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PubMed1987

Electrophysiological evidence suggests a defective Ca2+ control mechanism in a new Paramecium mutant.

A new mutant of Paramecium tetraurelia, k-shyA, was characterized behaviorally and electrophysiologically. The mutant cell exhibited prolonged backward swimming episodes in response to depolarizing conditions. Electrophysiological comparison of k-shyA with wild type cells under voltage clamp revealed that the properties of three Ca2+-regulated currents were altered in the mutant. (i) The voltage-dependent Ca2+ current recovered from Ca2+-dependent inactivation two- to 10-fold more slowly than wild type. Ca2+ current amplitudes were also reduced in the mutant, but could be restored by EGTA injection. (ii) The decay of the Ca2+-dependent K+ tail current was slower in the mutant. (iii) The decay of the Ca2+-dependent Na+ tail current was also slower in the mutant. All other membrane properties studied, including the resting membrane potential and resistance and the voltage-sensitive K+ currents, were normal in k-shyA. Considered together, these observations are consistent with a defect in the ability of k-shyA to reduce the free intracellular Ca2+ concentration following stimulation. The possible targets of the genetic lesion and alternative explanations are discussed. The k-shy mutants may provide a useful tool for molecular and physiological analyses of the regulation of Ca2+ metabolism in Paramecium.

Animals↗
PubMed1997

Hyperpolarization- and depolarization-activated Ca2+ currents in Paramecium trigger behavioral changes and cGMP formation independently.

Using 5% ethanol as a deciliating agent, 20 mm colchicine to prevent reciliation and 1 mm amiloride to affect ion fluxes in Paramecium we examined the compartmentation and function of Ca2+ fluxes employing the biosynthesis of cGMP and the stereotypic swimming behavior as indicators for Ca2+ entry. As a function of extracellular Ca2+ Paramecia responded to colchicine and amiloride with a short-lived ciliary augmentation (fast swimming) which indicated hyperpolarization, and formation of cGMP, i.e., the reported hyperpolarization-activated Ca2+ inward current in the somatic membrane is coupled to intracellular generation of cGMP. This is comparable to the coupling of the depolarization-activated, ciliary Ca2+ inward current and ciliary cGMP formation.Ethanol-deciliated cells and ethanol-treated, yet ciliated control cells did not respond to a depolarization with backward swimming or formation of cGMP. Both responses recovered with similar kinetics. A persistent effect of an ethanol exposure on the axonemal apparatus or on guanylyl cyclase activity of ciliated control cells was excluded using permeabilized cells and cell-free enzyme, respectively. Further, in the presence of 20 mm colchicine ethanol-treated cells only recovered the depolarization-dependent avoiding reaction whereas the formation of cGMP remained depressed, i.e., the drug dissected both responses. Similarly, ethanol exposure of Paramecia did not affect the fast swimming response towards the hyperpolarizing agent amiloride whereas the cGMP formation was abrogated and recovered over a period of 7 hr, i.e., amiloride dissected the hyperpolarization-elicited behavioral response from the intracellular cGMP formation. The data demonstrate that in Paramecium depolarization- and hyperpolarization-stimulated behavioral responses and cGMP formation are not coupled. The behavioral changes are triggered by smaller Ca2+ inward currents than the formation of intracellular cGMP.

Amiloride↗
PubMed1997

Extracellular GTP causes membrane-potential oscillations through the parallel activation of Mg2+ and Na+ currents in Paramecium tetraurelia.

Paramecium tetraurelia responds to extracellular GTP (>/= 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg2+ or Na+ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg2+- and Na+-dependent, suggesting that GTP acts through Mg2+-specific (IMg) and Na+-specific (INa) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg2+ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks IMg ("eccentric"), while Na+ failed to support GTP responses in "fast-2," a mutant that specifically lacks INa. Both mutants responded normally to GTP if the alternative cation was provided. As IMg and INa are both Ca2+-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca2+ concentration. Indeed, applying GTP to cells in the absence of either Mg2+ or Na+ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca2+ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor-coupled Ca2+ conductance.

Animals↗
PubMed1998

Transmembrane Mg2+ currents and intracellular free Mg2+ concentration in Paramecium tetraurelia.

The properties of Mg2+ conductances in Paramecium tetraurelia were investigated under two-electrode voltage clamp. When bathed in physiological Mg2+ concentrations (0.5 mm), depolarizing steps from rest elicited a prominent Mg2+-specific current (IMg) that has been noted previously. The dependence of this current on extracellular Mg2+ approximated that of Mg2+-induced backward swimming, demonstrating that IMg contributes to normal membrane excitation and behavior in this ciliate. Closer analysis revealed that the Mg2+ current deactivated biphasically. While this might suggest the involvement of two Mg2+-specific pathways, both tail-current components were affected similarly by current-specific mutations and they had similar ion selectivities, suggesting a common pathway. In contrast, a Mg2+ current activated upon hyperpolarization could be separated into three components. The first, IMg, had similar properties to the current activated upon depolarization. The second was a nonspecific divalent cation current (INS) that was revealed following suppression of IMg by eccentric mutation. The final current was relatively minor and was revealed following suppression of IMg and INS by obstinate A gene mutation. Reversal-potential analyses suggested that IMg and INS define two intracellular compartments that contain, respectively, low (0.4 mM) and high (8 mM) concentrations of Mg2+. Measurement of intracellular free Mg2+ using the fluorescent dye, Mag-fura-2, suggested that bulk [Mg2+]i rests at around 0.4 mM in Paramecium.

Animals↗
PubMed1999

Thermoreception of Paramecium: different Ca2+ channels were activated by heating and cooling.

A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior. The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However, these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or became small in Ca2+-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward current under conditions where K+ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Sr2+, Mg2+, or Mn2+, and was lost upon replacing with equimolar concentration of Ni2+. On the other hand, the cold-induced inward current was not affected significantly by Ba2+, or Sr2+, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations of Mg2+, Mn2+, or Ni2+. These results indicate that Paramecium cells have heat-activated Ca2+ channels and cold-activated Ca2+ channels and that the cold-activated Ca2+ channel is different from the heat-activated Ca2+ channel in the ion selectivity and the calcium-dependent inactivation.

Animals↗
PubMed1999

Ca(2+) current-deficient pawn mutants are promoted to queens during chronic depolarization of Paramecium tetraurelia.

Chronic KCl-induced depolarization of Paramecium tetraurelia enhances Ca(2+)-dependent backward swimming behavior over a period of 8-24 hr. Here, we investigated the electrophysiological mechanisms underlying this adaptive phenomenon using voltage-clamp techniques. Cells that had been adapted to 20 mm KCl showed several significant changes in the properties of the Ca(2+) current that mediates ciliary reversal in Paramecium (I(Ca)), including a positive shift in voltage sensitivity and a significant slowing of inactivation. In seeking an explanation for these changes, we examined the effects of chronic depolarization on mutants that do not normally express a Ca(2+) current or swim backward. Surprisingly, pawn B mutant cells slowly regained the ability to reverse their cilia during KCl exposure with a time course that mirrored behavioral adaptation of the wild type. This behavior was accompanied by expression of a novel Ca(2+) current (I(QUEEN)) whose voltage sensitivity was shifted positive with respect to the wild-type Ca(2+) current and that was slow to inactivate. Coincidental expression of I(QUEEN) in the wild type during adaptation would readily explain the observed changes in I(Ca) kinetics. We also examined the effects of chronic depolarization on Dancer, a mutant suggested previously to have an I(Ca) inactivation defect. The mutant phenotype could be suppressed or exaggerated greatly by manipulating extracellular KCl concentration, suggesting that Dancer lesion instead causes inappropriate regulation of I(QUEEN).

Animals↗
PubMed2000

Programmed DNA under-amplification in Paramecium primaurelia.

Ciliates are unicellular eukaryotic organisms that contain two types of nuclei throughout their vegetative life, transcriptionally active macronuclei governing the cell phenotype, and transcriptionally inert micronuclei. Following sexual reproduction, new macronuclear genomes regularly develop from micronuclear genomes through programmed DNA rearrangements that include DNA splicing, DNA fragmentation and DNA amplification. In the course of characterization of the micronuclear version of the 9.0 kb G gene, which encodes the G surface antigen in Paramecium primaurelia, we characterized a G gene duplicate. Compared with the G gene, the G gene duplicate displays features identifying it as a psi G pseudogene. About 1.6 kb upstream from the G gene, we characterized a new gene, the P gene. A related psi P putative pseudogene lies 1.6 kb upstream from the psi G pseudogene, showing that the duplicated region extends over > 15 kb and putatively defining it as a pseudogene region. Within macronuclear genomes, this region is highly under-amplified; its level never exceeds 20% of that of the corresponding G gene/P gene region in the 11 cell clones we tested. Under-amplification of the psi G pseudogene/psi P putative pseudogene region could be due to its distal position on macronuclear chromosomes, the use of alternative DNA fragmentation domains being frequent in Paramecium species, or to intrinsically lower amplification of a large genomic region. Therefore, the psi G pseudogene/psi P putative pseudogene region presented in this study provides a useful tool for the analysis of DNA fragmentation and/or amplification in eukaryotic genomes.

Amino Acid Sequence↗
PubMed1997

Lectin-binding glycoconjugates in Paramecium primaurelia: changes with cellular age and starvation.

Lectins with different sugar specificities and binding to phagosome-lysosome systems as well as cell surface constituents were used to study glycoconjugate variation throughout culture and clonal life in Paramecium primaurelia, particularly during the transition period from logarithmic to stationary growth phase and in relation to clonal decline, respectively. These lectins include Griffonia simplicifolia agglutinin II (GS II), Ricinus communis agglutinin (RCA120). Arachis hypogea agglutinin (PNA), succinyl concanavalin A (succinyl-con A), and Triticum vulgaris agglutinin (WGA). The labeling obtained varies both according to the lectin used and to the culture and clonal age of the cells. Negative results were obtained in logarithmic growth phase cells and in clonal young cells by using lectin GS II. Conversely, lectins RCA120 and PNA bind to the cell surface, the oral region as well as cilia, and do not undergo modifications with culture or clonal age and after permeabilization. WGA binds to constituents of the cell surface, trichocyst tips, food vacuoles, the oral region, and cilia but the extent of labeling decreases as culture age increases; during clonal decline, cells show the same labeling pattern as starved cells. Finally, the lectin succinyl-con A shows a large amount of binding sites on the cell surface, on trichocyst tips, and in the oral region of logarithmic-phase cells, whereas the number of sites decreases in late stationary phase. The data obtained partly differ from those reported in the literature and the differences can be attributed to the culture conditions and species examined. Nevertheless, the assumption that a rearrangement of some glycoconjugates of membrane throughout culture and clonal life of Paramecium is confirmed.

Animals↗
PubMed2004

Flow cytometric studies of the host-regulated cell cycle in algae symbiotic with green paramecium.

Paramecium bursaria (green paramecium) possesses endosymbiotically growing chlorella-like green algae. An aposymbiotic cell line of P. bursaria (MBw-1) was prepared from the green MB-1 strain with the herbicide paraquat. The SA-2 clone of symbiotic algae was employed to reinfect MBw-1 cells and thus a regreened cell line (MBr-1) was obtained. The regreened paramecia were used to study the impact of the host's growth status on the life cycle of the symbiotic algae. Firstly, the relationship between the timing of algal propagation and the host cell division was investigated by counting the algal cells in single host cells during and after the host cell division and also in the stationary phase. Secondly, the changes in the endogenous chlorophyll level, DNA content, and cell size in the symbiotic algae were monitored by flow cytometry and fluorescence microscopy. The number of algae was shown to be doubled prior to or during the host cell division and the algal population in the two daughter cells is maintained at constant level until the host cell cycle reenters the cytokinesis, suggesting that algal propagation and cell cycle are dependent on the host's cell cycle. During the host's stationary growth, unicellular algal vegetatives with low chlorophyll content were dominant. In contrast, complexes of algal cells called sporangia (containing 1-4 autospores) were present in the logarithmically growing hosts, indicating that algal cell division leading to the formation of sporangia with multiple autospores is active in the dividing paramecia.

Animals↗
PubMed1985

Divalent cation-dependent ATPase activities in ciliary membranes and other surface structures in Paramecium tetraurelia: comparative in vitro studies.

Cilia membrane preparations from axenically grown Paramecium contain ATPase activities with distinct electrophoretic mobilities on Triton-polyacrylamide gels [M.J. Doughty and E.S. Kaneshiro (1983) J. Protozool. 30, 569-575]. Such gel analyses also show additional ATPase activity bands associated with ciliary axonemes (dyneins), cell pellicles, exocytotic trichocysts, and the external cell surface (ectoenzyme). In the present report, the in vitro properties of these activities in various cell fractions were compared. The activity in ciliary membranes was stimulated by Ca2+ greater than Mg2+, in pellicles by Ca2+ greater than Mg2+, and in trichocysts by Ca2+ = Mg2+. The ecto-ATPase was strictly Ca2+ dependent. Determination of the affinities for various phosphate-containing substrates showed that the activities in all fractions were nucleoside triphosphate phosphohydrolases. Unlike the axonemal dynein ATPases, all other fractions were vanadate- and p-chloromercuribenzoate-insensitive. Activities in all cell fractions were sensitive to ruthenium red, the ciliary membrane being the most sensitive (Ki = 4 microM). The ciliary membrane Ca2+ ATPase activity exhibited an apparent affinity for CaATP2- of 9 microM and was inhibited by other divalent cations, La3+, and phosphate, but not by ADP or AMP. The kinetic properties of the ciliary membrane Ca2+ ATPase activity in wild type and several behavioral mutants were similar except for those in the pawn mutant, d495, and the paranoiac mutant, d490, both of which had lower specific activities. These studies support the finding that the ciliary membrane ATPase activity of Paramecium is a specific Ca2+-dependent ATPase distinct from other divalent cation-dependent ATPase activities found in either the cilia or other cell surface structures.

Adenosine Triphosphatases↗
PubMed1981

Electrically induced Ca2+ transport across the membrane of Paramecium caudatum measured by means of flow-through technique.

Transmembrane calcium fluxes related to excitation were studied in Paramecium caudatum. Radioactive (45Ca2+) or inactive solution was flowed through a dense suspension of unlabelled or labelled cells, and radioactivity was monitored in the solution. The organisms were electrically stimulated by means of extracellular electrodes. As a result of stimulation Ca2+ uptake and release was measured. The uptake response dropped with increasing number of successive stimulation periods and increased with growing stimulus amplitude and duration. Maximum uptake was obtained at 20 V/cm and at least 60 s duration and for temperatures between 10 and 15 degrees C. A Ca2+ influx of 700 pmol/1000 cells upon 1 min stimulation was measured at 15 degrees C. This corresponds to an increment of the intraciliary [Ca2+] of about 5 x 10(-4) M. Ca2+ release was dependent on the stimulus amplitude in a similar manner as was Ca2+ uptake. Photographic recordings of the swimming behaviour of the organisms were used to interpret the flux data. At temperatures up to 15 degrees C the cells swam backward perpendicular to the applied electric field of 10 to 20 V/cm. At 25 degrees C they showed forward spiralling movement. For the first time evidence was brought for stimulated Ca2+ influx in Paramecium at physiological temperatures. It is concluded from the results that a strong active Ca2+ extrusion from the intraciliary space counteracts the influx. The Ca2+ pump rate must be at least 8 x 10(12) calcium ions per s per cm2 ciliary surface.

Animals↗
PubMed1984

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium.

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15-52 degrees C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37 degrees C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca2+ flux into ciliary membrane vesicles of Paramecium as measured with the Ca2+ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35 degrees C with a minimum around 15 degrees C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca2+ remained unaffected by temperature. The differences in temperature dependency of Ca2+ conductance and membrane fluidity indicate that the Ca2+ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.

Animals↗
PubMed1988

Neomycin inhibits the calcium current of Paramecium.

The duration of high-K+ stimulated backward swimming is a commonly used bioassay for estimating the amplitude of the inward calcium current of Paramecium. Electrophysiological analysis confirmed that concentrations of neomycin which decreased the duration of stimulated backward swimming also reduced the isolated inward calcium current. Other polycations were also effective in this bioassay and their effectiveness was correlated with the number of their positive charges. Paramecium is therefore a convenient model system for studying the effects of compounds such as neomycin on calcium currents as well as their mechanisms of action.

Animals↗
PubMed1989

Porin of Paramecium mitochondria isolation, characterization and ion selectivity of the closed state.

Porin was isolated and purified from mitochondria of Paramecium tetraurelia. The protein showed a single band of apparent Mr 37,000 on sodium dodecyl sulfate polyacrylamide electrophoretograms. The reconstitution of the protein into artificial lipid bilayer membranes revealed it to be a porin giving pores with an average single-channel conductance of 0.26 nS in 0.1 M KCl. This conductance is about half of that of other eukaryotic porins studied to date. The pore formed by the mitochondrial porin of Paramecium was found to be voltage-dependent and switched to a defined substrate at membrane voltages larger than 20 mV. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.3 nm. The properties of the low conductance state of the pore were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. The possible role of the low-conductance state in the regulation of transport processes across the outer mitochondrial membrane and in mitochondrial metabolism is discussed.

Animals↗
PubMed1990

Purification and identification of a growth factor produced by Paramecium tetraurelia.

We previously reported that the jumyo mutant of a cilate protozoan Paramecium tetraurelia excretes into the medium a factor which promotes its own cell division. Here, the factor was purified to electrophoretic homogeneity through a series of liquid chromatographic procedures. This substance is a protein with a molecular weight of 17,000 which at concentrations of 1 X 10(-9) M (17 ng/ml) or more results in the recovery of the cell division rate of the jumyo mutant to the level of the wild type. The factor is therefore considered to be a growth factor and was named Paramecium growth factor (ParGF). This is the first report of direct proof for the production of a growth factor in an organism other than vertebrates.

Animals↗
PubMed1986

Control of cell division in Paramecium tetraurelia. Effects of abrupt changes in nutrient level on accumulation of macronuclear DNA and cell mass.

In the cell cycle of Paramecium there are three points of interaction between cell growth-related processes and the processes of macronuclear DNA replication and cell division: initiation of DNA synthesis, regulation of the rates of growth and DNA accumulation, and initiation of cell division. This study examines the regulation of the latter two processes by analysis of the response of each to abrupt changes in nutrient level brought about either by transferring dividing cells from a steady-state chemostat culture to medium with unlimited food, or by transferring well-fed dividing cells to exhausted medium. The rates of DNA accumulation and cell growth respond quickly to changes in nutrient level. The amounts of these cell components accumulated during the cell cycle following a shift in nutrient level are typical of those occurring during equilibrium growth under post-shift conditions. Commitment to division occurs at a fixed interval prior to fission that is similar in well-fed and nutrient-limited cells. Initiation of cell division in Paramecium is associated with accumulation of a threshold DNA increment, whose level is largely independent of nutritive conditions. The amount of DNA accumulated during the cell cycle varies with nutritional conditions because the rates of growth and DNA accumulation are affected by nutrient level; slowly growing cells accumulated relatively little DNA during the fixed interval between commitment to cell division and fission.

Animals↗
PubMed1989

Propulsive force of Paramecium as revealed by the video centrifuge microscope.

Using the video centrifuge microscope we constructed, we observed the behavior of Paramecium cells in a solution of graded densities under centrifugal acceleration. Beyond 300g, they not only gather in the zone where the density is closest to theirs, but also orient themselves with their longitudinal axis parallel to the direction of centrifugation turning their anterior ends toward either centripetal or centrifugal direction. Since all of them retain still active swimming capacity, it is possible to calculate their propulsive force from the difference in density between theirs (1.04 g cm-3) and that of the upper or lower layer which they can reach. The propulsive force of single Paramecium cells thus obtained was calculated to be about 7 x 10(-4) dyn.

Animals↗
PubMed1986

Nucleotide sequence of the Paramecium primaurelia G surface protein. A huge protein with a highly periodic structure.

The complete DNA sequence of the G surface protein of Paramecium primaurelia has been determined. It contains an open reading frame of 8145 nucleotides devoid of introns and coding for a protein of 329,000 Mr. Analysis of the deduced amino acid sequence reveals remarkable features such as important internal homologies and a periodic structure, which could be dictated in part by the rigid scaffolding of cysteine residues. The predicted secondary structure shows a quasi absence of alpha-helix and an abundance of beta-pleated sheets and random coils. The monotony of the amino acid sequence is in favour of a structural role for the protein. Interestingly, homologies with other proteins are limited to surface antigens of trypanosomes. Finally, our data are consistent with a genetic code for paramecium which differs from the "universal" code by the assignment of TAA and TAG codons to glutamine.

Amino Acid Sequence↗