Inner arm dynein 1 is essential for Ca++-dependent ciliary reversals in Tetrahymena thermophila

Cilia in many organisms undergo a phenomenon called ciliary reversal during which the cilia reverse the beat direction, and the cell swims backwards. Ciliary reversal is typically caused by a depolarizing stimulus that ultimately leads to a rise in intraciliary Ca++ levels. It is this increase in intraciliary Ca++ that triggers ciliary reversal. However, the mechanism by which an increase in intraciliary Ca++ causes ciliary reversal is not known. We have previously mutated the DYH6 gene of Tetrahymena thermophila by targeted gene knockout and shown that the knockout mutants (KO6 mutants) are missing inner arm dynein 1 (I1). In this study, we show that KO6 mutants do not swim backward in response to depolarizing stimuli. In addition to being unable to swim backwards, KO6 mutants swim forward at approximately one half the velocity of wild-type cells. However, the ciliary beat frequency in KO6 mutants is indistinguishable from that of wild-type cells, suggesting that the slow forward swimming of KO6 mutants is caused by an altered waveform rather than an altered beat frequency. Live KO6 cells are also able to increase and decrease their swim speeds in response to stimuli, suggesting that some aspects of their swim speed regulation mechanisms are intact. Detergent-permeabilized KO6 mutants fail to undergo Ca++-dependent ciliary reversals and do not show Ca++-dependent changes in swim speed after MgATP reactivation, indicating that the axonemal machinery required for these responses is insensitive to Ca++ in KO6 mutants. We conclude that Tetrahymena inner arm dynein 1 is not only an essential part of the Ca++-dependent ciliary reversal mechanism but it also may contribute to Ca++-dependent changes in swim speed and to the formation of normal waveform during forward swimming.     

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

In many organisms, depolarizing stimuli cause an increase in intraciliary Ca2+, which results in reversal of ciliary beat direction and backward swimming. The mechanism by which an increase in intraciliary Ca2+ causes ciliary reversal is not known. Here we show that Tetrahymena cells treated with okadaic acid or cantharidin to inhibit protein phosphatases do not swim backwards in response to depolarizing stimuli. We also show that both okadaic acid and cantharidin inhibit backward swimming in reactivated, extracted cell models treated with Ca2+. In contrast, treatment of whole cells or extracted cell models with protein kinase inhibitors has no effect on backward swimming. These results suggest that a component of the axonemal machinery is dephosphorylated during ciliary reversal. The phosphorylation state of inner arm dynein 1 (I1) was determined before and after cells were exposed to depolarizing conditions that induce ciliary reversal. An I1 intermediate chain is phosphorylated in forward swimming cells but is dephosphorylated in cells treated with a depolarizing stimulus. Our results suggest that dephosphorylation of Tetrahymena inner arm dynein 1 may be an essential part of the mechanism of ciliary reversal in response to increased intraciliary Ca2+.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Certain components of lettuce juice modify the thermoresponse of the ciliated protozoan Blepharisma japonicum

When the cells of Blepharisma cultured in lettuce juice were transferred to media containing lettuce juice with temperature gradient of 20–30°C, they accumulated in a region corresponding to about 25°C. The cells washed in a saline solution, however, accumulated in the region above 30°C. The results indicate that certain components contained in lettuce juice change the thermosensitivity of the cells. When the temperature was suddenly changed, a transient decrease or increase in the frequency of ciliary reversal was observed. The response of the cells incubated in a saline solution without lettuce juice was different from those in the saline solution containing lettuce juice above 25°C. Above 25°C, the cells incubated in a saline solution without lettuce juice responded by increased frequency of ciliary reversal only to step-down in temperature and by repression of ciliary reversal to a step-up in temperature, while the cells incubated in the same saline solution containing lettuce juice responded by increased frequency of ciliary reversal to a step-up in temperature and by repression of ciliary reversal to a step-down in temperature. The thermal response was examined in bisected fragments of the cells. The difference in response between the saline solution without lettuce juice and the solution containing lettuce juice was detected only in posterior fragments above 25°C. Above 25°C, the posterior fragments incubated in the solution without lettuce juice did not respond to sudden temperature changes, whereas the fragments kept in the medium containing lettuce juice responded (step-up thermophobic response and step-down repression of ciliary reversal) in the presence of lettuce juice.     

The involvement of cyclic adenosine monophosphate in the control of schistosome miracidium cilia

This study examined the possible involvement of cyclic adenosine monophosphate (cAMP) in the control of ciliary action of Schistosoma mansoni miracidia. Miracidia immobilized in hypertonic NaCl solution were treated with 3 compounds that are known to increase intracellular cAMP concentrations. Forskolin, at a concentration of 50 microM, induced 50.1% of the miracidia to swim in hypertonic solution. The corresponding values obtained for 3-isobutyl-1-methylxanthine (IBMX) at 1 mM and 8-bromo-cAMP at 10 mM were 42.2 and 50.4%, respectively. The motility-enhancing effect of these compounds was dose dependent. Nevertheless, the swimming speed of miracidia activated in this way was only 10% of that observed in artificial pond water (APW). Cholera toxin had no apparent effect on miracidia swimming in hypertonic NaCl solution. Likewise, swimming in APW treated with forskolin at 50 microM, IBMX at 1 mM, or 8-bromo-cAMP at 10 mM did not induce any apparent change in motility. Miracidia swimming in APW were then treated with 3 compounds that decrease the intracellular concentration of cAMP. MDL-12,330A, at a concentration of 250 microM, caused a dramatic decrease in swimming over a period of 1 hr. Likewise, SQ22536 and imidazole, at concentrations of 20 and 50 mM, respectively, caused 36.5 and 73.4% decreases in swimming under the same conditions. Finally, inhibitors of cAMP-dependent protein kinase, i.e., PKI(14-22)amide, H89, and H88, completely inhibited miracidia swimming in APW at concentrations of 25, 50, and 100 microM, respectively. These results suggest that cAMP and cAMP-dependent protein kinase are involved in osmosis-controlled ciliary motion of schistosome miracidia.     

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH.

Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0. Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium. The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi). The cytosolic acidification by M. taylorii at alkaline pH values was accompanied by K+ extrusion. The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values. High external K+ concentrations (210 mM or higher) inhibited the growth of M. taylorii at alkaline pH values, perhaps by preventing K+ extrusion. Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol. The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M. taylorii only at alkaline pH values. Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis. Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values. Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl. The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M. taylorii growing at alkaline pH values.     

环境pH及渗透压对玫瑰无须鲃精子运动的影响

以玫瑰无须鲃Puntius conchonius精子为材料,应用计算机辅助精子分析系统(CASA),研究了精子在不同pH和不同渗透压的NaCl溶液中的运动百分率、运动时间和运动速率。结果表明,酸性(pH<7.0)或碱性较强(pH>9.0)的溶液均不利于精子运动,而弱碱性(pH7.5～8.5)的溶液较适合精子的运动;在渗透压较低(<75mOsm/kg)或较高(>175mOsm/kg)的NaCl溶液中,精子的运动时间和运动百分率都显著较100～150mOsm/kg渗透压溶液中的短或低(P<0.05);而运动时间最长,并且运动百分率最高的条件为pH8.0和125mOsm/kg渗透压的溶液环境。     

An automated assay for quantifying the swimming behavior of Paramecium and its use to study cation responses

Paramecium tetraurelia is a ciliated protist that alters its swimming behavior in response to various stimuli. Like the sensory responses of many organisms, these responses in Paramecium show adaptation to continued stimulation. For quantitative studies of the initial response to stimulation, and of the time course of adaptation, we have developed a computerized motion analysis assay that can detect deviations from the normal swimming pattern in a population of cells. The motion of an average of ten cells was quantified during periods ranging from 15 to 60 seconds, with a time resolution of 1/15 seconds. During normal forward swimming, the maximum deviation from a straight-line path was less than 17 degrees. Path deviations above this threshold value were defined as changes in swimming direction. The percentage of total path time that cells spent deviating from forward swimming was defined as percent directional changes (PDC). This parameter was used to construct dose-response curves for the behavioral effects of various externally added cations known to induce behavioral changes and also to show the time course of adaptation to a depolarizing K+ stimulus. This assay is a valuable tool for studies of chemoeffectors or mutations that alter the swimming behavior of Paramecium and may also be applicable to other motile organisms.     

Membrane potential responses of paramecium caudatum to external Na+

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.     

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.     

Temperature-Sensitive Responses in Blepharisma

Temperature sensitivity of Blepharisma cultured at 23°C was investigated in a temperature range between 18.5°C and 33.5°C. The cells accumulated in an optimal temperature (ca. 27°C) region when they were placed in a chamber with a temperature gradient, although a certain population of the cells accumulated at much higher temperatures. The quantitative analysis of behavioral responses exhibited by the cells revealed that three types of thermal response were responsible for thermoaccumulation of the cells in an optimal temperature: (1) an increase in the frequency of thermophobic response in the cells swimming away from the optimal temperature region; (2) acceleration of forward swimming velocity of the cells swimming toward the optimal temperature region; and (3) higher frequency of spontaneous ciliary reversal of the cells in higher temperature regions.     

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.     

Electrophysiological Control of Reversed Ciliary Beating in Paramecium

Quantitative relations between ciliary reversal and membrane responses were examined in electrically stimulated paramecia. Specimens bathed in 1 mM CaCl₂, 1 mM KCl, and 1 mM Tris-HCl, pH 7.2, were filmed at 250 frames per second while depolarizing current pulses were injected. At current intensities producing only electrotonic shifts the cilia failed to respond. Stimuli which elicited a regenerative response were followed by a period of reversed ciliary beating. With increasing stimulus intensities the latency of ciliary reversal dropped from 30 to 4 ms or less, and the duration of reversal increased from 50 ms to 2.4 s or more; the corresponding regenerative responses increased in amplitude and rate of rise. With progressively larger intracellular positive pulses, electric stimulation became less effective, producing responses with a progressive increase in latency and decrease in duration of reversed beating of the cilia. When 100-ms pulses shifted the membrane potential to +70 mV or more, ciliary reversal was suppressed until the end of the pulse. "Off" responses then occurred with a latency of 2–4 ms independent of further increases in positive potential displacement. These results suggest that ciliary reversal is coupled to membrane depolarization by the influx of ions which produces the regenerative depolarization of the surface membrane. According to this view suppression of the ciliary response during stimulation occurs when the membrane potential approaches the equilibrium potential of the coupling ion, thereby retarding its influx. Previous data together with the present findings suggest that this ion is Ca²⁺.     

Okadaic acid, an inhibitor of protein phosphatase 1 in Paramecium, causes sustained Ca2(+)-dependent backward swimming in response to depolarizing stimuli.

Backward swimming is a stereotypic behavioural response of Paramecium. It is triggered by depolarizing stimuli, which open calcium channels in the excitable ciliary membrane. The influx of Ca2+ causes the reversal of ciliary beat and initiates backward swimming. Here, we demonstrate that the protein phosphatase inhibitor okadaic acid does not affect the normal forward swimming pattern of Paramecium, but greatly extends the duration of backward swimming as initiated by depolarization caused by a rise in extracellular K+. Chelation of external Ca2+ results in an immediate resumption of forward swimming. The results suggest that the voltage-operated calcium channel is inactivated by a dephosphorylation event, and that okadaic acid blocks this dephosphorylation without any effect on the motile apparatus of the cilia. In addition, Paramecium is unique among eukaryotic cells, in that okadaic acid inhibits just one protein phosphatase, namely a type 1 enzyme, 75% of which is tightly associated with the excitable ciliary membrane. The type 2A protein phosphatases in Paramecium are unaffected by okadaic acid. The results indicate that protein phosphatase 1 is the enzyme responsible for the dephosphorylation and closure of the calcium channel in Paramecium.     

The membrane potential ofMethanobacterium thermoautotrophicum under different external conditions

The membrane potential (delta psi) of whole cells of Methanobacterium thermoautotrophicum strain delta H was estimated under different external conditions using a TPP(+)-sensitive electrode. The results show that the delta psi values of M. thermoautotrophicum at alkaline pHout (8.5) are comparable with delta psi values under slightly acidic conditions (pH 6.8; 230 and 205 mV, respectively). On the other hand, the size of colonies on Petri dishes was remarkably smaller at pH 8.5 than at 6.8. The delta psi was insensitive to relevant ATPase inhibitors. At pH 6.8, the protonophore 3,3',4',5-tetrachlorosalicylanilide (TCS) strongly inhibited delta psi formation and ATP synthesis driven by methanogenic electron transport. On the other hand, at pH 8.5 the CH4 formation and ATP synthesis were insensitive to TCS and a protonophore-resistant delta psi of approximately 150 mV was determined. The finding of a protonophore-resistant delta psi at pH 8.5 indicates that at alkaline pHout these cells can switch from H(+)-energetics to Na(+)-energetics, when the delta [symbol: see text] H+ becomes limited. The results strongly support the hypothesis that at alkaline pHout Na+ ions might fully substitute for H+ in these cells as the coupling ions.     

Na<Superscript>+</Superscript> and flagella-dependent swimming of alkaliphilic <Emphasis Type="Italic">Bacillus pseudofirmus</Emphasis> OF4: a basis for poor motility at low pH and enhancement in viscous media in an “up-motile” variant

Flagella-based motility of extremely alkaliphilic Bacillus species is completely dependent upon Na⁺. Little motility is observed at pH values < ∼8.0. Here we examine the number of flagella/cell as a function of growth pH in the facultative alkaliphile Bacillus pseudofirmus OF4 and a derivative selected for increased motility on soft agar plates. Flagella were produced by both strains during growth in a pH range from 7.5 to 10.3. The number of flagella/cell and flagellin levels of cells were not strongly dependent on growth pH over this range in either strain although both of these parameters were higher in the up-motile strain. Assays of the swimming speed indicated no motility at pH < 8 with 10 mM Na⁺, but significant motility at pH 7 at much higher Na⁺ concentrations. At pH 8–10, the swimming speed increased with the increase of Na⁺ concentration up to 230 mM, with fastest swimming at pH 10. Motility of the up-motile strain was greatly increased relative to wild-type on soft agar at alkaline pH but not in liquid except when polyvinylpyrrolidone was added to increase viscosity. The up-motile phenotype, with increased flagella/cell may support bundle formation that particularly enhances motility under a subset of conditions with specific challenges.     

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.     

Cloning and characterization of vmaA,the gene encoding a 69-kDa catalytic subunit of the vacuolar H+-ATPase during alkaline pH mediated growth of Aspergillus oryzae

Screening of a cDNA library constructed under alkaline pH mediated growth of Aspergillus oryzae implicated a vacuolar H+-ATPase gene (vmaA) as a putative candidate involved in alkaline pH adaptation. A. oryzae vmaA genomic DNA extended to 2072 bp including three introns and encoded a protein of 605 amino acids. VmaAp was homologous to Vma-1p from Neurospora crassa (71%), Vma1p from Saccharomyces cerevisiae (69%) and ATP6A2 from human (49%). The vmaA cDNA complemented S. cerevisiae V-ATPase disrupted strain (Deltavma1) was viable at alkaline pH 8.0 and in the presence of CaCl(2) (100 mM). Northern analysis revealed an enhanced expression of vmaA during growth of A. oryzae in alkaline medium (pH 10.0). The A. oryzae vmaA disruptant exhibited abnormally shrunken vacuoles and hyphal walls at pH 8.5 and a growth defect at pH 10.0, implicating an alkaline pH stress responsive role for vmaA in A. oryzae.     

Stimulation of canine kidney BBMV ATPase activity by acidic pH in the presence of Zn2+: an ATPase activity distinct from transport ATPases and alkaline phosphatase that may be an ecto-ATPase

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.