Nonbehavioral selection for pawns, mutants of Paramecium aurelia with decreased excitability.
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The reversal response in Paramecium aurelia is mediated by calcium which carries the inward current during excitation. Electrophysiological studies indicate that strontium and barium can also carry the inward current. Exposure to high concentrations of barium rapidly paralyzes and later kills wild-type paramecia. Following mutagenesis with nitrosoguanidine, seven mutants which continued to swim in the ;high-barium' solution were selected. All of the mutants show decreased reversal behavior, with phenotypes ranging from extremely non-reversing (;extreme' pawns) to nearly wild-type reversal behavior (;partial' pawns). The mutations fall into three complementation groups, identical to the pwA, pwB, and pwC genes of Kunget al. (1975). All of the pwA and pwB mutants withstand longer exposure to barium, the pwB mutants surviving longer than the pwA mutants. Among mutants of each gene, survival is correlated with loss of reversal behavior. Double mutants (A-B, A-C, B-C), identified in the exautogamous progeny of crosses between ;partial' mutants, exhibited a more extreme non-reversing phenotype than either of their single-mutant (;partial' pawn) parents.---Inability to reverse could be expected from an alteration in the calcium-activated reversal mechanism or in excitation. A normal calcium-activated structure was demonstrated in all pawns by chlorpromazine treatment. In a separate report (Schein, Bennett and Katz 1976) the results of electrophysiological investigations directly demonstrate decreased excitability in all of the mutants, a decrease due to an altered calcium activation. The studies of the genetics, the survival in barium and the electro-physiology of the pawns demonstrate that the pwA and pwB genes have different effects on calcium activation. (+info)
Interactions of membrane potential and cations in regulation of ciliary activity in Paramecium.
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Ciliary activity in Paramecium was investigated in different external solutions using techniques of voltage clamp and high frequency cinematography. An increase in the external concentration of K, Ca or Mg ions decreased the resting potential. It had no effect on ciliary activity. When the membrane potential was fixed, an increase in external Ca or Mg and, to a lesser extent, an increase in K concentration, raised the frequency of normal beating or decreased the frequency of reversed beating of the cilia. Similar effects resulted from membrane hyperpolarization with constant ionic conditions. Increase in concentration of Ca, but not of Mg or K, enhanced hyperpolarization-induced augmentation of ciliary frequency. Increase in Ca concentration also specifically augmented the delayed increase in inward current during rapid hyperpolarizing clamp. The results support the view that [Ca]i regulates the frequency and direction of ciliary beating. It is suggested that the insensitivity of the ciliary motor system to elevations of the external concentrations of ions results from compensation of their effects on [Ca]i. Depolarization itself appears to increase [Ca]i while elevation of the external ion concentrations at a fixed membrane potential appears to decrease [Ca]i. (+info)
Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.
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An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation. (+info)
Cell division: The renaissance of the centriole.
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Centrioles are located at the center of the cytoskeleton and duplicate exactly once per cell cycle. Recent studies suggest that centrioles are required for the organization of a functional centrosome and that centriole assembly requires both gamma- and delta-tubulin. (+info)
An electrophysiological study of the regulation of ciliary beating frequency in Paramecium.
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1. The role of the surface membrane in the control of ciliary beat frequency in Paramecium was examined by intracellular electrophysiological techniques and pressure injection of Ca2+ and EGTA. Experiments were done on wild type P. caudatum and on both the wild type and a pawn mutant of P. tetraurelia. 2. The increased frequency of beating that accompanies reversal of power stroke orientation in response to depolarization in the wild type fails to occur during depolarization in the mutant pawn, which also fails to exhibit ciliary reversal upon depolarization. 3. Injection of moderate amounts of EGTA blocked the frequency increase without interfering with reversal of the beat in response to depolarization of the wild type. Larger injection of EGTA also prevented reversed beating. 4. The beat frequency in the normal (forward-swimming) direction increased during hyperpolarization in pawn. The hyperpolarizing frequency-voltage relations were quantitatively similar to those of the wild type. 5. Injection of EGTA to a final concentration of 10 mM into wild type cells neither modified the resting frequency nor blocked the frequency increase which normally accompanies hyperpolarization. 6. Transient ciliary reversal in both pawn and wild type produced by injection of Ca2+ could be terminated by the passage of inward current. The power stroke returned to the normal forward-swimming direction and the ciliary beating frequency increased. Upon termination of the inward current the cilia of Ca2+-injected cells again beat in reverse for many seconds. 7. The results support previous reports that increased frequency of beating and ciliary reversal seen in response to depolarization both require the entry of Ca2+ through the surface membrane. On the other hand, the results indicate that frequency increase with hyperpolarization is independent of an altered rate of Ca2+ entry. 8. Increased frequency during hyperpolarization appears to be related more closely to electrotonic membrane current than to membrane potential. It is proposed that inward current might activate high frequency beating by altering the ionic environment of the axoneme within the restricted volume of the cilium by electrophoretic means. (+info)
Altered calcium conductance in pawns, behavioural mutants of Paramecium aurelia.
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Pawns are behavioural mutants (in one of three genes) of Paramecium aurelia that have lost, to varying degrees, the reversal response which is thought to depend on the calcium influx during excitation. This report shows that all of the single and double mutants have reduced active inward (calcium) current, the reduction correlating with the degree of behavioural deficit. All of the mutants display normal resting potential, input impedance and delayed rectification. Mutants in genes pwA and pwC show normal anomalous rectification, but pwB mutants do not show anomalous rectification until the membrane is hyperpolarized further. We suggest that the pwA gene plays a role in depolarization sensitivity (the 'gate') and the pwB gene a role affecting either the wall of the channel itself or the total number of channels. (+info)
Calcium channel stability measured by gradual loss of excitability in pawn mutants of Paramecium aurelia.
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Mutants of Paramecium aurelia that are unable to reverse swimming direction are called pawns. They lack the inward ionic (calcium) current required for the upstroke of the electrically excitable membrane response. By following the progressive loss of reversal response and excitability in cells that are suddenly changed from a heterozygous (wild-type) state to a homozygous mutant state, an estimate of the stability and mean lifetime of the calcium channel has been obtained. During rapid growth, channel dilution due to division occurred, but no channel decay was observed. Under conditions of slow growth, decay could also be observed; channel lifetime was found to be from 5 to 8 days. (+info)
Guanylyl cyclases with the topology of mammalian adenylyl cyclases and an N-terminal P-type ATPase-like domain in Paramecium, Tetrahymena and Plasmodium.
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We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related. (+info)