Ionic mechanisms of depolarizing and hyperpolarizing quinine receptor potentials in Paramecium caudatum

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca²⁺ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K⁺ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca²⁺ channels, and the hyperpolarizing quinine receptor potential by an activation of K⁺ channels. Accepted: 3 October 1997     

Membrane potential responses controlling chemodispersal of Paramecium caudatum from quinine

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca²⁺ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.     

Genetic control of lactate dehydrogenase isozymes in Paramecium caudatum

The LDH isozymes were surveyed in bacterized cultures of syngens 1, 3, 12, and 13 of Paramecium caudatum by polyacrylamide gel electrophoresis. All the examined stocks of different syngens except one stock in syngen 3 had a single common LDH isozyme, and intra- and intersyngenic variation was not observed except for the one stock in syngen 3. Breeding data using the exceptional stock indicated that the LDH isozymes of P. caudatum are controlled by two codominant alleles at a single locus whose products aggregate randomly, forming a dimer.     

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.     

Insights into Three Whole-Genome Duplications Gleaned from the Paramecium caudatum Genome Sequence

Paramecium has long been a model eukaryote. The sequence of the Paramecium tetraurelia genome reveals a history of three successive whole-genome duplications (WGDs), and the sequences of P. biaurelia and P. sexaurelia suggest that these WGDs are shared by all members of the aurelia species complex. Here, we present the genome sequence of P. caudatum, a species closely related to the P. aurelia species group. P. caudatum shares only the most ancient of the three WGDs with the aurelia complex. We found that P. caudatum maintains twice as many paralogs from this early event as the P. aurelia species, suggesting that post-WGD gene retention is influenced by subsequent WGDs and supporting the importance of selection for dosage in gene retention. The availability of P. caudatum as an outgroup allows an expanded analysis of the aurelia intermediate and recent WGD events. Both the Guanine+Cytosine (GC) content and the expression level of preduplication genes are significant predictors of duplicate retention. We find widespread asymmetrical evolution among aurelia paralogs, which is likely caused by gradual pseudogenization rather than by neofunctionalization. Finally, cases of divergent resolution of intermediate WGD duplicates between aurelia species implicate this process acts as an ongoing reinforcement mechanism of reproductive isolation long after a WGD event.     

Inefficient serotype knock down leads to stable coexistence of different surface antigens on the outer membrane in Paramecium tetraurelia

Expression of surface antigens is usually mutually exclusive, meaning that only one protein is present on the cell surface. With the RNAi feeding technology we induce serotype shifts in Paramecium tetraurelia which are demonstrated to be incomplete, meaning that the cells remain in a shifting state. The coexpression of "old" and "new" protein on the surface can be detected to be stable for more than 15 divisions over a 5-day feeding procedure, a time period different from that reported for temperature-induced shifts. A characteristic heterogenic distribution of the different surface antigens is demonstrated by double indirect-immunofluorescent-staining and we show antigen transport mechanisms related to the tips of cilia. Therefore, we discuss release mechanisms, potential sorting mechanisms for glycosylphosphatidylinositol-anchored proteins and the localizations of surface antigens, which are important for the reported classical immobilization reaction.     

Electric-field effects on gravikinesis in Paramecium

Equilibrated Paramecium caudatum cells exposed to a constant DC gradient reorient with their depolarized anterior ends toward the cathode (galvanotaxis). Voltage gradients were applied to cells swimming either horizontally or vertically. Their velocity and orientation were recorded and compared to unstimulated cells. The DC field increased the horizontal velocity (= reference) up to 175% (galvanokinesis). Swimming velocities saturated after 1 min and were unchanged during the following 4 min. The upward and downward swimming velocities of stimulated cells were below those of horizontal swimmers. The difference in vertical rates (determining gravikinesis) was independent of variations in absolute velocity. Normalization of vertical velocities to horizontal velocities (= 100%) separated DC-field dependent changes from gravity-induced changes in velocities. A weak voltage gradient (0.3 V/cm) was most effective in raising downward gravikinesis up to threefold (-202 m/s) above the unstimulated reference (-66 m/s) and to change sign of gravikinesis in upward swimmers (-43 m/s +33 m/s). We conclude that DC-field stimulation is equivalent to a depolarizing bias on gravikinetic responses of Paramecium. The stimulation does not directly interfere with mechanoreception, but modulates somatic Ca²⁺ entry to induce contraction of the cell soma. This presumably affects the gating of gravisensory transduction channels.     

Thermal performance curves of Paramecium caudatum: a model selection approach

The ongoing climate change has motivated numerous studies investigating the temperature response of various organisms, especially that of ectotherms. To correctly describe the thermal performance of these organisms, functions are needed which sufficiently fit to the complete optimum curve. Surprisingly, model-comparisons for the temperature-dependence of population growth rates of an important ectothermic group, the protozoa, are still missing. In this study, temperature reaction norms of natural isolates of the freshwater protist Paramecium caudatum were investigated, considering nearly the entire temperature range. These reaction norms were used to estimate thermal performance curves by applying a set of commonly used model functions. An information theory approach was used to compare models and to identify the best ones for describing these data. Our results indicate that the models which can describe negative growth at the high- and low-temperature branch of an optimum curve are preferable. This is a prerequisite for accurately calculating the critical upper and lower thermal limits. While we detected a temperature optimum of around 29 °C for all investigated clonal strains, the critical thermal limits were considerably different between individual clones. Here, the tropical clone showed the narrowest thermal tolerance, with a shift of its critical thermal limits to higher temperatures.     

Ca2+ transport and chemoreception in Paramecium

Intracellular Ca²⁺ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca²⁺ pump and that a Ca²⁺-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca²⁺ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca²⁺ homeostasis and pump activity.Abbreviations EGTA ethyleneglycol tetra-acetate - ER endoplasmic reticulum - IBMX isobutyl methylxanthine - I _che index of chemokinesis - Mops 3-[N-morpholino] propanesulfonic acid - PEI phosphoenzyme intermediate - STEN sucrose, TRIS, EDTA, sodium chloride - TCA trichloroacetic acid - TRIS tris[hydroxymethyl] aminomethane     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

Cationic inhibition of Ca current and Ca-dependent ciliary responses in Paramecium

The Paramecium cell membrane was voltage-clamped under K current suppression conditions. Ciliary beating was registered using high-speed video microscopy. Depolarizing step pulses activated a transient inward current and induced reversed ciliary beating. Very strong positive steps inhibited ciliary reversal during the pulse suggesting inhibition of the Ca influx. We call the potential, which is sufficiently positive to induce transition from reversed to normal ciliary beating, the transition potential. The transition potential rose with increasing external Ca²⁺ showing saturation beyond 1 mM Ca²⁺. Addition of Mg²⁺, Ba²⁺ or K⁺ to the 1 mM Ca²⁺ bathing solution depressed the transition potential in a concentration-dependent manner. The depolarization-activated inward Ca current increased with rising external Ca concentration, and addition of either Mg²⁺, Ba²⁺ or K²⁺ diminished the inward Ca current. The diverging results of Ca²⁺-dependent positive shifts, and Mg²⁺-(Ba²⁺-, K⁺-) dependent negative shifts in transition potential are compared with shifts of V_Imax. It is concluded that external cations bind competitively — in addition to membrane surface charges — to affinity sites of Ca channel, where they specifically modulate permeation of calcium.     

Comparison of the evolutionary distances among syngens and sibling species of Paramecium

The morphospecies of the genus Paramecium have several mating type groups, so-called syngens, composed of cells of complementary mating types. The Paramecium aurelia complex is composed of 15 sibling species assigned to the species from the syngen. To increase our understanding of the evolutionary relationships among syngen and sibling species of the genus Paramecium, we investigated the gene sequences of cytosol-type hsp70 from 7 syngens of Paramecium caudatum and 15 sibling species of P. aurelia. Molecular phylogenetic trees indicated that the P. aurelia complex could be divided into four lineages and separated into each sibling species. However, we did not find any obvious genetic distance among syngens of P. caudatum, and they could only be separated into two closely related groups. These results indicated that the concept of syngens in P. caudatum differs quite markedly from that of the P. aurelia complex. In addition, we also discuss the relationships among these species and other species, Paramecium jenningsi and Paramecium multimicronucleatum, which were once classified as varieties of P. aurelia.     

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum

Summary. Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria. Correspondence and reprints: Department of Cytology and Histology, Faculty of Biology and Soil Sciences, Saint Petersburg State University, Universitetskaya naberezhnaya 7/9, 199034 Saint Petersburg, Russia.     

A physical model for galvanotaxis of Paramecium cell

We propose a qualitative physical model of galvanotaxis of Paramecium cells using a bottom-up approach to link the microscopic ciliary motion and the macroscopic behavior of the cells. From the characteristic pattern of ciliary motion called the Ludloff phenomenon, the torque that orients the cell toward the cathode is derived mathematically. Dynamical equations of motion are derived and their stability is discussed. In numerical simulations using our model, cells exhibit realistic behavior, such as U-turns, like real cells.     

Detection of NADPH-diaphorase activity in Paramecium primaurelia

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.     

Distinct localization of a beta-tubulin epitope in the Tetrahymena thermophila and Paramecium caudatum cortex

Summary. Many of the highly organized microtubular arrangements in ciliates are located in the cortical area containing membrane vesicles and vacuoles. In Tetrahymena thermophila and Paramecium caudatum, immunofluorescence microscopy with the monoclonal antibody TU-06, directed against β-tubulin, revealed distinct staining of this cortical region alone, while the cilia and other microtubular structures were unstained. The specificity of the antibody was confirmed by immunoblotting and by preabsorption of the antibody with purified tubulin. Double-label immunofluorescence with antibodies against γ-tubulin, detyrosinated α-tubulin, and centrin showed that the TU-06 epitope is localized outside the basal body region. This was also confirmed by immunogold electron microscopy of thin sections. Proteolytic digestion of porcine brain β-tubulin combined with a peptide scan of immobilized, overlapping peptides disclosed that the epitope was in the β-tubulin region β81–95, a region which is phylogenetically highly conserved. As known posttranslational modifications of β-tubulin are located outside this area, the observed staining pattern cannot be interpreted as evidence of subcellular sequestration of modified tubulin. The limited distribution of the epitope could rather reflect the dependence of TU-06 epitope exposition on conformations of tubulin molecules in microtubule arrangements or on differential masking by interacting proteins. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

K<Superscript>+</Superscript>-induced Ca<Superscript>2+</Superscript> Conductance Responsible for the Prolonged Backward Swimming in K<Superscript>+</Superscript>-agitated Mutant of <Emphasis Type="Italic">Paramecium caudatum</Emphasis>

The K⁺-agitated (Kag) mutant of Paramecium caudatum shows prolonged backward swimming in K⁺-rich solution. To understand the regulation mechanisms of the ciliary motility in P. caudatum, we examined the membrane electrical properties of the Kag mutant. The duration of the backward swimming of the Kag in K⁺-rich solution was about 10 times longer than that of the wild type. In response to an injection of the outward current, the wild type produced an initial action potential and a subsequent membrane depolarization due to I-R potential drop, while the Kag exhibited repetitive action potentials during the depolarization. Under voltage-clamp conditions, the depolarization-activated transient inward current exhibited by the Kag was slightly smaller than that exhibited by the wild type. In response to an application of K⁺-rich solution, both the wild type and the Kag exhibited a depolarizing afterpotential representing the activation of the K⁺-induced Ca²⁺ conductance. The inactivation time course of the K⁺-induced Ca²⁺ conductance of Kag was about 10 times longer than that of the wild type. This difference corresponds well with the difference in behavioral responses between Kag and wild type to K⁺-rich solution. We conclude that the overreaction of the Kag mutant to the K⁺-rich solution is caused by slowing down of the inactivation of the K⁺-induced Ca²⁺ conductance.     

Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

Oxidants act as chemorepellents in Paramecium by stimulating an electrogenic plasma membrane reductase activity

Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 M, 2.2 M and 100 M respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca+ or Mg+ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NADPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.Abbreviations bmv Body plasma membrane vesicles - BPS Bathophenanthroline disulfonate - cAMP Cyclic adenosine monophosphate - cmv Ciliary membrane vesicles - cyt c Cytochrome c - DPI Diphenyleneiodonium - EC ₅₀ Concentration for 50% effectiveness - FeCN Ferricyanide [Fe(CN)₆–3] - FeEDTA Ethylenediaminetetracetic acid (ferric-sodium salt) - GTP Guanosine 5-triphosphate - KCN Potassium cyanide - mM Millimolar - MOPS 3-(N-morpholino) propanesulfonic acid - mV Millivolts - NADH Nicotinamide adenine dinucleotide (reduced form) - NADPH Nicotinamide adenine dinucleotide phosphate (reduced form) - NBT Nitro blue tetrazolium - nm Nanometer - pCMB p-Chloromercuribenzoate - PMA Phorbol 12-myristate 13-acetate - s.d. Standard deviation - SOD Superoxide dismutase - Tris Tris(hydroxymethyl)aminomethane - M Micromolar