PHOTOTACTIC BEHAVIOR OF INDIVIDUAL CELLS OF CRYPTOMONAS SP. IN RESPONSE TO CONTINUOUS AND INTERMITTENT LIGHT STIMULI

Abstract— The phototactic response of cells of Cryptomonas sp. to stimulation with continuous or intermittent lateral light was determined by an individual cell method using photomicrography and videomicrography. The cells showed positive phototaxis under the conditions studied. The phototactic orientation of individual cells was induced most effectively by irradiation with light of 570 nm; blue light was less effective, and no orientation was found in red light. An intermittent stimulus regime with a long dark interval (250 ms) elicited a weaker phototactic orientation than did a regime with a short dark interval (63 ms) irrespective of the duration of light pulses (16, 250 and 1000 ms). The swimming rate was ca. 240 ums ^-1 and the rotation period ca. 450 ms in the dark, neither of which was greatly affected by stimulation with continuous or intermittent light. Neither step-up nor step-down photophobic responses were observed at the time of onset or removal of the light stimulus under the experimental conditions. The swimming direction of individual cells became gradually oriented toward the light source. Phototactic response was detectable within 4 s after the onset of light stimulation, reaching a saturation level after more than 30 s.     

PHOTOTACTIC RESPONSE OF CHLAMYDOMON AS TO FLASHES OF LIGHT-I. RESPONSE OF CELL POPULATIONS

Abstract— Chlamydomonas reinhardi responds phototactically to a single, very short flash of blue light (6-4 μs). Net oriented response of a cell population is monitored photometrically, using the "population system" of Feinleib and Curry (1967). A single high-intensity flash elicits a small, but definite net movement away from the stimulus source. Repetitive flashing at low frequency (between 8 and 60 flashes per min) and at the same intensity elicits a prolonged response in the same direction. Net phototactic response to single or repetitive flashes varies with stimulus intensity in the same way as does response to continuous light (Feinleib and Curry, 1971b); response is positive at low intensity and negative at high intensity. These data indicate that at least some cells become oriented in response to a short flash. The occurrence of such a response has implications for the mechanism of phototactic orientation. If almost all the cells responded, one would assume that Chlamydomonas perceives light direction instantaneously by detecting an absorption gradient within the cell. Unequivocal interpretation of the short-flash response requires examination of the behavior of individual cells.     

PHOTOTACTIC RESPONSE OF CHLAMYDOMONAS TO FLASHES OF LIGHT –II. RESPONSE OF INDIVIDUAL CELLS

Abstract— –Video-microscope studies provide further evidence that Chlamydomonas can become oriented in response to a single short flash of light. Following a flash, 50% of the cells in a negatively phototactic population undergo a transient deflection in swimming path ('turn response'), 10% show a 'stop response', and 40% continue to swim straight ahead. The direction of turning is related to the direction of the stimulus; a majority of cells turn away from the flash source. Repetitive flashing at 60 per s elicits oriented swimming, indistinguishable from that observed with continuous light. Responses at the onset of repetitive flashing resemble single-flash responses, reinforcing the idea that response to a single flash corresponds to the initial stages of orientation to continuous light. A stop response sometimes occurs at the onset of orientation to repetitive flashing, but it is apparently not an essential component of orientation. The fact that only 60% of the cells turn or stop in response to a flash is consistent with the hypothesis that light direction is perceived by comparing light absorbed in one photoreceptive region at two instants in time (before and during the flash). The only cells to turn or to stop would be those in which the photoreceptor organelle is appropriately oriented at the instant of the flash.     

Phototactic orientation mechanism in the ciliate Fabrea salina, as inferred from numerical simulations

The marine ciliate Fabrea salina shows a clear positive phototaxis, but the mechanism by which a single cell is able to detect the direction of light and orient its swimming accordingly is still unknown. A simple model of phototaxis is that of a biased random walk, where the bias due to light can affect one or more of the parameters that characterize a random walk, i.e., the mean speed, the frequency distribution of the angles of directional changes and the frequency of directional changes. Since experimental evidence has shown no effect of light on the mean speed of Fabrea salina, we have excluded models depending on this parameter. We have, therefore, investigated the phototactic orientation of Fabrea salina by computer simulation of two simple models, the first where light affects the frequency distribution of the angles of directional changes (model M1) and the second where the light bias modifies the frequency of directional changes (model M2). Simulated M1 cells directly orient their swimming towards the direction of light, regardless of their current swimming orientation; simulated M2 cells, on the contrary, are unable to actively orient their motion, but remain locked along the light direction once they find it by chance. The simulations show that these two orientation models lead to different macroscopic behaviours of the simulated cell populations. By comparing the results of the simulations with the experimental ones, we have found that the phototactic behaviour of real cells is more similar to that of the M2 model.     

Flagellar Motions in Phototactic Steering in a Brown Algal Swarmer

Using infrared high-speed video microscopy, we observed light-triggered transitory flagellar motions in flagellate reproductive cells (swarmers) of a brown alga, Scytosiphon lomentaria, under primary helical swimming conditions before and during negative phototactic orientation to unilateral actinic light. The posterior flagellum, which is autofluorescent and thought to be light-sensing, was passively dragged in the dark and exhibited one to several rapid lateral beats during orientation changes for phototactic steering. Notably, a brief cessation of anterior flagellar beating was occasionally observed concomitantly with rapid beats of the posterior flagellum. This behavior caused a pause in helical body rotation, which may contribute to the accuracy of phototactic steering. Thus, coordinated regulation of the movement of the two flagella plays a crucial role in phototactic steering.     

CALCIUM CONTROL OF PHOTOTACTIC ORIENTATION IN Chlamydomonas reinhardtii: SIGN AND STRENGTH OF RESPONSE

Abstract— Chlamydomonas reinhardtii responds to a blue light stimulus by an oriented swimming (phototaxis) toward or away from the stimulus source. In this study it is established that the sign and strength of the phototactic response are a complex function of extracellular [Ca²⁺], stimulus fluence rate, time of analysis after onset of stimulation and light pretreatment. At very low extracellular [Ca²⁺] the response is weak and usually negative. At [Ca²⁺] close to the preconditioning level, phototactic response becomes stronger and positive. As [Ca²⁺] is raised further, the initial (2 s) response remains positive but the long term (20 s) becomes negative and very strong. At extremely high [Ca²⁺] the cells become immobile. This bimodal behavior suggests that two different mechanisms determine the direction of the turn. Data cannot be explained in terms of a simple model. The model which accounts for most of the details of the behavior is that of Kamiya and Witman (1984), which proposes that positive response is triggered by a transient increase in intracellular [Ca²⁺] and negative response by a decrease below unstimulated level of Ca²⁺, at least in the range of 10^-9-10^-6 M [Ca²⁺]. The strong negative orientation which follows an initial positive response above this level of [Ca²⁺], in these experiments, is best explained by an adaptation of the cells due to an increased (on average) intracellular [Ca²⁺].     

THE ACTION SPECTRUM OF DAPHNIA MAGNA (CRUSTACEA) PHOTOTAXIS IN A SIMULATED NATURAL ENVIRONMENT

Abstract— The action spectrum of phototaxis in Daphnia magna (Crustacea) was measured in a chamber which simulated a natural angular distribution of underwater light. A 17% step-down in irradiance was used to stimulate the phototactic response at all wavelengths and irradiances tested. Peaks in the spectral response curves depended on the fluence rate to which the zooplankton were acclimated. The wavelength of maximum response (Z_max) shifted from yellow-green at the highest acclimation fluence rate (5.1 × 10⁻² Wm⁻²) to blue-violet at moderate rates. At low acclimation fluence rates, the blue-violet maximum was retained and another maximum developed in the red. At the lowest fluence rate (1.6 × 10⁻⁵ Wm⁻²), the blue-violet and red maxima were lost and another maximum developed in the near ultraviolet. The action spectrum indicates the presence of three, and possibly four, photopigments with Z_max, at ∼405, 440, 570 and 690nm. The 440 and 690nm maxima may belong to the same photopigment; however, this was not tested. Changes in zooplankton swimming speed, caused either by large changes in irradiance or by mechanical stimuli, were accompanied by changes in the strength of the phototactic response to the −17% stimulus at any irradiance level for white and monochromatic light, and indicated the presence of a mechanism connecting swimming speed and photosensitivity.     

REVERSIBLE BLEACHING OF Chlamydomonas reinhardtii RHODOPSIN in vivo

Abstract— The effect of hydroxylamine on the phototactic activity of Chlamydomonas reinhardtii was investigated. The following results were obtained: (1) wild type cells, irradiated for 10 min with green light immediately after addition of 1 mM hydroxylamine, showed a 20 min transient loss of phototactic activity, (2) irradiation of cells, preincubated in the dark with 4 mM. hydroxylamine for 30 min, diminished the phototactic sensitivity permanently by more than 100-fold without loss of cell motility. (3) The phototactic sensitivity completely recovered within 3(1 min of the removal of hydroxylamin from carotenoid-containing cells or from carotenoid-negative cells upon addition of 11- cis or all- trans retinal. Our explanation is bleaching of rhodopsin by more than 99% and reconstitution by de novo synthesized or by added retinal.     

FLAGELLUM AUTOFLUORESCENCE and PHOTOACCUMULATION IN HETEROKONT ALGAE

Zoids of various brown algae capable of positive or negative phototaxis show strong autofluorescence of their posterior fiagellum and flagellar swelling when excited with blue light of 450 nm. This effect is lacking in zoids without stigma and phototactic capability. Fluorescence studies on living cells point to a flavin as photoreceptor pigment. The spectral sensitivity curve of photoaccumul-ation suggests a carotenoid pigment (maximum 430–450 nm) being involved, with no effect below 380 nm and above 540 nm. The results obtained in this study indicate a flavin as photoreceptor pigment, which is periodically shaded by a carotenoid stigma.     

EFFECTS OF SOLAR AND ARTIFICIAL UV IRRADIATION ON MOTILITY AND PHOTOTAXIS IN THE FLAGELLATE, Euglena gracilis

The effect of solar irradiation on the percentage of motile cells, their average speed and their phototactic orientation to white actinic light was studied in the flagellate, Euglena gracilis. Unfiltered solar radiation in midsummer during mid-day at a location near Lisboa, Portugal, was found to impair motility within 2 h. This effect is exclusively due to the UV-B component of the radiation and not due to UV-A, visible light or a temperature increase. Likewise, phototactic orientation was drastically impaired. Reduction of the solar UV-B irradiation by insertion of an ozone-Hooded plexiglass cuvette partially reduced the inhibition and covering the cuvettes with glass prevented any decrease in motility and photoorientation. Similar results were found with artificial irradiation (Xe lamps). After inoculation the motility of the population follows an optimum curve (optimum at 8 days). Also, the UV-B effect on motility was smallest after about one week and increased for younger and older cultures.     

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.     

Effect of preillumination on photomotile responses of the marine ciliate Fabrea salina

Fabrea salina is a marine ciliate that shows photomotile responses such as positive phototaxis and a step-down photophobic reaction. We found that preilluminated F. salina cells show a phototactic response significantly greater than that of dark-adapted cells when exposed to the same phototactic light stimulus. In particular, positive phototaxis is strongly enhanced by preillumination. This enhancement effect depends on the preillumination light irradiance, on the total preillumination dose, and on the duration of the dark interval between preillumination and the phototaxis measurement. Our results show that the determining factor is the total preillumination dose given to the sample. The enhancement effect shows an asymptotic behavior over a certain range of energy values (10-200 W/m2). Further, the effect is transient; after 120 s in the dark, the cells lose any memory of the preillumination, independent of the preillumination energy received. These results are tentatively discussed in terms of light-driven membrane potential or membrane channel conductances.     

ACTION SPECTRA FOR PHOTOACCUMULATION OF GREEN AND COLORLESS EUGLENA: EVIDENCE FOR IDENTIFICATION OF RECEPTOR PIGMENTS

Abstract —Phototaxis action spectra have been determined in three different types of Euglena gracilis ; green and dark-bleached cells containing the stigma and streptomycin-treated white mutant with an absorptionless stigma. In all three types of microorganisms the flavin containing paraflagellar body is present. The shape of the action spectrum is the same for the three types of cells and proportional to the absorption spectrum of flavoproteins. It has been shown that the structure of the action spectrum does not depend on the presence of screening organelles, on which, instead, depends the direction of the response to the light stimulus. It is concluded that the flavin chromophores present in the photoreceptor are the pigments responsible for phototaxis in Euglena gracilis.     

PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS: A GENETIC APPROACH*

The photocontrol of anthocyanin synthesis in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) has been studied in an aurea (au) mutant which is deficient in the labile type of phytochrome, a high pigment (hp) mutant which has the wild-type level of phytochrome and the double mutant au/hp , as well as the wild type. The hp mutant demonstrates phytochrome control of anthocyanin synthesis in response to a single red light (RL) pulse, whereas there is no measurable response in the wild type and au mutant. After pretreatment with 12 h blue light (BL) the phytochrome regulation of anthocyanin synthesis is 10-fold higher in the hp mutant than in the wild type, whilst no anthocyanin is detectable in the au mutant, thus suggesting that it is the labile pool of phytochrome which regulates anthocyanin synthesis. The au/hp double mutant exhibits a small (3% of that in the hp mutant) RL/far-red light (FR)-reversible regulation of anthocyanin synthesis following a BL pretreatment. It is proposed that the hp mutant is hypersensitive to the FR-absorbing form of phytochrome (P_fr) and that this (hypersensitivity) establishes response to the low level of P_fl. (below detection limits in phytochrome assays) in the au/hp double mutant.     

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)     

Images of Azurin Protein Studied by AFM

Azurins, a wild type and a genetically mutant K27 altered one. were immobilized on annealed gold sur-face and investigated by means of atomic force microscopy. It was found that the surface coverage and height distribution of the adsorbed protein molecules are different from each other, which is possibly the result of the different orientation on the surface. It is believed that the wild type azurin is connected to gold surface by the disulphide bridge;while the mutant, K27C, might be through the thiol groups of the cysteine residues on their surface.     

RESPONSE OF THE WILD-TYPE and HIGH LIGHT-TOLERANT MUTANT OF Anacystis nidulans AGAINST PHOTOOXIDATIVE DAMAGE: DIFFERENTIAL MECHANISM OF HIGH LIGHT TOLERANCE

Abstract— A high light-tolerant mutant of Anacystis was able to tolerate about three-fold higher light energy irradiance (30 W m^-2) than the wild type (10 W m^-2). The loss of sulfhydryl content and rate of lipid peroxidation in the wild-type cells is lower than in the mutant cells at high light irradiance. This phenomenon in the wild type is probably due to high light-induced severe photoinhibitory conditions resulting in a decreased rate of O₂ evolution. Results on the bleaching of the N, N '-dimethyl- p -nitrosoaniline at high light irradiance show a higher rate of bleaching in the wild-type than in the mutant cells. Further, results on the rate of N, N '-dimethyl- p -nitrosoani)ine bleaching in the presence of radical scavengers like sodium azide, histidine and sodium formate (10 m M , each) suggest that singlet oxygen is the predominant oxygen species produced in both the wild-type and mutant cells under high light. However, a similar quenching effect of formate in the mutant cells is indicative of increased formation of hydroxyl radicals. This observation is further corroborated by higher rate of lipid peroxidation. In addition to this, the superoxide dismutase activity is higher in the mutant (1.2 unit) than in the wild type. Taken together, these results suggest that the cells of the high light-tolerant mutant have an efficient intracellular mechanism to transform the free oxygen radicals.     

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.     

ACTION SPECTRA FOR INHIBITION OF HYPOCOTYL GROWTH OF WILD-TYPE PLANTS AND OF THE hy2 LONG-HYPOCOTYL MUTANT OF Arabidopsis thaliana L.

Abstract— An analysis was made by action spectroscopy, using the Okazaki Large Spectrograph, of the inhibition of hypocotyl elongation of wild-type plants and the hy2 mutant of Arabidopsis thaliana. Two day old etiolated seedlings were irradiated for 8 h with monochromatic light and left in the dark for 16 h before measurement of hypocotyl length. Spectrophotometric measurement showed that levels of phytochrome in the etiolated tissue of the hy2 mutant were less than 9% of those in the wild type. The action spectra of the wild type looked like those of high irradiance response and showed peaks at 375, 450, 625 and 725 nm, whereas the action spectra of hy2 showed only the peaks at 375 and 450 nm. Monochromatic light of wavelengths longer than 500 nm had no significant inhibitory effects on hy2 plants. Blue and UV-A light were about five times more effective in the wild type than in hy2 plants. Severe inhibitory effects were observed with UV-B light. It is concluded that inhibition of the growth of the hypocotyl involves combined actions of phytochrome and a putative blue/UV-A photoreceptor(s).     

Semibiological molecular machine with an implemented "AND" logic gate for regulation of protein folding

A semibiological molecular machine with an implemented "AND" logic gate was developed, which was capable of controlling the folding process of proteins in response to ATP and light as input stimuli. The molecular design made use of a genetically engineered chaperonin GroEL bearing, at both entrance parts of its cylindrical cavity, cysteine residues, which were functionalized by an azobenzene derivative to construct photoresponsive mechanical gates (azo-GroEL). This engineered chaperonin trapped denatured green fluorescent protein (GFP(denat)) and prohibited its refolding. However, when hosting azo-GroEL detected ATP (input stimulus 1) and UV light (input stimulus 2) at the same time, it quickly released GFP(denat) to allow its refolding. In contrast, reception of either input stimulus 1 or 2 resulted in only very slow or no substantial refolding of GFP(denat). Implementation of such "AND" logic gate mechanisms in mechanically driven biomolecular systems is an important step toward the design of secured drug delivery systems.