Chemotaxis in the green flagellate alga Chlamydomonas.

Behavior of the green flagellate alga Chlamydomonas changes in response to a number of chemical stimuli. Specific sensitivity of the cells to different substances might appear only at certain stages of the life cycle. The heterogamous species C. allensworthii demonstrates chemotaxis of male gametes towards pheromones excreted by female gametes. In C. reinhardtii chemotaxis towards tryptone occurs only in gametes, whereas chemotaxis towards ammonium, on the contrary, only in vegetative cells. Chemotaxis to different chemical stimuli might involve different mechanisms of reception and signal transduction, elucidation of which has only recently begun. Indirect evidences show that the cells likely respond to tryptone with changes in the membrane electrical conductance. The recently completed project of sequencing the whole nuclear genome of C. reinhardtii provides the basis for future identification of molecular elements of the chemosensory cascade in this alga.

[Phototaxis of the green algae: the new class of rhodopsin receptors]. / Fototaksis zelenykh vodoroslei: novyi klass rodopsinovykh retseptorov.

Photomotility behavior in green flagellate algae is mediated by rhodopsin-like receptors, which was initially suggested on the basis of physiological evidence. The cascade of rapid Ca(2+)-dependent electrical responses in the plasma membrane plays a key role in the signal transduction chain during both phototaxis and the photophobic response. The photoreceptor current through the plasma membrane is the earliest detectable event upon photoexcitation of the photoreceptors. Analysis of this current revealed that it consists of at least two components with different characteristics. Genes encoding two archaeal-type rhodopsins (type I rhodopsins) were recently identified in the genome of Chlamydomonas reinhardtii and named (Chlamydomonas Sensory Rhodopsins A and B CSRA and CSRB). The measurements of photoelectric and motor responses in genetic transformants of C. reinhardtii enriched in each of these receptor proteins showed that the two components of the photoreceptor current are mediated by the two rhodopsins, and that both CSRA and CSRB are involved in phototaxis and the photophobic response. The CSRA-mediated current dominates at high light intensities and contributes primarily to the photophobic response. The CSRB-initiated transduction involves an efficient amplification cascade and mediates the highly sensitive phototaxis at low light intensities. CSRA and CSRB expressed heterologously in oocytes of Xenopus laevis act as light-gated proton channels, although it is unclear whether this channel activity plays a functional role in the initiation of motor responses and/or occurs in the native system.

Rhodopsin receptors of phototaxis in green flagellate algae.

Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the signal transduction chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suitable approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique rhodopsin-type protein. It shares common chromophore properties with retinal proteins from archaea. However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual rhodopsins. The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinal-binding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential function of this protein as the phototaxis receptor has been questioned in recent time.

Photoreceptor current and photoorientation in chlamydomonas mediated by 9-demethylchlamyrhodopsin.

Green flagellates possess rhodopsin-like photoreceptors involved in control of their behavior via generation of photocurrents across the plasma membrane. Chlamydomonas mutants blocked in retinal biosynthesis are "blind," but they can be rescued by the addition of exogenous retinoids. Photosignaling by chlamyrhodopsin regenerated with 9-demethylretinal was investigated by recording photocurrents from single cells and cell suspensions, and by measuring phototactic orientation. The addition of a saturating concentration of this analog led to reconstitution of all receptor molecules. However, sensitivity of the photoreceptor current in cells reconstituted with the analog was smaller compared with retinal-reconstituted cells, indicating a decreased signaling efficiency of the analog receptor protein. Suppression of the photoreceptor current in double-flash experiments was smaller and its recovery faster with 9-demethylretinal than with retinal, as it would be expected from a decreased PC amplitude in the analog-reconstituted cells. Cells reconstituted with either retinal or the analog displayed negative phototaxis at low light and switched to positive one upon an increase in stimulus intensity, as opposed to the wild type. The reversal of the phototaxis direction in analog-reconstituted cells was shifted to a higher fluence rate compared with cells reconstituted with retinal, which corresponded to the decreased signaling efficiency of 9-demethylchlamyrhodopsin.

A novel express bioassay for detecting toxic substances in water by recording rhodopsin-mediated photoelectric responses in Chlamydomonas cell suspensions.

The influence of Cu2+, Zn2+, Cd2+, Pb2+ and formaldehyde on rhodopsin-mediated photoelectric responses in the green flagellate Chlamydomonas reinhardtii was investigated using three modifications of a recently developed population method for electrical recording (in nonoriented, phototactically preoriented (PO) and gravitactically preoriented cell suspensions). The addition of the heavy metal ions at concentrations several times lower than those known to affect swimming velocity and other physiological parameters in photosynthetic flagellates led to a rapid (one to several minutes) inhibition of the responses. Formaldehyde induced a significant temporary increase in the gravi-orientation of the cells simultaneously with an inhibition of their photoelectric cascade, photo-orientation and motility. The signals recorded in PO suspensions were more sensitive to all tested toxic substances than those recorded from nonoriented cells and indicated a switch from negative to positive phototaxis in the presence of the toxic substances. Of the two major components of the photoelectric cascade, the regenerative response was more sensitive to the tested heavy metal ions, but not to formaldehyde, than the photoreceptor current. The results obtained show that measurement of the photoinduced electrical responses in Chlamydomonas cell suspensions is a powerful novel bioassay for testing environmental pollutants in water samples.

Isolation and characterization of novel Chlamydomonas mutants that display phototaxis but not photophobic response.

The unicellular green alga Chlamydomonas displays two distinct kinds of behavioral response to light: phototaxis, in which cells swim toward or away from the light source under constant illumination; and photophobic responses (also called stop responses or photoshock responses), in which cells transiently convert their flagellar waveform and swim backward upon sudden increase in light intensity. It has been suggested that the two responses partly share a common signal transduction pathway, but exactly how the different responses are produced has not been established. In this study, to help understand the molecular and cellular mechanisms that bring about the photophobic response, we isolated novel mutants (ppr1, ppr2, ppr3, and ppr4) that do not show the photophobic response. Importantly, these mutants retain the ability to display phototaxis, with almost the same sensitivities as in the wild type cell. Demembranated and reactivated flagellar axonemes of the ppr mutants were found to convert the bending patterns depending on the Ca2+ concentration, indicating that the axonemal mechanism for waveform conversion required for the photophobic response was unaffected by the mutations. In addition, measurements of electric currents in cell suspensions showed that these mutants generate normal photoreceptor currents (PRC) upon photostimulation, suggesting that they retain the normal activity of photoreception and the ionic channels that produce PRCs. However, the all-or-none flagellar current (FC), a Ca2+ current generated by PRC-induced depolarization of flagellar membrane, was absent or seriously impaired in the mutants. These findings clearly indicate that the all-or-none FC is necessary for the photophobic response but not for phototaxis. The isolation of the four genetically independent ppr mutants suggests that the generation of the FC is based on multiple components that are not used in the mechanism for phototaxis, and implies that the Chlamydomonas flagellar membrane possesses a voltage-dependent Ca2+-channel specifically used for generation of photophobic responses.

Desensitization and Dark Recovery of the Photoreceptor Current in Chlamydomonas reinhardtii.

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. The 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 novel component of the rhodopsin-mediated signaling system in C. reinhardtii that is involved in the process of dark adaptation of the system.

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.

Photoinduced electric currents in carotenoid-deficient Chlamydomonas mutants reconstituted with retinal and its analogs.

Reconstitution of the photoelectric responses involved in photosensory transduction in "blind" cells of Chlamydomonas reinhardtii carotenoid-deficient mutants was studied by means of a recently developed population method. Both the photoreceptor current and the regenerative response can be restored by addition of all-trans-retinal, 9-demethyl-retinal, or dimethyl-octatrienal, while the retinal analogs prevented from 13-cis/trans isomerization, 13-demethyl-retinal and citral, are not effective. Fluence dependence, spectral sensitivity, and effect of hydroxylamine treatment on retinal-induced photoelectric responses are similar to those found earlier in green strains of Chlamydomonas, although an alternative mechanism of antenna directivity in white cells of reconstituted "blind" mutants (likely based on the focusing effect of the transparent cell bodies) leads to the reversed sign of phototaxis in mutant cells under the same conditions. The results obtained indicate that both photoreceptor current and regenerative response are initiated by the same or similar rhodopsins with arhaebacterial-like chromophore(s) and prove directly the earlier suggested identity of the photoreceptor pigment(s) involved in photomotile and photoelectric responses in flagellated algae.

Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt (-).

The function of the eyespot in phototaxis of the flagellate green alga Chlamydomonas reinhardtii Dangeard was studied using quantitative reflection confocal laser scanning microscopy and photoelectric measurements. The reflective properties of the eyespot and the photoreceptor current of the C. reinhardtii eyespot mutant ey 627, mt (-) were compared with those of Chlamydomonas strains possessing a well-developed eyespot. Under growth conditions in which strongly disorganized eyespots were observed in the mutant by electron microscopy, there was a significant reduction in the reflection intensity of the eyespot and in the amplitude ratio (500∶440 nm) of photoreceptor currents induced by flashes of 500- and 440-nm light in non-oriented cells. Photoelectrical responses of pre-oriented cells revealed that the latter effect is caused by an altered directional sensitivity of the antenna complex, whereas the functional state of the photoreceptor pigment is not strongly affected in mutant cells. Both the reflection intensity and the amplitude ratio of photoreceptor currents increased to the level of reference strains under conditions supporting the development of a well-organized eyespot in the mutant. Furthermore, incubation of the mutant with high concentrations of all-trans-retinal (10 µM), independent of whether carotenoid biosynthesis was inhibited or not, was found to increase the reflection intensity of the eyespot. An increase in the rate of photoorientation of the mutant occurred concomitant with the increase in the reflective properties of the mutant eyespot. These observations demonstrate the importance of an intact eyespot for interference reflection and absorption of phototactically active light, and thus for the directional sensitivity of the eyespot apparatus.

Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis.

The kinetics of the photoreceptor potential of phototaxis in biflagellated green alga Haematococcus pluvialis in response to a 10-ns laser pulse of three wavelengths (465, 550, and 590 nm) were measured in single cells with 30 mus time resolution. The rise and the decay of photoinduced potential are both at least biphasic. The first component of the rise is very stable and has no measurable (<30 mus) time delay. The second component is triggered after a 120-400-mus lag period, depending on flash intensity. Its appearance is sensitive to the physiological state of the cell and the amplitude can be increased by phototactically ineffective red background illumination. The electrical generators for both components are localized in the same region of the cell membrane (on the stigma-bearing side) and these components have the same depolarizing sign. The results indicate that the photoreceptor potential in phototaxis comprises two components, which could be interpreted as light-induced charge movement within the photoreceptor molecules and changes in ion permeability of the cell membrane.

[Light-induced changes in quantum yields of the photochemical cycle of conversion of bacteriorhodopsin and transmembrane proton transfer in cells of Halobacterium halobium]. / Fotoindutsirovannye izmeneniia kvantovykh vykhodov fotokhimicheskogo tsikla prevrashchenii bakteriorodopsina i transmembrannogo perenosa protonov v kletkakh Halobacterium halobium.

It was found that the rate of proton efflux from the cells, vH+, and the turnover number of bacteriorhodopsin photoconversion cycle per second, vM, are gradually decreased during continuous illumination of Halobacterium halobium with orange light (550-650 nm, 10(3) W/m2). The steady-state value of vH+ after 3 min of illumination is 3 times lower than vH+ at the 1st second of illumination. At the same time the concentration of the photochemical cycle intermediate, M412, the quantum yield of its formation, phi M, and the quantum efficiency of proton efflux, phi H+, are decreased (phi M and phi H+ up to 3-4-fold), whereas the concentration of the initial form of bacteriorhodopsin, BR570, and lifetime of M412, tau, are increased. These light-induced effects are abolished by uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP) and are enhanced by the phosphorylation inhibitor, N,N'-dicyclohexylcarbodiimide (DCCD). This suggests that the observed changes of M412 and BR570 concentrations, tau, VH+, VM, phi M and phi H+ are due to the light-induced changes in the transmembrane electric potential, delta psi, and in pH inside and outside the cell, which control the photochemical cycle reactions. The decrease of phi M and phi H+ is discussed on the basis of an earlier proposed scheme of the branched photochemical cycle. It is assumed that the back reactions of the cycle (shunts) not coupled with the transmembrane proton translocation, e.g. conversion of L550 (P550) into BR570 without M412 formation, etc., become more probably at high values of the proton electrochemical potential gradient.

[Induction of fluorescence of a single chloroplast in an entire cell of the green alga Haematococcus pluvialis]. / Induktsiia fluorestsentssi odinochnogo khloroplasta v tseloi kletke zelenoi vodorosli Haematococcus pluvialis

The fluorescense induction time-course of a single chloroplast in an intact cell of the green alga Haematococcus pluvialis has a line structure, which could not be observed in a suspension because of the superposition of the emissions of many cells. The time-course of the induction includes 12 well separated parts in the time range from several milliseconds to several minutes. Parameters of the fine structure depends on the physiological state of the cell, intensity and wave-length of the exciting light.