisted of an initial membrane depolarization (photoreceptor potential) followed by an action potential and maintaining phase of depolarization (afterdepolarization). The amplitude of the receptor potetial is dependent on the intensity of light stimulus and the action potetials appears with a lag period (latency) after the onset of light stimuklus. The extent of the membrane established between the latency for te action poitential and the onset of ciliary reversal (stop responses). A time correlation was also observed between the duration of the membrane afterdepolarization and the duration of backward swimming. the action spectrum for the photoreceptor potential amplitude of Stentor resembled the action spectra for the latency of ciliary reversal and the photoresponsiveness, iondicating that the photomovement response and membrane potential changes are coupled through the same photosensor system. A hypothesis on the photosensory transduction chain in Stentor is discussed according to ehich the photoreceptors and the ciliary apparatus is mediated by the membrane potential canges.     

CAFFEINE-ENHANCED PHOTOMOVEMENT IN THE CILIATE, STENTOR COERULEUS

The effects of caffeine, ionophores and calcium flux blockers on the step-up photophobic response, phototactic orientation and the intracellularly recorded, light-induced electrical action potential were studied in the ciliate, Stentor coeruleus . Caffeine alters the absorption and CD spectra and enhances the fluorescence of the photoreceptor pigment, stentorin. Independent of its effects on the spectroscopic properties of the photoreceptor pigment, caffeine shortens the photophobic response time by enhancing the Ca²⁺ conductivity of membranes, while Ca²⁺ flux blockers (LaCI₃ or ruthenium red) prolong it; both effects cancel each other. Evidence is presented that phototactic orientation is brought about by repetitive photophobic responses, since a change in the phobic response time results in a decreased accuracy of phototaxis.     

EFFECTS OF K+ AND Ca2+ IONS ON MOTILITY AND PHOTOSENSORY RESPONSES OF STENTOR COERULEUS

Extracellular K⁺ ions above a critical concentration induce ciliary reversal in unstimulated Stentor coeruleus and suppress step-up photophobic response. This threshold concentration of K⁺ ions depends on the extracellular Ca²⁺ concentration, and the subsequent backward gyration and light-sensitivity suppression seem to depend on the relative concentrations of K⁺ and Ca²⁺. The concentration of Ca²⁺ necessary to overcome K⁺-mediated inhibition of phobic response and backward swimming increases non-linearly with increasing K⁺ concentration. The Ca²⁺-blocking agent. D-600, selectively inhibits photophobic responses of Stentor , thus further confirming the role of Ca²⁺ ions in photosensory transduction of this ciliate.     

A videomicroscopic study of the effect of l-cis-diltiazem on the photobehavior of Stentor coeruleus

The protozoan ciliate Stentor coeruleus displays a step-up photophobic response to an increase in light intensity in its environment. The motile response consists of a delayed stop of ciliary beating and transient ciliary reversal period. Such light-avoiding behavior was significantly influenced by an incubation of cells with l-cis-diltiazem, a common blocker of cyclic guanosine monophosphate (cGMP)-gated ion channel conductance. The introduction of l-cis-diltiazem to the medium induced ciliary reversal in control cells, mimicking the step-up photophobic response. In light-stimulated ciliates, the presence of this inhibitor caused a substantial decrease of the latency of ciliary stop response, prolongation of the ciliary reversal duration and also an increase of cell photoresponsiveness in a dose- and time-dependent manner. The obtained behavioral results support the suggestion that the photosensitive ciliate S. coeruleus possesses cGMP-gated channels, which may be involved in the process of light signal transduction for the motile photophobic response.     

PHOTODYNAMIC ACTION IN Stentor coeruleus SENSITIZED BY ENDOGENOUS PIGMENT STENTORIN

Abstract— Stentorin acts as the photoreceptor for the step-up photophobic and negative phototactic responses in Stentor coeruleus . The chromophore of stentorin appears to be hypericin which is linked to apoprotein. In addition to the photomovement responses of the organism, S. coeruleus was found to be photodynamically sensitive to light absorbed by the hypericin chromophore, as the apparent action spectrum for the photodynamic killing matches the absorption spectrum of stentorin. The protective effect of β-carotene and crocetin on the photodynamic killing of S. coeruleus suggests that singlet oxygen generated by the stentorin-sensitization plays an important role, according to the so-called Type II mechanism of photosensitization. The generation of singlet oxygen via hypericin triplet was confirmed by in vitro photooxidation of tryptophan as a substrate. The photodynamic killing was more effective in deuterium oxide than in H₂O in both the photosensitization by stentorin (endogenous) and added hypericin (exogenous). These results are consistent with the involvement of singlet oxygen in the photodynamic killing of S. coeruleus .     

Stentor coeruleus SHOWS POSITIVE PHOTOKINESIS

Abstract— Stentor coeruleus responds to a sudden increase in light intensity with a step-up photophobic response (avoiding reaction), and to collimated light with negative phototaxis. The peaks of the action spectra for the photophobic response and for the phototaxis are in common, 610 nm.
5. coeruleus showed changes in its steady-state swimming velocity induced with varying intensities of light (photokinesis). The cells swam fast in light regions but slowly in dark ones (positive photokinesis); the mean velocity of swimming was about 0.6 mm/s at 100 lx but reached about 1.0 mm/s at 50000 lx. The peak of the action spectrum for this photokinesis was about 680 nm.
The organism is the first protozoan cell reported to show three types of photoresponse: photophobic response, phototaxis and photokinesis.     

Behaviors producing photodispersal in Stentor coeruleus

The ciliate Stentor coeruleus exhibits photodispersal, that is, these cells swim away from light sources and collect in dimly lighted areas. We imaged and reconstructed the tracks of 48 Stentor to determine which swimming behaviors produced their photodispersal. We observed that their photodispersal is not due to a change in their swimming speed but rather to a change in the frequency with which they reorient their swimming direction. Therefore, their photodispersal must be due to either (1) a gradual reorientation of the organism's swimming direction determined by the direction of the light beam (phototaxis) or (2) multiple randomly directed reorientations in swimming direction that occur less frequently when the cell is swimming away from the light source (biased random walk). Sixteen (19%) of the 83 observed forward swimming tracks lasting three or more seconds exhibited a gradual bending away from the light source consistent with a phototaxis. However, most tracks were interrupted repeatedly by abrupt reorientations resulting from ciliary reversals and "smooth turns" that caused cells to reorient through 5.4 times as many degrees as were needed to direct them away from the light source. When cells were swimming away from the light source, their probability of reorienting was reduced and photodispersal resulted.     

Maristentorin, a novel pigment from the positively phototactic marine ciliate Maristentor dinoferus, is structurally related to hypericin and stentorin

The photoreceptor pigment of the heterotrich ciliate, Maristentor dinoferus, has been characterized. It is structurally similar to those of Stentor coeruleus and Blepharisma japonicum but differs significantly in that it bears no aromatic hydrogens. The structure of the pigment, maristentorin, is based upon the hypericin skeleton, and its spectra are nearly identical to those of hypericin but shifted toward the red. Within experimental error, its fluorescence lifetime is identical to that of hypericin, approximately 5.5 ns in dimethylsulfoxide. It is remarkable that while the pigments are structurally similar in S. coeruleus and M. dinoferus, in the former there is an abrupt photophobic response, whereas in the latter there is a slow response toward light. The roles of the hypericin-like pigments in the heterotrich ciliates are discussed as potentially analogous in Maristentor.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. III. THE TEMPORAL RELATION BETWEEN MEMBRANE POTENTIALS AND PHOTOMOTILE RESPONSES IN Blepharisma japonic urn

Abstract— Blepharisma japonicum exhibits a step-up photophobic response when subjected to an increase in light stimulus intensity. This response is characterized by the stop reaction after a period of delay followed by backward swimming (lateral rotation). The latency of the stop response decreased and duration of the lateral rotation increased as the intensity of light stimuli was raised. A step-increase in light intensity elicited a graded membrane depolarization (photic receptor potential), as measured by intracellular microelectrode. When the amplitude of receptor potential exceeded a threshold depolarization for membrane excitation (15–25 mV), an all-or-none action potential of 50–65 mV in amplitude was evoked which also occurred with some latency. Light stimuli of higher intensity (suprathreshold) elicited action potential which was followed by a membrane after-depolarization. Increasing the intensity of stimuli caused generation of an action potential with shorter lag period and prolonged after-depolarization. The action spectra for the latency of stop reaction, receptor potential amplitude and cell photoresponsiveness showed maxima at 460, 530 and 580 nm. The analysis of temporal relationships between the electrophysiological responses and the motile events showed that latency of an action potential, induced by the receptor potential, correlates well with the latency of a cell stop response. Also the duration of membrane after-depolarization resembled the time period of the cell's backward swimming (cell rotation). The data obtained indicate that the primary reaction initiated by light absorption in the photoreceptor pigment (blepharismin) is converted into the observed electrical potential changes, which in turn results in the photomotile response of Blepharisma cells.     

Photoreceptor pigments for photomovement of microorganisms: some spectroscopic and related studies

Optical spectroscopy of photoreceptor pigments can substantially contribute to our understanding of the molecular processes which are the basis of photoreception and sensory transduction in photomotile microorganisms. The main spectroscopic techniques are briefly illustrated, together with the most significant types of progress that can be achieved. A few "case examples" are discussed in some detail: Halobacterium, with particular attention to the contribution of flash photolysis studies to the identification and characterization of sensory rhodopsins; Euglena, and the role of in vivo microspectrofluorometry in confirming the flavin nature of its photoreceptor pigment; the first suggestions on the rhodopsin-like nature of the Chlamydomonas photosensing system; Stentor and Blepharisma and the contribution of static and time-resolved fluorescence studies to a molecular model of the primary events in their photoreceptor pigments (stentorin and blepharismin) and systems.     

Action Spectrum for the Photophobic Response of Ciona intestinalis (Ascidieacea, Urochordata) Larvae Implicates Retinal Protein

Ascidian tadpole larvae change swimming behavior during the course of development. The photic behavior of the larvae of Ciona intestinalis was monitored by a computerized cell-tracking system with a time resolution of 0.1 s. Newly hatched larvae swim at an average speed of 1.4 mm/s but show no response to light stimuli. The swimming speed of the larvae became slower (0.4 mm/s) 3 h after hatching and they were induced to swim more rapidly by a sudden decrease in light intensity 4 h after hatching. During the course of development, the maximal speed of swimming behavior increased with time until 8 h after hatching and then plateaued. The action spectrum for the step-down photophobic response of the larvae was determined at around 8 h after hatching and was fitted to Dartnall's nomogram with the absorbance maximum of the pigment located at 505 nm. These results suggest retinal proteins in the ocellus of the larvae are the photoreceptors for the photobehavior.     

PHOTOSENSORY BEHAVIOR OF A BACTERIORHODOPSIN-DEFICIENT MUTANT, ET-15, OF Halobacterium halobium

– Halobacterium halobium , strain ET-15, which does not contain detectable amounts of bacteriorhodopsin (BR) shows behavioral responses to UV and yellow-green light. Attractant stimuli. i.e. light-increases in the yellow-green range or light-decreases in the UV, suppress the spontaneous reversals of the swimming direction for a certain time. Repellent stimuli, i.e. light-decreases in the yellow-green range or light-increases in the UV, elicit an additional reversal response after a few seconds. Action spectra of both sensory photosystems, PS 370 and PS 565, were measured with attractant as well as with repellent stimuli. As in BR-containing cells, maximal sensitivity was always found at 370 nm for the UV-system and at 565 nm for the long-wavelength system. Fluence-response curves at 370 and 565 nm obtained with strain ET-15 and with a BR-containing strain show that the sensitivity of both photosystems is not reduced in the absence of BR. It is concluded that BR is required neither for PS 565 nor for PS 370. Instead retinal-containing pigments different from BR have to be assumed to mediate photosensory behavior.     

Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented.     

LIGHT REACTIONS OF THE CILIATE Stentor coeruleus-A THREE-DIMENSIONAL ANALYSIS

A computer-controlled three-dimensional tracking and motion analysis system was developed to study the responses of Stentor coeruleus to short light pulses and to evaluate its distribution patterns. In addition to photokinesis and phototaxis, the step-up photophobic response was analyzed, which includes a gravity-controlled component at higher fluence rates and a light direction-dependent component at lower fluence rates.     

PHOTOTACTIC ORIENTATION BY THE CILIATE, STENTOR COERULEUS

Stentor coeruleus exhibits negative phototaxis to visible light, in addition to a step-up photophobic response. The negative phototaxis was established by demonstrating the swimming of Stentor toward a focused beam away from the light source. The action spectrum showed a maximum at 610–620 nm and is essentially identical to that of the step-up photophobic response. Proton uncouplers such as micromolar concentrations of FCCP and TPMP⁺ inhibited the negative phototaxis.     

TRANSFORMATION OF A BOP-HOP-SOP-I-SOP-II-Halobacterium halobium MUTANT TO BOP+: EFFECTS OF BACTERIORHODOPSIN PHOTOACTIVATION ON CELLULAR PROTON FLUXES AND SWIMMING BEHAVIOR

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE PHOTOMOVEMENT OF Caenorhabditis elegans, A NEMATODE WHICH LACKS OCELLI. PROOF THAT THE RESPONSE IS TO LIGHT NOT RADIANT HEATING

Abstract— Caenorhabditis elegans adults were tested at constant temperature with 10 s periods of monochromatic light alternated with 20 s dark periods. Stimuli at effective intensities and wavelengths caused an increase in the frequency of ecclitic (phobic, avoidance) responses, which was measured as an increase in the probability of a temporary reversal in direction of movement. For monochromatic stimuli ranging from 420 to 680 nm at a constant 56 picoeinsteins s^-1 cm^-2, only those at520–600 nm elicited significant responses. At 540 nm the threshold fluence rate was approximately 30 pE s^-1 cm^-2. At saturating intensities the mean reversal probability was increased to 0.20 in 10 s from a background level of 0.12. approximately.
Because C. elegans lacks ocelli and is very sensitive to temperature, possible sources of radiant heating were considered in detail, including (a) infrared present in the stimuli, (b) absorption of light by the arena, and (c) absorption of light by a nematode pigment. All possible sources were found to cause a negligible temperature rise, on the order of or less than the natural temperature fluctuations inside the worm, 1.5 times 10^-6°C. A 2 times 10^-4°C temperature rise produced by a 1230 nm infrared stimulus had no significant effect on reversal frequency. It was concluded that the response to illumination must have been to light, and not to temperature changes.
Large, + or - 2 °C changes from the acclimation temperature caused significant increases in the background frequency of ecclitic responses (a thermoecclisis or thermoklinokinesis). However, neither the threshold nor the saturation level of light-induced responses was affected by the ± 2°C changes.     

Phototaxis in the ciliated protozoan Ophryoglena flava: dose-effect curves and action spectrum determination

The sensitivity of positive phototactic orientation of cells of the ciliated protozoan Ophryoglena flava has been measured for white light, broad-band blue and red light, and narrow-band monochromatic light, using a laboratory-developed computer aided system. The white-light fluence rate-response curve shows that there is no negative phototaxis in the fluence rate range investigated (0-15 W/m2) and no adaptation phenomena; it is very well fitted by a hyperbolic function; the fluence rate curves under broad band blue and red light (full width at half maximum, FWHM= 100 nm) can be fitted by the same model. The saturation level is, within experimental errors, the same for the three curves, indicating that there are no chromaticity effects and that if there is more than one photoreceptor pigment, they act independently of each other. The fluence rate-response curves determined under narrow band monochromatic light (FWHM = 10 nm) can also be fitted by the same model and show, within experimental errors, the same saturation level. An action spectrum for positive phototaxis at 10-nm intervals has been calculated from fluence rate-response curves: it shows three maxima, at 420, 540 and 590 nm. This action spectrum is significantly different from the ones for photomotile responses in Blepharisma japonicum, Stentor coeruleus and Chlamydodon mnemosyne, whereas it resembles the ones of Paramecium bursaria and Fabrea salina.     

ACTION SPECTRUM FOR THE PHOTOINHIBITION OF BIOLUMINESCENCE IN THE MARINE DINOFLAGELLATE DISSODINIUM LUNULA

Abstract— The fractional photoinhibition of the mechanically stimulable bioluminescence in the vacuolar dinoflagellate Dissodinium lunula is proportional to the logarithm of the exposure. The action spectrum for this photoinhibition has been determined by measuring threshold exposures in absolute units of photons cm⁻². The threshold exposure at the wavelength of maximum sensitivity, 450 nm, was 2 ± 10⁻² photons cm⁻². The action spectrum is consistent with absorption by a blue light receptor pigment shielded by a nonphotoactive pigment which absorbs in the region of the bioluminescence emission spectrum. It is suggested that there may be some selective advantage for this absorbing pigment in the vacuolar dinoflagellates in order to prevent the organisms from being photoinhibited by their own bioluminescence.