Behavioral genetics of Paramecium.
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Animals↗
Biomedical subjects
Y Saimi
Publications and source records attributed to Y Saimi.
Ion channels in yeast.
Voltage-dependent ion channels have been found in the plasma membrane of the yeast Saccharomyces cerevisiae. Ion channel activities were recorded from spheroplasts or patches of plasma membrane with the patch-clamp technique. The most prominent activities came from a set of potassium channels with the properties of activation by positive but not negative voltages, high selectivity for potassium over sodium ion, unit conductance of 20 picosiemens, inhibition by tetraethylammonium or barium ions, and bursting kinetics.
Cell Membrane↗
A mutation that increases a novel calcium-activated potassium conductance of Paramecium tetraurelia.
Under two-electrode voltage clamp, a mutant of P. tetraurelia, restless (rst/rst), showed a large increase in induced current and an outward tail current when compared to the wild-type cell for hyperpolarizing voltage steps. An increase in the induced and tail currents is also observed for depolarizing voltage steps. The larger current during voltage steps and tail in the mutant were eliminated by the use of CsCl-filled electrodes and tetraethylammonium ion (TEA+) in the bath solution, characterizing the lesion as affecting a K+ conductance. Ionophoretic injection of ethylene glycol bis-(beta-aminoethyl ether) n,n,n',n-tetraacetic acid (EGTA) to buffer internal Ca2+ concentration reduced the increased K+ current and tail of the restless cell, indicating Ca2+ activation of the K+ current. Time course and amplitude of remaining currents after blockage of K+ conductances with Cs+ and TEA+ were similar in wild-type and restless cells suggesting no restless defect in entry of calcium. The Ca2+-activated sodium current was similar in the mutant to that in wild type arguing against a defect in calcium regulation activating the K+ channel in the restless cell. We conclude that the restless mutation alters a Ca2+-activated potassium conductance other than the one previously described. The multiplicity of Ca2+-activated potassium conductances in Paramecium is discussed.
Animals↗
Genetic analysis of mutants with a reduced Ca2+-dependent K+ current in Paramecium tetraurelia.
Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.
Animals↗
Mutants in paramecium tetraurelia defective in their axonemal response to calcium.
Six mutants of Paramecium tetraurelia, which display altered axonemal responses to Ca++, are described. The mutants, designated atalantas, are impaired in their ability to swim backward when stimulated by ions or heat; instead they spin very rapidly in one place. Three mutants, ataA1-3, are completely unable to swim backward. The three lines, however, can be distinguished from one another by their forward swimming velocities. The remaining three mutants are leaky. ataB swims backward briefly when stimulated, then stops and spins in place. ataC and ataD are extremely leaky and only display the spinning phenotype at elevated temperatures. An electrophysiological analysis reveals that all six mutants have normal membrane properties, including the Ca++ inward current under voltage clamp. When the membrane is disrupted so as to allow the axoneme free access to Ca++, wild-type cells swim backward, but the mutants do not. These data indicate the site(s) of lesion in the mutants is in the axoneme or in some step linking Ca++ influx and the axoneme, not within the ciliary membrane. These mutants may be useful in investigating the role of Ca++ in the regulation of axonemal motion.
Animals↗
Mutants with altered Ca2+-channel properties in Paramecium tetraurelia: isolation, characterization and genetic analysis.
Dancers are a group of mutants in Paramecium tetraurelia whose Ca2+ current inactivates poorly and are likely to be defective in the structure of their Ca2+ channels. These mutants show prolonged backward swimming in response to K+ and Ba2+ in the medium and were selected by this property in a galvanotactic trough. The dancer mutants are semidominant, and all isolated mutants belong to one complementation group; they are not allelic to any of the previously isolated behavioral mutants of P. tetraurelia. The phenotypic change from the homozygous parent to heterozygous F1 generation takes three to five fissions. There is no evidence of a cytoplasmic factor capable of converting the dancer to the wild-type phenotype, as has been demonstrated in the mutants pawn and cnr. We suggest that the dancer locus is a structural gene for the Ca2+ channel.
Barium↗
A mutation that alters properties of the calcium channel in Paramecium tetraurelia.
The membrane properties of a new mutant of Paramecium tetraurelia, dancer, were compared under voltage clamp with those of the wild type. The Ca2+ current was isolated and examined using CsCl-filled electrodes and tetraethylammonium in the bath solution to block K+ channels. The amplitude of the Ca2+ transient was not altered by the mutation. However, the Ca2+ current in the mutant inactivated more slowly and less extensively: hence a larger sustained Ca2+ current remained in the mutant. A change in the time course of the deactivation of the Ba2+ current was observed in the mutant. This mutational change is not likely to be the consequence of the Ca2+-channel inactivation because it is seen in the Ba2+ solution where there is little inactivation of the current. Other measured properties of the Ca2+ channel, the voltage-dependent K+ current, and the resting properties of the membrane were normal in the mutant. The Ca2+-activated K+ current and the Ca2+-activated Na+ current were larger in the mutant than in the wild type, consistent with a greater elevation of free intracellular Ca2+ during depolarization in the mutant. It is likely that the mutation causes an alteration in the Ca2+-channel structure or in its immediate environment and thereby affects the inactivation and deactivation processes of the Ca2+ channel. As would be expected from the greater Ca2+ current, the mutant tends to generate all-or-none Ca action potentials as opposed to the graded action potentials in the wild type.
Action Potentials↗
Characterization of cytoplasmic factors which complement Ca2+ channel mutations in Paramecium tetraurelia.
The analysis of Ca2+-channel function in the single-celled eukaryote Paramecium can be extended to a biochemical based on recent observations that transfer of cytoplasm from wild-type cells into mutants lacking Ca2+-channel function ("pawn" mutants) causes the mutant cells to regain Ca2-channel activity. Using a convenient behavioral assay for Ca2+-channel function, we have used microinjection of cytoplasmic fractions into mutant cells to enrich for and characterize those components from wild-type cytoplasm which can "cure" cells carrying mutations in the 3 different pawn genes affecting Ca2+-channel activity (pwA,pwB, and pwC). In each case, the curing factor appears to be a protein component of an intracellular membrane. They are distinguishable on the basis of thermal, pH and divalent ion sensitivities. In addition, the factor curing the pwC mutational defect has been purified more than 180-fold. Furthermore, the pwB curing activity appears to be amplified during sequential transfer between pwB cells.
Animals↗
Mutant analysis shows that the Ca2+-induced K+ current shuts off one type of excitation in Paramecium.
Two mutants of Paramecium tetraurelia, called "pantophobiacs," were found to lack most of the slow Ca2+-induced K+ outward current. Passive properties, the transient Ca2+ inward current, and the fast depolarization-induced K+ outward current remain normal. The mutant defect reduces the ability to shut off a normal, excited state of the membrane and results in repeated, long backward swimming instead of the wild-type jerks in response to a variety of ions, to heat, and to touch.
Action Potentials↗
Intra- and interspecific complementation of membrane-inexcitable mutants of Paramecium.
Membrane excitation was the basis for backward swimming of Paramecium facing stimulus. According to standard genetic tests, inexcitable mutants fell into three complementation groups for both Paramecium tetraurelia (pwA, pwB, and pwC) and Paramecium caudatum (cnrA, cnrB, and cnrC). Cytoplasm from a wild type transferred to a mutant through microinjection restored the excitability. Transfusions between genetically defined complementation groups of the same species effected curing, whereas transfusions between different mutants (alleles) of the same group or between sister cells of the same mutant clone did not. Cytoplasmic transfers of all combinations among the six groups of mutants of the two species showed that any cytoplasm, except those from the same group, was able to cure. Since the pawns and the caudatum nonreversals complement one another through transfusion, they appeared to belong to six different complementation groups. The extent of curing, the amount of transfer needed to cure, and the time course of curing were characteristic of the group that received the transfusion. Variations in these parameters further suggested that the six groups represented six different genes. Because the donor cytoplasms from either species were equally effective quantitatively in curing a given mutant, the curing factors were not species specific. These factors are discussed.
Animals↗
Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability.
Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i-antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.
Animals↗
Are ions involved in the gating of calcium channels?
The rates of activation and deactivation of the currents carried by calcium, strontium, or barium ions through the voltage-sensitive calcium channel of Paramecium are different. The differences cannot be attributed to complications due to internal ion concentration, calcium channel inactivation, potassium current activation, surface charge effects, or incomplete space clamping. The findings indicate participation of the divalent cations in the voltage-driven calcium channel gating process.
Animals↗
Microinjection of cytoplasm as a test of complementation in Paramecium.
Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.
Action Potentials↗
The physiological basis of taxes in Paramecium.
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Action Potentials↗
A Ca-induced Na-current in Paramecium.
Under a voltage clamp, step depolarization and repolarization can induce a sustained inward current and a tail inward current in Paramecium tetraurelia bathed in a solution containing 8 mM-Na+. These currents are best seen in the 'paranoiac' mutant. The I-V plot of the sustained inward current can have a region of negative resistance around -20 mV. This current is absent when Na+ is excluded from the bath solution, and it increases as the Na+ concentration increases from 2 to 8 mM. Injection of Na+ into the cell suppresses this inward current. This current develops very slowly, reaching its maximum seconds after the step depolarization and decays with a time constant of hundreds of milliseconds after the repolarization. This slow current is dependent on Ca2+. It can be suppressed by reduction or deletion of external Ca2+ or by iontophoretic injection of EGTA. 'Pawn' mutants with defective Ca-conductance also lack this current. We conclude that Paramecium has a Ca-induced conductance through which the Na-current flows. Although more prominent in the 'paranoiac' mutant, this Ca-induced Na-current is also seen in the wild type. This conductance may function in generating plateau depolarizations lasting seconds or even minutes and the corresponding prolonged backward swimming away from sources of irritation and stress.
Animals↗
In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli.
Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.
Amino Acid Sequence↗
Induction of antibiotic resistance in Paramecium tetraurelia by the bacterial gene APH-3'-II.
We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at > or = 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.
Animals↗
Solute sensing vs. solvent sensing, a speculation.
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Animals↗