A microspectrophotometric study of the shielding properties of eyespot and cell body in Chlamydomonas.

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.     

A gap isolation method to investigate electrical and mechanical properties of fully contracting skeletal muscle fibers.

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 microseconds, 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.     

Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly

The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and is associated with the daughter four-membered (D4) microtubule rootlet, comprises an elliptical patch of rhodopsin photoreceptors on the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. EYE2 is positioned along, and adjacent to, the D4 rootlet in the absence of pigment granules. The eyespot pigment granule array is required for maintenance of the elliptical shape of both the overlying EYE2 and channelrhodopsin-1 photoreceptor patches. We propose a model of eyespot assembly wherein rootlet and photoreceptor direct EYE2 to an area of the chloroplast envelope, where it acts to facilitate assembly of pigment granule arrays, and EYE3 plays a role in the biogenesis of the pigment granules.     

Phototropin Influence on Eyespot Development and Regulation of Phototactic Behavior in Chlamydomonas reinhardtii

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity–dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.     

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 (500440 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 M), 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.Abbreviations HSM high-salt medium This study was supported by the Deutsche Forschungsgemeinschaft. O. A. Sineshchekov was supported by a Research Fellowship from the Alexander von Humboldt Foundation. The authors wish to thank U. Powalowski (Botanisches Institut, Universität zu Köln) for help with electron microscopy.     

Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization

The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells.     

Reflection confocal laser scanning microscopy of eyespots in flagellated green algae

The reflection properties of different types of eyespots in three unicellular, flagellated green algae (Tetraselmis chui, Chlamydomonas eugametos, Hafniomonas reticulata) were investigated using confocal laser scanning microscopy in the epireflection mode. The eyespots differed with respect to the number of eyespot lipid globule layers and surface appearance (concave/convex). A strong reflection signal was observed in all eyespots, and a detailed quantitative analysis by optical xy (horizontal) and xz (vertical) sectioning was performed. By applying both sectioning capabilities, multi- and single/double-layered eyespots as well as concave and convex eyespot surfaces could be distinguished using living, immobilized cells. Focusing of the reflected light was only observed in eyespots with concave surfaces. In xz series of multi-layered eyespots at reduced laser intensities (0.01%), the intensity profiles of the reflection revealed a series of alternating maxima and minima with increasing reflection intensities toward the cell surface. At very low laser intensities (0.001%), multi-layered eyespots exhibited about twice the reflection intensity at the presumptive photoreceptor site compared to single/double-layered eyespots. Our results provide the first experimental evidence to support the proposal that multi-layered eyespots act as interference reflectors in photoaxis of green algae.     

Control of phobic behavioral responses by rhodopsin-induced photocurrents in Chlamydomonas.

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).     

Characterization of the Eyespot Regions of "Blind"Chlamydomonas Mutants after Restoration of Photophobic Responses

Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2–4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance. Consistent with these findings, several laboratories have shown that the full range of phototactic responses can be reconstituted in FN68 and CC-2359, but that negative phototaxis requires a significantly stronger light stimulus in the latter strain. We confirm the suggestion that the eyespot is not necessary for the photophobic response, and is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Furthermore, the locus of reconstitution of the functional receptor is not the eyespot. Because of the definitive demonstration of the absence of the eyespot in CC-2359, however, the eyespot may play a role in negative phototaxis.     

Intrinsic difference in beat frequency between the two flagella of Chlamydomonas reinhardtii

The flagellar beat frequency of the biflagellated green alga Chlamydomonas reinhardtii was measured by fast Fourier transform analysis of the light intensity fluctuation in microscope images of swimming cells. Live cells had a mean beat frequency of 48-53 Hz at 20 degrees C. However, detergent-extracted "cell models," when reactivated in the presence of 1 mM ATP, appeared to have two different beat frequencies of about 30 and 45 Hz. Measurements in cell models in which only one of the two flagella was beating indicated that the lower and higher frequencies most likely represented the beat frequency of the flagellum nearer to the eyespot (the cis-flagellum) and that of the flagellum farther from it (the trans-flagellum), respectively. In live cells also, the trans-flagellum beat at a frequency about 30% higher than that of the cis-flagellum when the cells were rendered uniflagellated by mechanical treatment, whereas both flagella beat at the frequency of the cis-flagellum under normal conditions. These observations suggest that the two flagella of Chlamydomonas have different intrinsic beat frequencies but that they are somehow synchronized when beating together on a live swimming cell.     

Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii.

The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.     

Polarizing microscopy of eyespot of Chlamydomonas: in situ observation of its location, orientation, and multiplication

The eyespot in the cell of Chlamydomonas reinhardtii has been found to appear as a bright spot under the cross polar setting of a polarizing microscope. This was confirmed by isolating the eyespot from a homogenate of wall-deficient mutant cw-15, and by observing it under a polarizing microscope. Thus, the eyespot was proved to be a strongly birefringent body. Next, gametes (mt+ and mt-) of 137c strain were prepared by cultivating it in a low-nitrogen (NH4Cl) medium. Here, every cell shows only one (and never more than one) birefringent spot. The birefringent eyespot was located always near the surface on the "equator," that is, at the farthest point from the "meridionial" cell-axis that is defined as the bisector of the two flagella projected out from the cell surface. It was shown, in addition, that the optic axis of this birefringent eyespot is oriented in the cell always along the parallel direction of the cell axis defined above. Thus, the polarizing microscopy has been shown to provide a powerful method for in vivo, in situ pursuit of the eyespot of Chlamydomonas.     

Effect of Light Intensity on the Phase and Period Response Curves in the Nocturnal Field Mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Chlamyrhodopsin represents a new type of sensory photoreceptor.

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.     

Sinusoidal and delta function responses of visual cells of the Limulus eye

Dynamic responses of visual cells of the Limulus eye to stimuli of sinusoids and narrow pulses of light superimposed on a nonzero mean level have been obtained. Amplitudes and phase angles of averaged sinusoidal generator potential are plotted with respect to frequency of intensity modulation for different mean levels of light adaptation. At frequencies above 10 CPS, generator potential amplitudes decrease sharply and phase lag angle increases. At frequencies below 1 CPS, amplitude decreases. A maximum of amplitude in the region of 1 to 2 CPS is apparent with increased mean intensity. The generator potential responses are compared with those of differential equation models. Variation of gain with mean intensity for incremental stimuli is consistent with logarithmic sensitivity of the photoreceptor. Frequency response of the photoreceptor derived from narrow pulses of light predicts the frequency response obtained with sinusoidal stimuli, and the photoreceptor is linear for small signals in the light-adapted state.     

Effect of light intensity on the phase and period response curves in the nocturnal field mouse Mus booduga

The effect of light intensity on the phase response curve (PRC) and the period response curve (τRC) of the nocturnal field mouse Mus booduga was studied. PRCs and τRCs were constructed by exposing animals free-running in constant darkness (DD), to fluorescent light pulses (LPs) of 100 lux and 1000 lux intensities for 15min duration. The waveform of the PRCs and τRCs evoked by high light intensity (1000 lux) stimuli was significantly different compared to those constructed using low light intensity (100 lux). Moreover, a weak but significant correlation was observed between phase shifts and period changes when light stimuli of 1000 lux intensity were used; however, the phase shifts and period changes in the 100 lux PRC and τRC were not correlated. This suggests that the intensity of light stimuli affects both phase and period responses in the locomotor activity rhythm of the nocturnal field mouse M. booduga. These results indicate that complex mechanisms are involved in entrainment of circadian clocks, even in nocturnal rodents, in which PRC, τRC, and dose responses play a significant role.     

Eyespot placement and assembly in the green alga Chlamydomonas

The eyespot organelle of the green alga Chlamydomonas allows the cell to phototax toward (or away) from light to maximize the light intensity for photosynthesis and minimize photo-damage. At cytokinesis, the eyespot is resorbed at the cleavage furrow and two new eyespots form in the daughter cells 180 degrees from each other. The eyespots are positioned asymmetrically with respect to the microtubule cytoskeleton. Eyespots are assembled from all three chloroplast membranes and carotenoid-filled granules, which form a sandwich structure overlaid by the tightly apposed plasma membrane. This review describes (1) my interest in cellular asymmetry and organelle biology, (2) isolation of mutations that describe four genes governing eyespot placement and assembly, (3) the characterization of the EYE2 gene, which encodes a thioredoxin superfamily member, and (4) the characterization of the MIN1 gene, which is required for the layered organization of granules and membranes in the eyespot. BioEssays 25:410-416, 2003.     

In vitro identification of rhodopsin in the green alga Chlamydomonas

The unicellular alga Chlamydomonas can detect both intensity and direction of the ambient light and adjust its swimming speed and direction accordingly. On the basis of physiological experiments, the functional photoreceptor for this visual process has recently shown to be a rhodopsin. We here report the in vitro identification of endogenous retinal and a rhodopsin in Chlamydomonas cell extracts and purified membrane preparations. The rhodopsin absorption spectrum has fine structure with the maximum at 495 nm and matches the action spectra for the behavioral light responses. The rhodopsin can be bleached and subsequently reconstituted with exogenous retinal. Labeling with [3H]retinal occurs in the final preparation only with a single protein with a molecular weight of 32,000. We conclude that this protein is the visual photoreceptor in Chlamydomonas.     

Light Adaptation in the Ventral Photoreceptor of Limulus

Light adaptation in both the ventral photoreceptor and the lateral eye photoreceptor is a complex process consisting of at least two phases. One phase, which we call the rapid phase of adaptation, occurs whenever there is temporal overlap of the discrete waves that compose a light response. The recovery from the rapid phase of adaptation follows an exponential time-course with a time constant of approximately 75 ms at 21°C. The rapid phase of adaptation occurs at light intensities barely above discrete wave threshold as well as at substantially higher light intensities with the same recovery time-course at all intensities. It occurs in voltage-clamped and unclamped photoreceptors. The kinetics of the rapid phase of adaptation is closely correlated to the photocurrent which appears to initiate it after a short delay. The rapid phase of adaptation is probably identical to what is called the "adapting bump" process. At light intensities greater than about 10 times discrete wave threshold another phase of light adaptation occurs. It develops slowly over a period of ½ s or so, and decays even more slowly over a period of several seconds. It is graded with light intensity and occurs in both voltage-clamped and unclamped photoreceptors. We call this the slow phase of light adaptation.     

Linear Relations between Stimulus Amplitudes and Amplitudes of Retinal Action Potentials from the Eye of the Wolf Spider

Incremental photic stimuli have been used to elicit small amplitude retinal action potentials from light-adapted ocelli of the wolf spider, Lycosa baltimoriana (Keyserling) in order to see whether or not the amplitudes of these potentials are linearly related to the stimulus amplitudes. Sine wave variations of light intensity around a mean elicit sine wave variations in potential which contain inappreciable harmonics of the stimulus frequency and whose amplitudes are linearly related to the stimulus amplitudes. Likewise, the responses to the first two periodic Fourier components of incremental rectangular wave stimuli of variable duty cycle are directly proportional to the amplitudes of these components and have phases dependent only on the frequencies and phases of these components. Thirdly, a linear transfer function can be found which describes the amplitudes and phases of responses recorded at different frequencies of sine wave stimulation and this transfer function is sufficient to predict the responses to incremental step stimuli. Finally, it is shown that flash response amplitudes are linearly related to incremental flash intensities at all levels of adaptation. The relations of these linear responses to non-linear responses and to physiological mechanisms of the eye are discussed.