Selective inhibition of flagellar activity in Chlamydomonas by nickel.

Flagellar activity in the biflagellate chlorophyte Chlamydomonas reinhardtii is selectively inhibited by Ni2+ or by treatment with Ca2+-chelating agents. Inhibitions of swimming speed, geotaxis, phototaxis, and pattern swimming result from qualitative and quantitative losses in the activity of individual flagella and in the coordination of activity between the 2 flagella of each cell. Addition of Ca2+ (a) prevents inhibition and (b) restores normal flagellar activity in inhibited cells. Mg2+ is partially effective in reversal of inhibition. Other ions do not cause similar inhibition or reversal of nickel inhibition. The characteristics of inhibition and reversal suggest that the primary target for nickel is a component of the flagellar apparatus, and that this component uses Ca2+ to perform its normal function in the regulation of flagellar activity. A 2nd target for nickel is a Ca-requiring process specific to phototaxis (and not involved in the photophobic response).     

Effects of mechanical and chemical disruption on the ATP-phosphohydrolase activity and ultrastructure of sperm flagella.

The Ca²⁺- and Mg²⁺-ATP-phosphohydrolase (ATPase) activities measurable in suspensions of and extracts from bull epididymal spermatozoa flagella, sonicated for 2 min at 8 W, were compared with those of control flagella after 0, 3, and 18 h dialysis against an alkaline 0.5 M KCl extracting solution. Activity was measured in the absence of and in the presence of oligomycin. The effects of sonication and extraction on structural components within the flagella were visualized electron microscopically. Sonication caused fragmentation of flagella, extensive disruption of mitochondria and an immediate (0 h) increase in both Ca²⁺- and Mg²⁺-ATP-ase activity. Prolonged dialysis resulted in solubilization of specific flagellar structures, partial disruption of mitochondrial integrity, and increases in ATPase activity. Mg²⁺-ATPases of flagellar suspensions and extracts were greater than Ca²⁺-ATPases, and a part of this Mg²⁺-ATPase activity was inhibited by oligomycin. Therefore, Mg²⁺-ATPases from disrupted mitochondria contribute to the Mg²⁺-ATPase activities measurable in suspensions of and extracts from bull sperm flagella. This study emphasizes the necessity of evaluating the effects of the mechanical and chemical treatments used in fractionating cells before interpreting the biochemical information derived from their isolated components.     

Effect of calcium on phototactic orientation of individual cryptomonas cells

The effect of Ca²⁺ ions on the phototactic orientation of Cryptomonas cells was determined by observation of the direction of swimming of individual cells using infrared video-micrography. When 2 mM EGTA was added to the medium, changes in the swimming direction of cells became smaller, consequently the phototactic orientation was inhibited. The EGTA-induced inhibition was almost completely overcome by the addition of 2 mM CaCl₂. Consequently the presence of Ca²⁺ ions appears to be crucial for the orientation that is necessary during phototaxis of Cryptomonas cells.     

Inverse relationship of Ca<Superscript>2+</Superscript>-dependent flagellar response between animal sperm and prasinophyte algae

Symmetry/asymmetry conversion of eukaryotic flagellar waveform is caused by the changes in intracellular Ca²⁺. Animal sperm flagella show symmetric or asymmetric waveform at lower or higher concentration of intracellular Ca²⁺, respectively. In Chlamydomonas, high Ca²⁺ induces conversion of flagellar waveform from asymmetric to symmetry, resulting in the backward movement. This mirror image relationship between animal sperm and Chlamydomonas could be explained by the distinct calcium sensors used to regulate the outer arm dyneins (Inaba 2015). Here we analyze the flagellar Ca²⁺-response of the prasinophyte Pterosperma cristatum, which shows backward movement by undulating four flagella, the appearance similar to animal sperm. The moving path of Pterosperma shows relatively straight in artificial seawater (ASW) or ASW in the presence of a Ca²⁺ ionophore A23187, whereas it becomes circular in a low Ca²⁺ solution. Analysis of flagellar waveform reveals symmetric or asymmetric waveform propagation in ASW or a low Ca²⁺ solution, respectively. These patterns of flagellar responses are completely opposite to those in sperm flagella of the sea urchin Anthocidaris crassispina, supporting the idea previously proposed that the difference in flagellar response to Ca²⁺ attributes to the evolutional innovation of calcium sensors of outer arm dynein in opisthokont or bikont lineage.     

Distinct regulation of two flagella by calcium during chemotaxis of male gametes in the brown alga Mutimo cylindricus (Cutleriaceae,Tilopteridales)

Brown algal male gametes show chemotaxis to the sex pheromone that is released from female gametes. The chemotactic behavior of the male gametes is controlled by the changes in the beating of two flagella known as the anterior and posterior flagellum. Our previous study using Mutimo cylindricus showed that the sex pheromone induced an increment in both the deflection angle of the anterior flagellum and sustained unilateral bend of the posterior flagellum, but the mechanisms regulating these two flagellar waveforms were not fully revealed. In this study, we analyzed the changes in swimming path and flagellar waveforms with a high-speed recording system under different calcium conditions. The extracellular Ca²⁺ concentration at 10⁻³ M caused an increment in the deflection angle of the anterior flagellum only when ionomycin was absent. No sustained unilateral bend of the posterior flagellum was induced either in the absence or presence of ionomycin in extracellular Ca²⁺ concentrations below 10⁻² M. Real-time Ca²⁺ imaging revealed that there is a spot near the basal part of anterior flagellum showing higher Ca²⁺ than in the other parts of the cell. The intensity of the spot slightly decreased when male gametes were treated with the sex pheromone. These results suggest that Ca²⁺-dependent changes in the anterior and posterior flagellum are regulated by distinct mechanisms and that the increase in the anterior flagellar deflection angle and sustained unilateral bend of the posterior flagellum may not be primarily induced by the Ca²⁺ concentration.     

A backward swimming mutant of Chlamydomonas reinhardii

A backward swimming mutant (RL-10) was isolated from Chlamydomonas reinhardii. In contrast to the wild-type flagellum which usually displays a ciliary type beating pattern, the flagella in the RL-10 cells always propagated such undulating waves as found in sperm flagella. This abnormal beating pattern was maintained after the cell was demembranated by a non-ionic detergent (Nonidet P40) and reactivated with ATP. Reactivated axonemes (demembranated flagella) of the wild-type cells changed the beating pattern from the ciliary type to the flagellar type when the Ca²⁺ concentration was increased from 10⁻⁷ to 10⁻⁶ M. However, the RL-10 axonemes did not show such a Ca-dependent change in the beating pattern. Hence the RL-10 flagella might carry defects in the controlling mechanisms of flagellar beating pattern, at sites other than the membrane.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

Effect of external factors on phototaxis of Chlamydomonas reinhardtii

The effects of several cations on phototaxis of Chlamydomonas reinhardtii have been studied with the aid of an automated phototaxis monitoring device, coupled with a continuous culture. Sodium, potassium and magnesium ions, if added to the complete nutrient medium, have only slight effects on phototaxis at lower concentrations (10^-3 mol), but inhibit at higher concentrations (10^-2 mol). This inhibitory effect is not specific because motility is also impaired. Addition of 10^-3 mol calcium enhances the phototactic reaction for some hours, but then the stimulation decreases gradually. Addition of 10^-2 mol calcium causes strong inhibition. However, the reactivity recovers gradually during the following hours. If 10^-3 mol potassium which does not influence phototaxis if added alone is applied simultaneously with calcium, the stimulation by calcium is enhanced. By the addition of 5·10^-4—2·10^-3 mol Ca²⁺ or Ca²⁺+K⁺ cicadian rhythms with an average period length of 24 h are initiated which damp out after 1–2 weeks. If the cells are grown in a calcium deficient medium or if calcium is removed, phototactic activity decreases to very low reaction values or to zero, but is drastically increased immediately after the addition of calcium. The stimulatory effect of Ca²⁺ ions is specific. Ca²⁺ cannot be fully substituted by Ba²⁺ or Sr²⁺, and phototaxis is reversibly inhibited by lanthanum which is known to inhibit the calcium pump.     

Induction of deflagellation by various local anesthetics in Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,Chlorophyceae)

Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod‐like shape to a disk‐like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell‐wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1‐h EC₅₀) for flagellar excision are as follows: dibucaine (1.37 × 10^?5 M) < tetracaine (3.16 × 10^?5 M) < bupivacaine (4.25 × 10^?4 M) < procaine (2.02 × 10^?3 M) < lidocaine (3.61 × 10^?3 M). In all cases, Ca²⁺ depletion from the solution inhibited flagellar excision. However, Ca²⁺‐channel blockers, IP3 receptor antagonists, and inhibitors of phospholipase C did not prevent excision. We suggest that the local anesthetics induce flagellar excision by increasing the fluidity of the flagellar/cell membrane, thereby allowing extracellular Ca²⁺ to flow into the cell and cause flagellar excision.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn^{2 +} (up to 2 mM) and activated above this concentration. Ca^{2 +} inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Calcium lonophore-induced stimulation of secretory activity in Chlamydomonas reinhardii

The cell-wall lysin in gametes from Chlamydomonas reinhardii which under normal mating conditions is activated by flagellar cell contact was found to be susceptible to stimulation by the antibiotic ionophore A 23187 provided that CA²⁺ was included in the medium. Ionophore-induced release of the cell-wall lysin did not deend on the mating type or the gametic state of the cells. Vegetative cells which normally do not exhibit any mating capacity reacted with cell-wall lysis like gametes stimulated by cell contact.Ionophore-dependent Ca²⁺-transfer across the cell membranes generated a signal for cell-wall lysis only in cells with intact flagella. Deflagellated cells did not respond to A 23187 before regeneration of the amputated organelles. Another indication for a possible role of flagella in Ca²⁺-mediated cell-wall lysis was obtained from a conditional flagellar-assembly mutant of C. reinhardii which had been isolated and described by Huang et al. (1977). Upon shift-up the mutant strain immediately became unresponsive to A 23187 and Ca²⁺ but regained susceptibility soon after being retransferred to permissive conditions (20°C).     

Inactivation of Ca2+-induced ciliary reversal by high-salt extraction in the cilia of Paramecium

Intracellular Ca²⁺ induces ciliary reversal and backward swimming in Paramecium. However, it is not known how the Ca²⁺ signal controls the motor machinery to induce ciliary reversal. We found that demembranated cilia on the ciliated cortical sheets from Paramecium caudatum lost the ability to undergo ciliary reversal after brief extraction with a solution containing 0.5 M KCl. KNO₃, which is similar to KCl with respect to chaotropic effect; it had the same effect as that of KCl on ciliary response. Cyclic AMP antagonizes Ca²⁺-induced ciliary reversal. Limited trypsin digestion prevents endogenous A-kinase and cAMP-dependent phosphorylation of an outer arm dynein light chain and induces ciliary reversal. However, the trypsin digestion prior to the high-salt extraction did not affect the inhibition of Ca²⁺-induced ciliary reversal caused by the high-salt extraction. Furthermore, during the course of the high-salt extraction, some axonemal proteins were extracted from ciliary axonemes, suggesting that they may be responsible for Ca²⁺-induced ciliary reversal.     

Vision in microalgae

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca²⁺ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca²⁺ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca²⁺ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals. Received: 10 March 1997 / Accepted: 18 April 1997     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Biophysical effects of the natural product euplotin C on the <Emphasis Type="Italic">Paramecium</Emphasis> membrane

The effect of euplotin C—a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus—on the voltage-dependent Ca²⁺ channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca²⁺ concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca²⁺ influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca²⁺ channel blockers, we demonstrate that euplotin C modulates Ca²⁺ channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca²⁺ channel functions through the reduction of cell membrane fluidity.     

Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia

Intracellular activities of K⁺, H⁺, Mg²⁺, Ca²⁺, and Cl^?, measured with ion selective microelectrodes in the oocyte and the nurse cells in ovarian follicles of Hyalophora cecropia, indicated that a Ca²⁺ current is a key component of the electrical potential that is maintained across the intercellular bridges connecting these two cells. In vitellogenic follicles, Ca²⁺ activity averaged 650 nM in the oocyte and 190 nM in the nurse cells, whereas activities of the other ions studied differed between these cells by no more than 6%. Incubation in 200 μM ammonium vanadate caused a reversal of electrical potential from 8.3 mV, nurse cell negative, to 3.0 mV, oocyte negative, and at the same time the Ca²⁺ gradient was reversed: activities rose to an average 3.0 μM in the nurse cells and 1.6 μM in the oocyte, whereas transbridge ratios of the other cations remained at 0–3%. In immature follicles that had not yet initiated their transbridge potentials, Ca²⁺ activities averaged ～? 2 μM in both oocyte and nurse cells. The results suggest that vitellogenic follicles possess a vanadatesensitive Ca²⁺ extrusion mechanism that is more powerful in the nurse cells than in the oocyte. © 1994 Wiley-Liss, Inc.     

Nifedipine impairs neutrophil respiratory burst by a mechanism other than calcium channel blockade

Summary Superoxide production by mice neutrophils was inhibited by nifedipine exposure in a dose dependent manner. The inhibition of Ca²⁺ uptake elicited by nifedipine did not appear to account for the observed effect as the extracellular Ca²⁺ enrichment and depletion did not produce a significant reversal of the inhibition. Cytosolic free Ca²⁺ as measured by Quin 2AM fluorescence did not show any significant change, indicating that the effect was independent of the inhibition of Ca²⁺ influx. In addition nifedipine caused a significant inhibition (p < 0.01) in NADPH oxidase activity. Our data indicates that nifedipine inhibits superoxide production independent of inhibiting Ca²⁺ inflow and supports the hypothesis that Ca²⁺ antagonists affect cellular functions by non Ca²⁺ mediated process as well.     

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.     

Effects of cGMP,calcium and reversible methylation on sensory signal processing in halobacteria

Cyclic GMP and Ca²⁺ change the following parameters of sensory behavior of Halobacterium halobium in opposite directions: the interval between two spontaneous reversals of the swimming direction, the phases of increasing and decreasing responsiveness to light stimuli during an interval and the length of the refractory period. Inhibition of the methylation of membrane proteins by blocking the methyltransferase increases the attractant response and prolongs the time during which successive stimuli are integrated. A decrease of the external Ca²⁺ concentration, which activates methylation, has the opposite effect. Inhibition of methylation also prevents the entrainment of the reversal frequency by rhythmic repellent stimuli. The result suggest that cGMP and Ca²⁺ may be antagonistic components of an oscillator, which generates the autonomous rhythm of flagellar reversals, while methylation determines the life-time of sensory signals at an integration link prior to the oscillator, and thereby allows adaptation. Ca²⁺ is thought to act on the methylation system in a feedback loop.     

Mechanosensitive Channels in the Cell Body of Chlamydomonas

Mechanosensitive channels appear ubiquitous but they have not been well characterized in cells directly responding to mechanical stimuli. Here, we identified tension-sensitive channel currents on the cell body of Chlamydomonas, a protist that shows a marked behavioral response to mechanical stimulation. When a negative pressure was applied to the cell body with a patch clamp electrode, single-ion-channel currents of 2.4 pA in amplitude were observed. The currents were inhibited by 10 μm gadolinium, a general blocker of mechanosensitive channels. The currents were most likely due to Ca²⁺ influxes because the current was absent in Ca²⁺-free solutions and the reversal potential was 98 mV positive to the resting potential. The distribution of channel-open times conformed to a single exponential component and that of closed times to two exponential components. This mechanosensitive channel was similar to the one found in the flagella in the following respects: both channels were inhibited by Gd³⁺ at 10 μm but not at 1 μm; both passed Ca²⁺ and Ba²⁺; their kinetic parameters for channel opening were similar. These observations raise the possibility that identical mechanosensitive channels may function both in the behavioral control through the mechanoreception by the flagella and in the regulation of cellular physiology in response to mechanical perturbation on the cell body. Received: 13 May 1998/Revised: 2 September 1998