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PubMed1999

Evidence for the presence of a mammalian-like cholinesterase in Paramecium primaurelia (Protista, Ciliophora) developmental cycle.

By histochemical and immunohistochemical methods, the presence of cholinergic-like molecules has previously been demonstrated in Paramecium primaurelia, and their functional role in mating-cell pairing was suggested. In this work, both true acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were electrophoretically investigated, and the presence of molecules immunologically related to BuChE was checked by immunoblotting. The AChE activity, shown in the membrane protein fraction of mating-competent cells and in the cytoplasmic fraction of immature cells, is due to a 260-kDa molecular form, similar to the membrane-bound tetrameric form present in human erythrocytes. This AChE activity does not appear in either the cytoplasmic fraction of mating-competent cells or in the membrane protein fraction of immature cells. No evidence was found for the presence or the activity of BuChE-like molecules. The role of AChE in P. primaurelia developmental cycle is discussed.

Acetylcholinesterase↗

PubMed1996

Purification, in vitro reassembly, and preliminary sequence analysis of epiplasmins, the major constituent of the membrane skeleton of Paramecium.

The epiplasmic layer, a continuous rigid granulo-fibrillar sheet directly subtending the surface membranes of Paramecium, is one of the outermost of the various cytoskeletal networks that compose it cortex. We have previously shown that the epiplasm consists of a set of 30 to 50 protein bands on SDS-PAGE in the range 50 to 33 kDa, the epiplasmins. We report a purification procedure for the set of epiplasmic proteins, a description of their physicochemical and reassembly properties, and a preliminary characterization of their sequence. The conditions for solubilization of the epiplasm and for in vitro reassembly of its purified constituents ar described. Reassembly of the entire set of proteins and of some (but not all) subsets are shown to yield filamentous aggregates. Microsequences of two purified bands of epiplasmins reveal a striking amino acid sequence consisting of heptad repeats of only three main amino acids, P, V, and Q. These repeats were confirmed by DNA sequencing of polymerase chain reaction products. The motif is QPVQ-h, in which h is a hydrophobic residue. This may constitute the core of the epiplasmin sequence and, in view of the tendency of such a sequence to form a coiled-coil, may account for the remarkable self-aggregation properties of epiplasmins.

Amino Acid Sequence↗

PubMed1997

Genetic evidence for a role of centrin-associated proteins in the organization and dynamics of the infraciliary lattice in Paramecium.

Within the superfamily of "EF-hand Ca2+-modulated proteins," centrins constitute a family of cytoskeletal proteins that are highly conserved from lower eukaryotes to man. Their cytoskeletal specialization is manifest in their capacity to form filamentous contractile arrays of various shapes and functions and by their association with microtubule organizing centres (MTOCs). While the latter property has been conserved throughout the evolution of eukaryotes, centrin-based contractile structures are only found in protists where they form arrays of widely diverse organization and function. In the ciliate Paramecium tetraurelia, three centrin genes have been characterized, which may be part of a larger centrin gene family [Madeddu et al., 1996: Eur J. Biochem. 238:121-128]. The products of these genes were originally identified as components of the infraciliary lattice, a contractile cytoskeletal network [Garreau de Loubresse et al., 1991: Biol. Cell 71:217-225]. We show here that centrins are localized not only in this lattice but also in basal bodies and in the cord, a filamentous structure associated with the oral apparatus. We demonstrate that in the infraciliary lattice, but not in basal bodies, centrins are associated with high-molecular-weight proteins (ca. 350 kD). Their role in the biogenesis of the infraciliary lattice is documented by cytological and biochemical properties of the mutant "démaillé" (dem1) characterized by altered centrin-associated proteins and abnormal organization and dynamics of the infraciliary lattice.

Animals↗

PubMed1998

Intracellular pH and chemoresponse to NH4+ in Paramecium.

Paramecium are attracted to ammonium chloride solutions relative to sodium chloride control solutions, but little is known about the mechanisms by which attraction is evoked. A known effect of ammonium solutions in other cell types is an alteration of intracellular pH. We show here that intracellular pH is elevated upon initial exposure to 5 mM NH4Cl, but appears to decline within 10 minutes, both in wild type cells and in two mutants which do not show sustained attraction to NH4Cl using the standard behavioral assay, the T-maze. We also present quantitative values of swimming parameters that underlie the response to NH4Cl.

Ammonium Chloride↗

PubMed1999

Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development.

BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities. Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells. The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids. METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states. RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells). These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells). In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells. CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity. A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells. CLSM allowed us to gather volume information about the lipid droplet distribution within the cell.

Animals↗

PubMed2000

Flow cytometry as a strategy to study the endosymbiosis of algae in Paramecium bursaria.

BACKGROUND: The stable symbiotic association between Paramecium bursaria and algae is of interest to study such mechanisms in biology as recognition, specificity, infection, and regulation. The combination of algae-free strains of P. bursaria, which have been recently established by treating their stocks of green paramecia with herbicide paraquat (Hosoya et al.: Zool Sci 12: 807-810, 1995), with the cloned symbiotic algae isolated from P. bursaria (Nishihara et al.: Protoplasma 203: 91-99, 1998), provides an excellent clue to gain fundamental understanding of these phenomena. METHODS: Flow cytometry and light microscopy have been employed to characterize the algal cells after they have been released from the paramecia by ultrasonic treatment. Algal optical properties such as light scattering and endogenous chlorophyll fluorescence intensity have been monitored for symbiotic and free-living strains, and strains at stages of interaction with a host. RESULTS: Neither algal morphology nor chlorophyll content has been found to be altered by sonication of green paramecia. This fact allows to interpret in adequate degree changes in the optical properties of symbiont that just has been released from the association with a host (decreased forward light scatter and chlorophyll fluorescence signals). Optical characterization of both symbiotic and free-living algal strains with respect to their ability to establish symbioses with P. bursaria showed that chlorophyll content per cell volume seems to be a valuable factor for predicting a favorable symbiotic relationship between P. bursaria and algae. CONCLUSIONS: Flow cytometry combined with algae-free paramecia and cloned symbiotic algae identifies algal populations that may be recognized by host cells for the establishment of symbioses.

Animals↗

PubMed1984

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↗

PubMed1988

Ultrastructure and motion analysis of permeabilized Paramecium capable of motility and regulation of motility.

Structural and behavioral features of intact and permeabilized Paramecium tetraurelia have been defined as a basis for study of Ca2+ control of ciliary reversal. Motion analysis of living paramecia shows that all the cells in a population swim forward with gently curving spirals at speeds averaging 369 +/- 19 microns/second. Ciliary reversal occurs in 10% of the cell population per second. Living paramecia, quick-fixed for scanning electron microscopy (SEM), show metachronal waves and an effective stroke obliquely toward the posterior end of the cell. Upon treatment with Triton X-100, swimming ceases and both scanning and transmission electron microscopy reveal cilia that uniformly project perpendicularly from the cell surface. Thin sections of these cells indicate that the ciliary, cell, and outer alveolar membranes are greatly disrupted or entirely missing and that the cytoplasm is also disrupted. These permeabilized paramecia can be reactivated and are capable of motility and regulation of motility. Motion analysis of cells reactivated with Mg2+ and ATP in low Ca2+ buffer (pCa greater than 7) shows that 71% swim forward in straight or curved paths at speeds averaging 221 +/- 20 microns/second. When these cells are quick-fixed for SEM the metachronal wave patterns of living, forward swimming cells reappear. Motion analysis of permeabilized cells reactivated in high Ca2+ buffers (pCa 5.5) shows that 94% swim backward in tight spirals at a velocity averaging 156 +/- 7 microns/second. SEM reveals a metachronal wave pattern with an effective stroke toward the anterior region. Although the permeabilized cells do not reverse spontaneously, the pCa response is preserved and the Ca2+ switch remains intact. The ciliary axonemes are largely exposed to the external environment. Therefore, the behavioral responses of these permeabilized cells depend on interaction of Ca2+ with molecules that remain bound to the axonemes throughout the extraction and reactivation procedures.

Animals↗

PubMed1993

Immunological comparison of 22S, 19S, and 12S dyneins from Paramecium cilia.

Three forms of dynein (22S, 19S, and 12S) were purified from Paramecium cilia. Two classes of monoclonal antibodies against purified 22S dynein were generated. One class reacted on immunoblots with the heavy chains of 22S, 19S, and 12S dyneins; the second class reacted with an 88 kD intermediate chain of 22S dynein. Polyclonal antiserum to the heavy chains of 22S dynein reacted with the alpha-heavy chain of 22S and 19S dyneins. A previously described antiserum raised against 22S dynein [Travis et al.: Biochim. Biophys. Acta 966:73-83, 1988] recognized the gamma-heavy chain of 22S dynein which was also present in 19S and 12S dyneins, along with the 88 and 76 kD intermediate chains of 22S dynein. This antiserum was also able to immunoprecipitate dynein from crude extracts of cilia. Electron microscopy revealed that the 22S dynein consisted mainly of two-headed particles with some three-headed particles present. The 12S dynein was mainly one-headed particles. The 19S dynein was a mixture of three-, two-, and one-headed particles. The immunological and electron microscopic studies showed that 19S dynein arises from 22S dynein, and that 12S dynein is heterogeneous, composed of the gamma-heavy chain of 22S dynein and a unique dynein ATPase. The polyclonal antibodies were also used to detect cross-reactive proteins in other organisms. Both the anti-heavy chain and the anti-22S dynein sera reacted strongly with 22S outer arm dynein of Tetrahymena, but not with the 14S dynein of this organism.

Animals↗

PubMed1992

Developmental expression of macronuclear specific antigen in Paramecium caudatum.

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudatum and P. dubosqui. Immunoblotting showed that the antigen was a polypeptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macronuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus.

Animals↗

PubMed1992

Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium.

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA+ or cnrB+) of the donor 9-24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum.

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.

Apoptosis↗

PubMed1992

The beta-tubulin genes of Paramecium are interrupted by two 27 bp introns.

In the ciliate Paramecium tetraurelia, the analysis of the tubulin gene family has revealed the existence of four alpha and three beta genes. We show here that the coding sequence of the first beta-tubulin gene to be cloned and sequenced is interrupted by two short non-coding sequences of 27 bp each, which present at their extremities the pairs GT/AG, characteristic of eukaryotic pre-mRNA introns, and the internal pentanucleotide TTAAT, consensual in Tetrahymena introns. We demonstrate by PCR experiments that the three macronuclear beta-tubulin genes contain these sequences in similar positions, thereby ruling out the possibility that these sequences are ciliate IES present in the micronucleus and eliminated in the transcriptionally active macronucleus. S1 mapping analysis and mRNA sequencing show that the sequences are absent from the beta-tubulin transcripts. These sequences are the first introns described in protein encoding genes in P. tetraurelia and the shortest known introns altogether.

Amino Acid Sequence↗

PubMed1993

Calmodulin is essential for assembling links necessary for exocytotic membrane fusion in Paramecium.

Calmodulin has long been suspected to be involved in calcium-regulated exocytosis but its precise site(s) of action has not yet been identified. In Paramecium, a genetic approach to the problem is possible as in vivo-selected mutations in the calmodulin gene that prevent the activation of some channels have been characterized. Three of these calmodulin mutants were examined for exocytotic capacity and the mutant cam1 was found to be defective for exocytosis at 35 degrees C. The loss of exocytotic capacity in cam1 cells can be restored by transformation with the wild-type calmodulin gene, demonstrating that its exocytotic lesion is indeed due to the mutation in the calmodulin gene. The cam1 mutant displays abnormal exocytotic sites at the non-permissive temperature: it lacks the links ('rosettes' of intramembranous particles in the plasma membrane and the fibrous 'connecting material') which normally connect plasma and trichocyst membranes. Upon shift of cam1 cells from the permissive to a non-permissive temperature, performed sites remain functional. These results demonstrate that calmodulin is necessary for the assembly of these links at the exocytotic site. These results do not, however, exclude the possibility of calmodulin also being involved in Ca(2+)-dependent steps of the stimulus-exocytosis coupling.

Animals↗

PubMed1995

Glycosylinositol-phosphoceramide in the free-living protozoan Paramecium primaurelia: modification of core glycans by mannosyl phosphate.

Glycolipids synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP-[3H]mannose and UDP-[3H]N-acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol-phosphoceramides (GIP-ceramides). The ceramide-based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP-[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP-ceramides were substituted with an acid-labile modification identified as mannosyl phosphate. The modification of the glycosylinositol-phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP-ceramide intermediates in P.primaurelia was studied by a pulse-chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP-ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate-6Man-alpha 1-2Man-alpha 1-6Man-(mannosyl phosphate)-alpha 1-4glucosamine-inositol-phosphoceramide.

Animals↗

PubMed2005

GABAB receptor intracellular trafficking after internalization in Paramecium.

The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively.

Animals↗

PubMed1976

Extra cytoproct mutant in Paramecium tetraurelia: morphogenetical analysis of proters and opisthes.

To account for the differences between proters and opisthes with regard to extra cytoproct morphogenesis in Paramecium tetraurelia, two hypotheses have been proposed and tested. (i) The differences may be a result of different actions of proter-macronucleus and opisthe-macronucleus. This hypothesis has been tested by cytoplasmically connecting the proter with the opisthe in the form of chains, some of which have only one macronucleus per chain. However, the connected proters and opisthes remain different in extra cytoproct morphogenesis, thus arguing against the hypothesis. (ii) The differences may be a result of differences between the proter and the opisthe with regard to the development of their posterior-ventral cortex: proters have a newly-developed posterior-ventral cortex whereas opisthes receive the posterior-ventral cortex from the pre-fission mother animal. This hypothesis has been tested by surgically producing an opisthe with a newly-regenerated posterior cortex. Such opisthes, however, remain different from proters in extra cytoproct morphogenesis. Thus no direct support for the second hypothesis is obtained. Also, proter-opisthe difference in morphogenesis may be understood in terms of Wolpert's positional information hypothesis, by assuming that the anterior and posterior ends of a dividing animal serve as reference points for establishing a gradient and that positional information before separation of the two daughter animals leads to differences in extra cytoproct morphogenesis between them after separation.

Animals↗

PubMed1979

Stomatogenesis during sexual and asexual reproduction in an amicronucleate strain of Paramecium caudatum.

Amicronucleate cells of Paramecium caudatum, whose micronuclei have been artifically removed by micropipetting, are characterized by the appearance of a deciliated area at the posterior part of the buccal opening. These cells form food vacuoles at a slightly lower rate than micronucleate cells. Their mean interfission time is longer than that in micronucleates. The exconjugants of amicronucleate cells can not form food vacuoles and eventually die witout fission, though conjugation proceeds normally in them as well as in their micronucleate mate. The oral apparatus of amicronucleate exconjugants seems to be shallower than that of micronucleates. The membranellar cilia, therefore, can be seen through the buccal overture by scanning electron microscope. The results obtained from the cross of micronucleate and amicronucleate strains and from the induction of autogamy in amicronucleate strains suggest that the micronucleus has a primary role in developing the normal oral apparatus after nuclear reorganization.

Animals↗