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PubMed1981

Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia.

The trichocysts of Paramecium tetraurelia constitute a favorable system for studying secretory process because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis. Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place. We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts. Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane.

Animals↗
PubMed1981

Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes.

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.

Animals↗
PubMed1983

Calmodulin antagonists inhibit secretion in Paramecium.

Secretion in Paramecium is Ca2+-dependent and involves exocytic release of the content of the secretory organelle, known as the trichocyst. The content, called the trichocyst matrix, undergoes a Ca2+-induced reordering of its paracrystalline structure during release, and we have defined three stages in this expansion process. The stage I, or fully condensed trichocyst, is the 4 microns-long membrane-bounded form existing prior to stimulation. Stage II, the partially expanded trichocyst, we define as an intermediate stage in the transition, preceding stage III, the fully expanded extruded form which is a 20-40 microns-long needlelike structure. These stages have been used to assay the effects of trifluoperazine (TFP) and W-7, calmodulin (CaM) antagonists, on trichocyst matrix expansion in vivo. TFP and W-7 are shown to reversibly block matrix release induced by picric acid. Ultra-structural examination reveals that one effect of this inhibition is reflected in the organelles themselves, which are prevented from undergoing the stage I-stage II transition by preincubation in 14 microM TFP or 35 microM W-7 before fixation. This inhibition of expansion by TFP can be moderated but not abolished by high extracellular Ca2+ (5 mM). The moderation by high Ca2+ can be eliminated by raising TFP concentration to 20 microM. A possible explanation for the ability to titrate the inhibition in this manner is that TFP is acting to block expansion by binding to the Ca2+-CaM complex. Brief exposure of cells to the Ca2+ ionophore A23187 and 5 mM Ca2+ following TFP treatment promotes matrix expansion, although in 14 microM TFP a residual level of inhibition remains. These results suggest that, following stimulation, CaM regulates secretion in Paramecium, possibly by controlling the Ca2+-dependent matrix expansion which accompanies exocytosis in these cells.

Animals↗
PubMed1983

Nonlysosomal vesicles (acidosomes) are involved in phagosome acidification in Paramecium.

Although acidification of phagocytic vacuoles has received a broadened interest with the development of pH-sensitive fluorescent probes to follow the pH changes of vacuoles and acidic vesicles in living cells, the mechanism responsible for the acidification of such vacuoles still remains in doubt. In previous studies of the digestive vacuole system in the ciliate Paramecium caudatum we observed and described a unique population of apparently nonlysosomal vesicles that quickly fused with the newly released vacuole before the vacuole became acid and before lysosomes fused with the vacuole. In this paper we report the following: (a) these vesicles, named acidosomes, are devoid of acid phosphatase; (b) these vesicles accumulate neutral red as well as acridine orange, two observations that demonstrate their acid content; (c) cytochalasin B given 15 s after exposure of the cells to indicator dye-stained yeast will inhibit the acidification of yeast-containing vacuoles; and that (d) we observed using electron microscopy, that fusion of acidosomes with the vacuole is inhibited by cytochalasin B. We conclude that the mechanism for acidification of phagocytic vacuoles in Paramecium resides, at least partially if not entirely, in the acidosomes.

Animals↗
PubMed1992

Calcium current activated upon hyperpolarization of Paramecium tetraurelia.

Hyperpolarization of Paramecium tetraurelia under conditions where K+ currents are suppressed elicits an inward current that activates rapidly toward a peak at 25-80 ms and decays thereafter. This peak current (Ihyp) is not affected by removing Cl ions from the microelectrodes used to clamp membrane potential, or by changing extracellular Cl- concentration, but is lost upon removing extracellular Ca2+. Ihyp is also lost upon replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Co2+, Mg2+, Mn2+, or Sr2+, suggesting that the permeability mechanism that mediates Ihyp is highly selective for Ca2+. Divalent cations also inhibit Ihyp when introduced extracellularly, in a concentration- and voltage-dependent manner. Ba2+ inhibits Ihyp with an apparent dissociation constant of 81 microM at -110 mV, and with an effective valence of 0.42. Ihyp is also inhibited reversibly by amiloride, with a dissociation constant of 0.4 mM. Ihyp is not affected significantly by changes in extracellular Na+, K+, or H+ concentration, or by EGTA injection. Also, it is unaffected by manipulations or mutations that suppress the depolarization-activated Ca2+ current or the various Ca(2+)-dependent currents of Paramecium. We suggest that Ihyp is mediated by a novel, hyperpolarization-activated calcium conductance that is distinct from the one activated by depolarization.

Animals↗
PubMed1995

The dynein genes of Paramecium tetraurelia: the structure and expression of the ciliary beta and cytoplasmic heavy chains.

The genes encoding two Paramecium dynein heavy chains, DHC-6 and DHC-8, have been cloned and sequenced. Sequence-specific antibodies demonstrate that DHC-6 encodes ciliary outer arm beta-chain and DHC-8 encodes a cytoplasmic dynein heavy chain. Therefore, this study is the first opportunity to compare the primary structures and expression of two heavy chains representing the two functional classes of dynein expressed in the same cell. Deciliation of paramecia results in the accumulation of mRNA from DHC-6, but not DHC-8. Nuclear run-on transcription experiments demonstrate that this increase in the steady state concentration of DHC-6 mRNA is a consequence of a rapid induction of transcription in response to deciliation. This is the first demonstration that dynein, like other axonemal components, is transcriptionally regulated during reciliation. Analyses of the sequences of the two Paramecium dyneins and the dynein heavy chains from other organisms indicate that the heavy chain can be divided into three regions: 1) the sequence of the central catalytic domain is conserved among all dyneins; 2) the tail domain sequence, consisting of the N-terminal 1200 residues, differentiates between axonemal and cytoplasmic dyneins; and 3) the N-terminal 200 residues are the most divergent and appear to classify the isoforms. The organization of the heavy chain predicts that the variable tail domain may be sufficient to target the dynein to the appropriate place in the cell.

Amino Acid Sequence↗
PubMed1995

A large multigene family codes for the polypeptides of the crystalline trichocyst matrix in Paramecium.

The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.

Amino Acid Sequence↗
PubMed1997

Genetic approach to regulated exocytosis using functional complementation in Paramecium: identification of the ND7 gene required for membrane fusion.

Paramecium is a unicellular organism that possesses a specialized pathway for regulated secretion that is amenable to genetic studies. Numerous mutations affecting the process have been isolated over the years, among which is a subclass blocking the terminal step of fusion of the secretory granule with the plasma membrane. We report herein the cloning by functional complementation of one such gene, ND7. The 506-amino acid polypeptide encoded by ND7 is predicted to be a type I integral membrane protein with a highly charged cytosolic domain featuring amphipathic and coiled-coil regions. This structure is compatible with the physiological data on the mutant nd7-1 suggesting that the protein is anchored in the membrane of the secretory granule and that it may interact with other proteins. This work presents the first identification by a genetic approach of a novel gene involved in regulated secretion and establishes Paramecium as a powerful model system for the genetic dissection of this process.

Amino Acid Sequence↗
PubMed1997

Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase.

Telomeric DNA at the ends of chromosomes consist of short, tandem repeat sequences. The telomeres of Paramecium tetraurelia are made up of variable repeats, whereas Paramecium caudatum telomeric repeats are largely invariant. To investigate variable repeat synthesis in P. tetraurelia, mutated telomerase RNA genes were expressed in vivo. We demonstrate that the P. caudatum telomerase RNA can participate in telomere synthesis when expressed in the P. tetraurelia macronucleus, despite 24% primary sequence divergence of the RNAs between the two species. De novo telomeric repeats from transformants indicate that P. tetraurelia telomerase fidelity is dramatically affected by template substitutions and that misincorporation at a single templating position is likely to account for the majority of P. tetraurelia telomeric DNA variability. Furthermore, we show that fidelity is not solely a function of the RNA moiety, as the P. caudatum telomerase RNA does not impart high fidelity to the chimeric enzyme.

Animals↗
PubMed1987

Factors determining the frequency of the killer trait within populations of the Paramecium aurelia complex.

The factors maintaining the cytoplasmically inherited killer trait in populations of Paramecium tetraurelia and Paramecium biaurelia were examined using, in part, computer simulation. Frequency of the K and k alleles, infection and loss of the endosymbionts, recombination during conjugation and autogamy, cytoplasmic exchange and natural selection were incorporated in a model. Infection during cytoplasmic exchange at conjugation and natural selection were factors that would increase the proportion of killers in a population. Conversely, k alleles reduced the proportion of killers in a population, acting through conjugation and autogamy. Field studies indicate that the odd mating type is prevalent in P. tetraurelia isolated from nature. Conjugation and therefore transmission by cytoplasmic transfer would be rare. Competition studies indicate a strong selective disadvantage for sensitives at concentrations found in nature. Natural selection must therefore be the factor maintaining the killer trait in P. tetraurelia.

Alleles↗
PubMed1991

An intragenic suppressor of a calmodulin mutation in Paramecium: genetic and biochemical characterization.

We describe a suppressor of the calmodulin mutant cam1 in Paramecium tetraurelia. The cam1 mutant, which has a SER----PHE change at residue 101 of the third calcium-binding domain, inhibits the activity of the Ca(2+)-dependent K+ current and causes exaggerated behavioral responses to most stimuli. An enrichment scheme, based on an increased sensitivity to Ba2+ in cam1 cells, was used to isolate suppressors. One such suppressor, designated cam101, restores both the activity of the Ca(2+)-dependent K+ current and behavioral responses of the cells. We show that the cam101 mutant is an intragenic suppressor of cam1, based on genetic and microinjection data. The cam101 calmodulin is shown to be similar to wild-type calmodulin in terms of its ability to stimulate calmodulin-dependent phosphodiesterase at low concentrations of free calcium. However, the cam101 calmodulin has a reduced affinity for a monoclonal antibody to wild-type Paramecium calmodulin, as does the parental cam1 calmodulin, and a different mobility on acid-urea gels relative to both wild-type and cam1 calmodulin. We have been able to demonstrate that the isolation of intragenic suppressors of a calmodulin mutation is possible, which allows for the further genetic analysis of structure-function relationships in the calmodulin molecule.

Animals↗
PubMed1993

Molecular and genetic analyses of the B type surface protein gene from Paramecium tetraurelia.

The gene encoding the B type variable surface protein from Paramecium tetraurelia stock 51 has been cloned and sequenced. The 7,182 nucleotide open reading frame contains no introns and encodes a cysteine-rich protein that has a periodic structure including three nearly perfect tandem repeats in the central region. Interestingly, the B gene is located near a macronuclear telomere as was shown previously for two other paramecium surface protein genes. In this paper, we characterize four independent mutants with complete macronuclear deletions of the B gene. Previous analysis of different macronuclear deletion mutants of the A surface protein gene demonstrated two types of inheritance: typical Mendelian segregation (as illustrated by d12) and cytoplasmic inheritance (shown by d48). F1 analysis of four B- mutants crossed with wild-type cells reveals heterozygous F1 cell lines derived from both parental cytoplasms contain approximately the same copy number of the B gene, as expected for a recessive Mendelian mutation. Analysis of F2 progeny from three of these four B- mutant crosses indicates that one of the three exhibits a Mendelian 1:1 segregation ratio of B+ and B- cell lines. The other two show a preponderance of B+ cells, but this is not correlated with the parental cytoplasmic type. In addition to having a large number of B+ individuals, the d12.144, A-, B- mutant produced some F2 progeny that stably maintain less than normal macronuclear amounts of the A gene and/or the B gene.

Animals↗
PubMed1994

Isolation and characterization of paramecium mutants defective in their response to magnesium.

Four mutant strains of Paramecium tetraurelia with a reduced ability to respond behaviorally to Mg2+ have been isolated. Voltage-clamp analyses showed that their Mg2+ insensitivity is associated with a reduced Ca(2+)-dependent Mg2+ current. The four mutants, which have been doubled "eccentric," result from recessive mutations in two unlinked loci, xntA and xntB. Further analysis of xntA1 showed it to be unlinked to any of the behavioral mutants of P. tetraurelia described previously, but it is allelic to d4-521, a "K(+)-resistant" strain, and d4-596, a "Ba(2+)-shy" mutant. The varied pleiotropic effects of xntA1, which include increased resistance to Ni2+ and Zn2+ poisoning, suggest that the locus encodes a central regulator of cell function in Paramecium.

Animals↗
PubMed1998

The cloning by complementation of the pawn-A gene in Paramecium.

The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.

Alleles↗
PubMed1999

A mutation in the flanking 5'-TA-3' dinucleotide prevents excision of an internal eliminated sequence from the Paramecium tetraurelia genome.

The germline chromosomes in Paramecium and other ciliated protozoa contain regions of DNA that are excised and eliminated during the development of a new macronuclear genome. Paramecium tetraurelia internal eliminated sequences (IESs) are invariably flanked by a 5'-TA-3' dinucleotide sequence that is part of a larger 8-bp terminal inverted-repeat consensus sequence. Both features, the absolutely conserved 5'-TA-3' and the remaining 6-bp terminal inverted repeat, are shared with the mariner/Tc1 class of transposons. In this article we describe a mutant cell line (AIM-2) defective in excision of a single IES from the coding region of the A51 surface antigen gene. Excision of the 370-bp IES6649 is prevented by a single A to G transition in the invariably conserved 5'-TA-3' dinucleotide. Failure to excise IES6649 also revealed a 29-bp IES located inside IES6649. Additional experiments with the previously isolated AIM-1 mutant, which also contains an internal IES, shows that alternate excision using the wild-type end of IES2591 with an end from the internal IES is extremely rare or nonexistent. These results indicate that IESs are discrete elements whose excision depends upon nucleotides located within the consensus sequence, but also suggest that additional information is required to match one end of an IES with its excision partner.

Animals↗
PubMed2001

K(+)-channel transgenes reduce K(+) currents in Paramecium, probably by a post-translational mechanism.

PAK11 is 1 of more than 15 members in a gene family that encodes K(+)-channel pore-forming subunits in Paramecium tetraurelia. Microinjection of PAK11 DNA into macronuclei of wild-type cells results in clonal transformants that exhibit hyperexcitable swimming behaviors reminiscent of certain loss-of-K(+)-current mutants. PAK2, a distant homolog of PAK11, does not have the same effect. But PAK1, a close homolog of PAK11, induces the same hyperexcitability. Cutting the PAK11 open reading frame (ORF) with restriction enzymes before injection removes this effect entirely. Microinjection of PAK11 ORF flanked by the calmodulin 5' and 3' UTRs also induces the same hyperexcitable phenotype. Direct examination of transformed cells under voltage clamp reveals that two different Ca(2+)-activated K(+)-specific currents are reduced in amplitude. This reduction does not correlate with a deficit of PAK11 message, since RNA is clearly produced from the injected transgenes. Insertion of a single nucleotide at the start of the PAK11 ORF does not affect the RNA level but completely abolishes the phenotypic transformation. Thus, the reduction of K(+) currents by the expression of the K(+)-channel transgenes reported here is likely to be the consequence of a post-translational event. The complexity of behavioral changes, possible mechanisms, and implications in Paramecium biology are discussed.

3' Untranslated Regions↗
PubMed1979

Absence of detectable mitochondrial recombination in Paramecium.

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.

Animals↗