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PubMed1992

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement.

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed.

Alleles↗
PubMed1994

DNA sequence requirements for the regulation of immobilization antigen A expression in Paramecium tetraurelia.

The Paramecium surface proteins (immobilization antigens) are expressed in a mutually exclusive manner; only one antigen is found on the cell surface at a time. Expression of these proteins is regulated in response to environmental cues such as temperature and pH. This regulation has been shown to be controlled at the level of mRNA abundance by transcriptional and post-transcriptional mechanisms. Here, we have studied the transcription and regulated expression of the immobilization antigen A gene in Paramecium tetraurelia by transforming an A-deficient strain, d12, with cloned portions of the A gene via microinjection. The A gene is approximately 8 kilobases (kb) long with the transcription start site at position -9 or -8 and the start of translation at position +1. Paramecia transformed with cloned DNA containing A-gene sequences beginning at position -264 and ending 63 base pairs (bp) past the gene's polyadenylation site show properly regulated expression of immobilization antigen A. Lines derived from paramecia transformed with a plasmid containing A-gene sequences starting at position -211, however, show markedly reduced A-gene mRNA levels, and rarely express the A antigen. Nevertheless, cells that do express the A protein exhibit mutual exclusion and normal responses to environmental stimuli. Thus, the 54 bp between -264 and -211, while important for transcription, are not involved in the control of mutual exclusion and responses to environmental changes. Further deletion to position -151 yields similar, but more extreme, results.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Interstitial telomeres are hotspots for illegitimate recombination with DNA molecules injected into the macronucleus of Paramecium primaurelia.

DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules. In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process. The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences. However, insertion sites were not totally at random. Roughly 30% of the molecules were integrated next to or in telomeric repeats. These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position. We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5 x 10(-4) and 3 x 10(-5).

Animals↗
PubMed1988

Aspects of signal transduction in stimulus exocytosis-coupling in Paramecium.

This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, II) the cytoplasms, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and III) the secretory vesicle itself. The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocytosis because this eukaryotic cell contains thousands of docked secretory vesicles (trichocysts) below the cell membrane which can be induced to release synchronously when triggered with secretagogue. This ensures a high signal-to-noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+-dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway. Aspects of the three components mentioned above that will be discussed here include a) the properties of the vesicle content, its pH, and its membrane; b) the role of phosphorylation/dephosphorylation of a cytosolic 63-kilodalton (kDa)Mr protein in membrane fusion; and c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).

Animals↗
PubMed1977

The in vivo effect of dimethyl sulphoxide (DMSO) on protein synthesis and the polyribosome profile in Paramecium.

When Paramecium tetraurelia in log phase growth is treated with 4% dimethyl sulphoxide (DMSO) for five minutes the amount of polyribosomes is reduced 3- to 4-fold while there is a corresponding increase in 80s ribosomal material. Reducing the concentration of DMSO to 1% allows immediate reversal of the condition. Paramecium polyribosomes subjected to 4% DMSO either in whole cell homogenates or during purification through sucrose density gradients appear unaffected while cycloheximide at concentrations up to 100 mug/ml did not prevent DMSO from exerting its effect in vivo. Analyses of 14C amino acid incorporation experiments indicated a strict correspondence between the effect of DMSO on polyribosomes and overall protein synthesis. The reduction of acid precipitable radioactivity in the polyribosomal region after DMSO treatment was associated with a corresponding increase in radioactivity in the 80s region. There was no comparable increase in the acid precipitable radioactivity in the soluble fraction. The overall results of the study suggest that DMSO acts on polyribosommes indirectly through some unknown primary reaction with cell constitutents, and that the mode of action is such as to cause the release of ribosomes from messenger RNA (mRNA) rather than to prevent initiation of the ribosome-mRNA complex. Our data suggest that the effect may be selective. Finally, it is of interest that high concentrations of DMSO (above 8%) appear to have the opposite effect of lower concentrations of DMSO, i.e., they appear to "freeze" the ribosomes to mRNA.

Animals↗
PubMed1979

Regulation of surface antigen expression in Paramecium primaurelia. II. Role of the surface antigen itself.

In the wild-type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed at medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G] while a small minority is phenotypically [156G-168G]. At high temperature, the antigens coded by another locus, generally the D locus, are expressed. This system, displaying both intergenic and interallelic exclusion, provides favourable material to analyze the respective roles of the genome, of the antigens expressed and of the environmental conditions, in particular temperature, on the regulation of the expression of surface antigens. This analysis was carried out by studying the variations of the expression of surface antigens as a function of temperature, culture medium and previously expressed antigens in different genetic situations (a) in homozygotes: the wild-type strains 156 and 168, and the isogenized strains "G156 isogenic 168 carrying the G156 allele in a 168 genetic background; (b) in heterozygotes of the two phenotypic classes of heterozygotes, [156G] and [156G-168G]. The results show that (1) the thermal stability of the expression of a given surface antigen and its rate of re-appearance at the cell surface depend on its own specificity; (2) in heterozygotes [156G-168G], the stability of the expression of the antigen 156G is modified and "adjusted" to that of the less stable surface antigen 168G, and (3) the surface antigen itself exerts a positive control on the maintenance of its own expression. An interpretative model of "transmembranous control" is proposed to account for the regulation of the expression of surface antigens in Paramecium.

Alleles↗
PubMed1982

Ion regulation in potassium-sensitive mutants of Paramecium tetraurelia.

Two recessive mutations of Paramecium tetraurelia confer sensitivity to potassium: While wild-type cells survive when up to 30 mM KCl is added to their growth medium, mutants cease to grow and die when levels of added KCl reach 20-25 mM. Similar sensitivities are seen to Rb+ and Cs+, but not to Na+. Swimming behavior of mutants is indistinguishable from wild type when placed in stimulating solutions containing Na+, K+, or Ba2+. Behavioral adaptation to low levels of K+ also is indistinguishable from wild type. Flame photometry reveals that one mutant is unable to keep out K+ and Na+ when those ions are at low levels in the medium. Both mutants have markedly lower internal Na+ than does wild type. Problems with K+ permeability account for the sensitivity of the one mutant to elevated external K+, but the basis of sensitivity in the other mutant is unclear. These mutants expand the range of ion regulation mutants in Paramecium and demonstrate that lesions in cellular ion regulation in this organism need not result in changes in swimming behavior.

Animals↗
PubMed1978

Internal calcium concentration and potassium permeability in Paramecium.

Ca or EGTA was ionophoretically injected into Paramecium tetraurelia to change [Ca]i. Ca decreased the resting membrane resistance and hyperpolarized the membrane. EGTA had the opposite effect. EGTA following TEA, which suppress GK, had little effect on resistance or resting potential. The I-V relation at steady state was studied before and after EGTA injection while the cell bathed in either K- or TEA-solution. The response to inward test pulses after EGTA injection was similar to that after TEA injection. These results show that [Ca]i controls a steady-state K permeability in Paramecium tetraurelia. A prolonged Ca-spike was recorded after EGTA injection. The plateau potentials in various Ca concentrations in a TEA-solution show the Nernst slope (29 mV for tenfold change in [Ca]o). This result suggests that the prolonged depolarization in this condition is due to a Ca current, after suppression of K-permeability and when [Ca]i is low. The difficulty of obtaining quantitative data on the internal Ca, and the difference between the effects of EGTA injection and TEA injection are discussed.

Calcium↗
PubMed1992

Primary mutations in calmodulin prevent activation of the Ca(++)-dependent Na+ channel in Paramecium.

Paramecium tetraurelia behavioral mutant cam12 displays a "fast-2" behavioral phenotype: it fails to respond to Na+ stimuli. Electrophysiologically, it lacks a Ca(++)-dependent Na+ current. Genetics and DNA sequencing showed the primary defect of cam12 to be in the calmodulin gene (Kink et al., 1990). To correlate calmodulin structure and function in Paramecium, we elucidated the primary structure of cam12 calmodulin. Peptide sequencing confirmed the two point mutations predicted by the DNA sequence: a glycine-to-glutamate substitution at position 40 and an aspartate-to-asparagine substitution at position 50. Our results further showed that lysine 13 and lysine 115 were methylated normally in cam12. It is likely that the electrophysiological abnormalities of cam12 are a direct reflection of the amino-acid substitutions, as opposed to improper posttranslational modification.

Amino Acid Sequence↗
PubMed2002

Development of periodic tension in the contractile vacuole complex membrane of paramecium governs its membrane dynamics.

The contractile vacuole complex is a membrane-bound osmoregulatory organelle of fresh water protozoa such as Paramecium. In Paramecium it consists of a central vacuole (the contractile vacuole) and 5-10 arms that radially extend from the vacuole into the cytosol (the radial arms). Excess cytosolic water, acquired osmotically, is segregated by the radial arms and enters the vacuole, so that the vacuole swells (the fluid-filling phase). The vacuole then rounds (the rounding phase) and the radial arms sever from the vacuole. The vacuole membrane then fuses with the plasma membrane at the pore region and the pore opens. The vacuole shrinks as its fluid is discharged through the pore (the fluid-discharging phase). The pore closes when the fluid has been discharged. The radial arms then reattach to the vacuole, so that the vacuole swells again as the fluid enters from the arms (the next fluid-filling phase). We found that the vacuole continued to show rounding and slackening even after it together with a small amount of cytosol had been isolated from the cell. Using a microcantilever placed on the surface of the vacuole the tension of the in vitro vacuole increased to 5 x 10(-3)N m(-1) as the vacuole rounds, and its lowest value was 1 x 10(-4)N m(-1) during slackening. We propose a hypothesis that an increase in the spontaneous curvature of the organelle's membrane leads to an increase in membrane tension and thus to the vacuole's rounding, severing of the radial arms from the vacuole, and opening of the pore. Conversely, a decrease in the spontaneous curvature accompanied by a decrease in membrane tension could lead to the closing of the pore and reattachment of the radial arm at the start of the fluid-filling phase.

Animals↗
PubMed1995

Lysozyme acts as a chemorepellent and secretagogue in Paramecium by activating a novel receptor-operated Ca++ conductance.

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 mM to 1.0 microM) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K+ channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca++ based depolarizations. Addition of either Na+ or Mg++ improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 microM neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 microM), lysozyme is a secretagogue. A transient inward current, recorded in Ca++ alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 mM) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca++ conductance. This receptor-operated Ca++ conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca++ influx at high concentrations to cause exocytosis.

Animals↗
PubMed1994

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels.

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mM veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10(-4) to 10(-3) M as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec. Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10(-2) M inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.

Aconitine↗
PubMed1995

Functional reconstitution of ion channels from Paramecium cortex into artificial liposomes.

Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless di- or trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca2+ release, and the prospects of their purification are discussed.

Animals↗
PubMed1995

Paramecium Na+ channels activated by Ca(2+)-calmodulin: calmodulin is the Ca2+ sensor in the channel gating mechanism.

Paramecium Na+ channels, which were Ca(2+)-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10(-5) to 10(-3) M Ca+ in the bath. They became much less active in the presence of 10(-6) M Ca2+, and their activity subsided completely at 10(-8) M Ca2+. A Hill plot showed a dissociation constant of 6 microM for Ca2+ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mM Mg2+. The channels also exhibited a mild voltage dependence. When exposed to 10(-8) M Ca2+ for an extended period of 2-4 min, channels were further inactivated even after bath Ca2+ was restored to 10(-4) M. Whereas neither high voltage (+100 mV) nor high Ca2+ (10(-3) M) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca2+ to the bath restored channel activity without a requirement of additional Mg2+ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca2+ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na+ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na+ channel activation, and is the Ca2+ sensor of the Na(+)-channel gating machinery.

Animals↗
PubMed1980

Malic dehydrogenase locus of Paramecium tetraurelia.

A search was undertaken for naturally occurring genetic markers for use in clonal aging studies of Paramecium tetraurelia. Clonal age is defined as the number of cell divisions since the last sexual process. Autogamy (self-fertilization) is a sexual process which can occur in aging lines, resulting in homozygosity and initiation of the next generation. Such "illicit" autogamies must be detected and eliminated from the aged clone. With codominant alleles, heterozygous aging lines can be established which will express a phenotype distinguishable from that of either parental type and autogamy can then be monitored by the appearance of either segregant homozygous phenotype. However, very few codominant alleles are available in this species. Electrophoretic mobilities of malic dehydrogenase (MDH) were assayed in 11 stocks of Paramecium tetraurelia by polyacrylamide gel electrophoresis. Nine stocks showed a single-banded "stock 51" type, while stock 174 and stock 29 each exhibited unique mobility. Crosses between stock 51 and the deviant stocks revealed distinct three-banded patterns indicative of heterozygosity of the F1 generation. In the autogamous F2 generation, 1:1 segregation of the parental types were recovered. The pattern of inheritance is consistent with codominant alleles and Mendelian inheritance. These naturally occurring biochemical markers are stable with increasing clonal age and are therefore useful genetic markers for studies of cellular aging.

Alleles↗
PubMed1987

Localization of the chemoreceptive properties of the surface membrane of Paramecium tetraurelia.

The plasma membrane of Paramecium tetraurelia comprises two morphologically distinct components; a membrane that encloses the cell body and a ciliary membrane. In order to investigate the relative contributions of the two membranes to attractant-induced membrane potential changes, cells were deciliated with ethanol and their subsequent responses to attractants examined. Deciliation did not significantly affect the magnitude of the hyperpolarizations evoked by acetic or lactic acids, and had no effect on the concentration dependence of responses to folic acid. We conclude that the components necessary for detection and response to attractants are not exclusive to the ciliary membrane of P. tetraurelia. Deciliation of Paramecium concomitantly permits localized chemical stimuli to be applied directly to the cell surface in the absence of strong fluid currents that are generated by the activity of the locomotory organelles. By systematically applying K2 folate to a number of sites on the cell surface, it has been possible to demonstrate an anterior-posterior gradient of chemosensitivity on the cell body of P. tetraurelia.

Acetates↗
PubMed1996

Annexins in Paramecium cells. Involvement in site-specific positioning of secretory organelles.

Annexins were isolated from Paramecium cell homogenates by standard ethylene glycol tetraacetic acid (EGTA) extraction and 100 000-g centrifugation. Two different antibodies (Abs) against synthetic peptides were used, Call-15 and B15, which in mammalian cells recognize a sequence of annexin II or a common sequence occurring in several annexins (except for annexin II), respectively. With anti-Call-15 Abs, western blots from EGTA extracts showed strongly reactive bands of 44.5 and 46 kDa and of higher values. Some of these bands bound to the 100 000-g pellet fraction when Ca(2+) was added. Immuno- and affinity labelling revealed selective, Ca(2+)-dependent labelling of the cell cortex, with enrichment around trichocyst docking sites (facing subplasmalemmal Ca(2+) stores). Cortical fluorescence labelling decreased in wild-type (7S) cells when trichocyst ghosts were detached after synchronous exocytosis. Similarly, cortical labelling was reduced when intact trichocysts were detached from the cell surface of non-discharge mutant cells (nd9-28 degrees C, showing identical bands on blots), which then contained numerous heavily labelled phagolysosomes. This strongly suggests annexin downregulation. All together, the dynamic labelling of cortical structures we observed strongly supports involvement of calpactin-like annexins in trichocyst docking. Anti-B15 Abs recognized a band of 51 kDa and some of higher values. These Abs selectively labelled the outlines of the cytoproct, the site of spent phagolysosome exocytosis. In conclusion, our data indicate involvement of specific sets of annexins in site-specific positioning and attachment of widely different secretory organelles at the cell surface in Paramecium cells.

Animals↗
PubMed1976

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria.

We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained from Paramecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0 M salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl- than to K+ and is impermeable to Ca++. The procedure for obtaining VDAC; AND THE PROPERTIES OF THE CHANNEL ARE HIGHLY REPRODUCIBLE. VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl- permeability suggest that there may indeed be a voltage-dependent Cl- permeability in mitochondria. The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.

Animals↗