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.     

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.     

Mutations in genes encoding inner arm dynein heavy chains in Tetrahymena thermophila lead to axonemal hypersensitivity to Ca2+

Calcium-dependent ciliary reversals are seen in ciliated protozoans such as Tetrahymena in response to depolarizing stimuli, but the axonemal mechanisms responsible for this response are not well understood. The model is that the outer arm dyneins (OADs) control the beating frequency while the inner arm dyneins (IADs) regulate ciliary waveform. Since ciliary reversal is a type of waveform change, the model would predict that IAD mutations could affect ciliary reversal. We have used gene disruption techniques to generate several behavioral mutants of Tetrahymena with functional disruptions of various IADs. One such mutant, called KO-6, is missing I1 (the two-headed IAD) and is unable to show ciliary reversals in response to any stimuli due to a loss of axonemal Ca2+ sensitivity [Eur J Cell Biol 80 (2001) 486-497; Cell Motil Cytoskeleton 53 (2002) 281-288.]. In contrast, disruption of 3 one-headed IADs [Liu et al., Cell Motil Cytoskeleton 59 (2004), 201-214] produced mutants, which showed over-responsiveness in bioassays measuring either their depolarization-induced avoiding reactions (AR) in Na+ and Ba2+ solutions or their duration of backward swimming (continuous ciliary reversal or CCR) in K+ solutions. Detergent-extracted and reactivated mutants also showed increased probabilities of CCR at lower Ca2+ concentrations suggesting that the behavioral over-responsiveness of these three mutants in vivo is due to increased axonemal Ca2+ sensitivity. Our data suggest the possibility that the one-headed IADs and the two-headed IAD act antagonistically in vivo and that loss of any one of the one-headed IADs leads to behavioral over-responsiveness due to less resistance to I1-induced reversals.     

Messenger role of calcium in ciliary electromotor coupling: A reassessment

Electrophysiological and cell reactivation studies in Paramecium and other ciliates have established that depolarizing stimulation opens voltage-sensitive ciliary Ca2+ channels leading to an elevation in intraciliary Ca2+, a rapid 'reversal' in sliding-microtubule based ciliary activity and backward swimming. Regulation of cilia by hyperpolarization modulates the pitch and rate of forward locomotion. The control of this predominant behaviour has been a matter of controversy because ciliary conductances do not change with negative shifts from the resting potential. Recordings of ciliary responses during electrophysiological manipulation of the Ca driving force in the ciliates Stylonychia and Didinium now suggests that a crucial step in hyperpolarization-induced ciliary activation (HCA) is a reduction in intraciliary Ca2+ from a resting steady-state level. The data are discussed with respect to previous hypotheses for the regulation of HCA.     

New Mutants of Paramecium Tetraurelia Defective in a Calcium Control Mechanism: Genetic and Behavioral Characterizations

The k-shy mutants of Paramecium tetraurelia are altered in several Ca2+-dependent functions which regulate ciliary motility. The isolation, genetics, and phenotypes of these mutants are described. Of six independent isolates, all contained recessive single-factor mutations and comprise two unlinked loci, ksA and ksB. All k-shy strains showed prolonged backward swimming responses to depolarizing stimuli, but gave infrequent responses to some stimuli. At least four k-shy strains displayed temperature sensitivity. Neither ksA nor ksB was allelic or linked to dancer, a mutation causing weak Ca2+ current inactivation and prolonged backward swimming. Analysis of ks+; Dn double mutants revealed synergism between the two mutations. The ksA mutant survived Ba2+ solutions longer than wild type, but was more sensitive to K+. Together with previous studies, these results are consistent with a defect in reducing intracellular Ca2+ causing both prolonged ciliary reversal and reduced Ca2+ channel activity due to more active Ca2+-dependent feedback mechanisms. The integration of the Ca2+-dependent stimulatory and inhibitory functions is therefore dependent on ks+ gene functions. The ksA mutant was rescued by microinjection of wild-type cytoplasm, suggesting a possible behavioral assay for factors related to the ksA+ gene product.     

Cloning,localization, and axonemal function of Tetrahymena centrin

Centrin, an EF hand Ca(2+) binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has > 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca(2+) control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca(2+) or the Ca(2+)-mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca(2+), increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca(2+) responses, including ciliary reversal or chemotaxis.     

Outer dynein arm light chain 1 is essential for controlling the ciliary response to cyclic AMP in Paramecium tetraurelia

The individual role of the outer dynein arm light chains in the molecular mechanisms of ciliary movements in response to second messengers, such as Ca(2+) and cyclic nucleotides, is unclear. We examined the role of the gene termed the outer dynein arm light chain 1 (LC1) gene of Paramecium tetraurelia (ODAL1), a homologue of the outer dynein arm LC1 gene of Chlamydomonas reinhardtii, in ciliary movements by RNA interference (RNAi) using a feeding method. The ODAL1-silenced (ODAL1-RNAi) cells swam slowly, and their swimming velocity did not increase in response to membrane-hyperpolarizing stimuli. Ciliary movements on the cortical sheets of ODAL1-RNAi cells revealed that the ciliary beat frequency was significantly lower than that of control cells in the presence of ≥ 1 mM Mg(2+)-ATP. In addition, the ciliary orientation of ODAL1-RNAi cells did not change in response to cyclic AMP (cAMP). A 29-kDa protein phosphorylated in a cAMP-dependent manner in the control cells disappeared in the axoneme of ODAL1-RNAi cells. These results indicate that ODAL1 is essential for controlling the ciliary response by cAMP-dependent phosphorylation.     

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.     

Visualization of calcium transients controlling orientation of ciliary beat

To image changes in intraciliary Ca controlling ciliary motility, we microinjected Ca Green dextran, a visible wavelength fluorescent Ca indicator, into eggs or two cell stages of the ctenophore Mnemiopsis leidyi. The embryos developed normally into free-swimming, approximately 0.5 mm cydippid larvae with cells and ciliary comb plates (approximately 100 microns long) loaded with the dye. Comb plates of larvae, like those of adult ctenophores, undergo spontaneous or electrically stimulated reversal of beat direction, triggered by Ca influx through voltage-sensitive Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluorescence intensity peaks rapidly (34-50 ms), then slowly falls to resting level in approximately 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulus pulse train, and slowly decline in 1-2 s. In both spontaneous and electrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms (1 video field) and to a similar relative extent above resting level from base to tip of the cilia. The decline of fluorescence intensity also begins simultaneously and proceeds at similar rates along the ciliary length. Ca-free sea water reversibly abolishes spontaneous and electrically stimulated Ca Green ciliary emissions as well as reversed beating. Calculations of Ca diffusion from the ciliary base show that Ca must enter the comb plate along the entire length of the ciliary membranes. The voltage-dependent Ca channels mediating changes in beat direction are therefore distributed over the length of the comb plate cilia. The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat cycle.     

Identification of the Ca2+ conductance responsible for K+-induced backward swimming in Paramecium caudatum

Membrane potential responses of Paramecium caudatum to an application of K+-rich solution were examined to understand the mechanisms underlying K+-induced backward swimming. A wild-type cell impaled by a microelectrode produced action potentials followed by a sustained depolarization in response to an application of a K+-rich test solution. After termination of the application, a prolongation of the depolarization (depolarizing after-potential) took place. Behavioral mutants incapable of exhibiting K+-induced backward swimming did not show depolarizing afterpotentials. Upon short application of K+-rich solution, the timing and duration of the ciliary reversal of the wild-type cell coincided well with the K+-induced depolarization. The duration of the depolarizing afterpotential decreased as the duration of the application increased. The depolarizing afterpotential recovered slowly after it had been suppressed by a preceding application of the K+-rich solution. By injection of an outward current into the wild-type cell, the action potentials were evoked normally during the period when the K+-induced depolarizing afterpotential was suppressed. We concluded that the prolongation of the depolarizing membrane potential response following the application of the K+-rich solution represents the Ca2+ conductance responsible for the K+-induced backward swimming in P. caudatum and that the characteristics of the K+-induced Ca2+ conductance are distinct from those of the Ca2+ conductance responsible for the action potentials.     

Responses of the ciliates tetrahymena and paramecium to vertebrate odorants and tastants

The ciliates Tetrahymena and Paramecium respond to strong depolarizing stimuli with Ca(2+)-based action potentials, ciliary reversals, and consequent bouts of backward and forward swimming called "avoidance reactions" (ARs). We found that several representative tastants and odorants cause repetitive ARs in Tetrahymena and Paramecium at low (nM to microM) concentrations. Tetrahymena responded well to capsaicin, quinine, quinacrine, denatonium benzoate, eugenol, piperine, chloroquine, carvacrol, allyl isothiocyanate (AITC), and menthol. Chemosensory adaptation was seen with carvacrol, eugenol, quinacrine, and capsaicin. Cross-adaptation was seen between some of these compounds, suggesting possible similarities in their chemosensory transduction or adaptation pathways. Paramecium only responded well to AITC, quinacrine, piperine, and eugenol (with the effective concentration for 50% response [EC(50)] values in the microM range) while chemosensory adaptation was only seen to eugenol in Paramecium, suggesting possible species differences. Tetrahymena and Paramecium may have primitive receptors that can recognize these and other compounds or some of these compounds can act independently of specific receptors.     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Identification of a 42 kDa protein as a substrate of protein phosphatase 1 in cilia from Paramecium.

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca-ATP as a substrate just as effectively as Mg-ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.     

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.     

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.     

Centrin is essential for the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels

Voltage-gated Ca(2+) channels play a critical role in controlling Ca(2+) entry in various cells. Ciliary reversal in Paramecium depends on the Ca(2+) influx through voltage-gated Ca(2+) channels on the ciliary membrane. One of the voltage-gated Ca(2+) channel mutants in Paramecium caudatum, cnrC, neither produces Ca(2+) action potentials nor responds to any depolarizing stimuli. Here, we report that the cnrC(+) gene product is P. caudatum centrin (Pccentrin1p), a member of the Ca(2+)-binding EF-hand protein superfamily. The Pccentrin1p gene of cnrC was found to contain a single-base deletion, a mutation that caused the loss of the fourth EF-hand of Pccentrin1p. Moreover, the wild-type Ca(2+) channel function was impaired by Pccentrin1p gene silencing, leading to the loss of current-evoked Ca(2+) action potentials and stimulated ciliary reversal. These results demonstrate that Pccentrin1p is indispensable for the activity of the voltage-gated Ca(2+) channels that control ciliary reversal in Paramecium.     

Targeted gene knockout of inner arm 1 in Tetrahymena thermophila

Cilia and flagella contain at least eight different types of dynein arms. It is not entirely clear how the different types of arms are organized along the axoneme. In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known. To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout. We have generated mutant cells that lack wild-type copies of the DYH6 gene. We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme. We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate.     

Motile detergent-extracted cells of Tetrahymena and Chlamydomonas

Tetrahymena and Chlamydomonas cells treated with high (0.25-0.5%) concentrations of the detergent Nonidet P-40 in appropriate buffers retain the shape of the intact cells but are devoid of any ciliary activity unless supplied with MgATP. ATP causes them to swim actively, with beat parameters and swimming patterns indistinguishable from those of intact cells. Both types of detergent-extracted cells are completely devoid of ciliary membranes. The Tetrahymena preparations also lack all cellular membranes, whereas cellular membranes remain intact in the Chlamydomonas preparations. Experiments demonstrating the effects of ATP, ADP, vanadate, erythro-9-[3-2-(hydroxynonyl)]-adenine, and Ca++ are described to illustrate the use of these detergent-extracted cells in research on ciliary motility.     

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.     

Light sensitivity of the ciliate Tetrahymena vorax induced by the fluorescent dye acridine orange.

The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately -55 mV and was blocked by 1 mmol l(-1) TEA. The rate of rise of electrically evoked Ca(2+)-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca(2+) concentration. This suggests that the hyperpolarisation may be caused by activation of Ca(2+)-sensitive K(+) channels. The depolarisation was abolished in Ca(2+)-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca(2+) influx through anterior channels, while Ca(2+) released from intracellular stores activates K(+) channels responsible for the delayed hyperpolarisation.