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PubMed1982

Nuclear transplant studies on the reduction in numbers of presumptive germ nuclei in exconjugants of Paramecium caudatum.

Nuclear differentiation in exconjugants of Paramecium caudatum is closely associated with a brief localization of the postzygotic nuclei near the opposite ends of the cell, with the germinal nucleus (micronucleus) in the anterior region and the somatic nuclei (macronuclei) in the posterior region. The posterior nuclei cannot regenerate to produce micronuclei when all four anterior nuclei are removed. There is no difference among the anterior four presumptive micronuclei, because, when any three of them were removed, the remaining nucleus was able to divide at each postconjugational fission and to persist as a micronucleus during the vegetative phase. This conclusion agrees with the results of transplanting a presumptive micronucleus into a vegetative cell. Cells during the vegetative phase, however, normally have only one micronucleus. Micronuclear number must be reduced to arrive at the uni-micronucleate condition after the stage of macro- and micronuclear differentiation. Elimination of supernumerary presumptive micronuclei, which had been indicated by morphological observations, was confirmed by the results of nuclear transplantation studies.

Animals↗
PubMed1982

Attachment of Paramecium to polystyrene surfaces: a model system for the analysis of sexual cell recognition and nuclear activation.

In the ciliate Paramecium caudatum, only mating-reactive cells can attach to the polystyrene surface of Petri dishes. The attachment of mating-reactive cells occurs exclusively at the tips of ventrally located cilia (mating-reactive cilia) and appears to involve a hydrophobic interaction. An increase in the hydrophilic nature of the polystyrene surface following treatment with sulphuric acid results in the loss of attachment of reactive cells. The presence or absence of Ca ions and varying the ionic strength of the medium have no effect upon attachment. Although attachment reactivity is closely correlated with mating reactivity, the attachment sites appear distinct from the mating recognition sites, as suggested by the following experimental results: (I) cells that lose mating reactivity following treatment with trypsin are still able to attach to the polystyrene surface; (2) the attachment of cells appears temperature-dependent over a range where mating reactivity remains uneffected; (3) mating reactivity is quickly lost as a result of attachment to the surface of the dish. While the mechanism of attachment is not the same as sexual recognition, the attachment to the polystyrene surface initiates the early stages of the conjugation process. Cells that attach show micronuclear migration while unattached cells do not. The maximum rate of micronuclear migration occurs approximately 30 min after attachment. Meiosis does not occur following migration in the attached cells as in cells that are sexually mating. The relationship between the mating substance and the attachment substance is discussed, while the utilization of the attachment phenomenon for other experimental procedures is proposed.

Animals↗
PubMed1982

Infections of Paramecium bursaria with bacteria and yeasts.

Infections of Paramecium bursaria with bacteria and yeasts are reported. Bacteria and yeasts multiply in the algae-free ciliate and are transmitted at various conditions as are symbiotic chlorellae. Like chlorellae, the bacteria and the yeast cells are situated in perisymbiont vacuoles. Both bacteria and yeasts maintain their capability for independent existence and can be grown on standard nutrient agar. Infection experiments show that aposymbiotic P. bursaria can be infected with Chlorella, bacteria and yeast. Chlorella-bearing P. bursaria cannot be infected with bacteria or yeast. Bacteria-bearing paramecia can be infected with Chlorella but not with yeast. Yeast-bearing paramecia can be infected with Chlorella but not with bacteria. Following infections with Chlorella the paramecia lose their bacteria or yeast symbionts. The bacteria found in P. bursaria probably belong to the genus Pseudomonas; the yeast has been identified as Rodutorula rubra.

Animals↗
PubMed1983

Microspectrophotometric and autoradiographic study of the timing and duration of pre-meiotic DNA synthesis in Paramecium caudatum.

Timing and duration of pre-meiotic G1, S and G2 phases in Paramecium caudatum have been clarified by microspectrophotometry of DNA content and autoradiography with [3H]thymidine ([3H]dThd) and [3H]deoxyuridinemonophosphate ([3H]dUMP). Microspectrophotometric measurement of DNA content showed that cells in stationary phase have a G1 micronucleus, and that pre-meiotic DNA synthesis begins with the micronuclear swelling and ends immediately before the nucleus enters meiotic prophase. The results indicate that the micronucleus is in G1 during the first 1.5 h after onset of mating reaction at 25 degrees C and in pre-meiotic S phase during the following 1 h, and that G2 phase is very short or lacking. Therefore, in P. caudatum, the transition point from pre-meiotic interphase to meiotic prophase is at or very close to the end of pre-meiotic DNA synthesis. Autoradiographic study showed that although [3H]dThd and [3H]dUMP were well utilized in pre-mitotic DNA synthesis, only [3H]dUMP was well utilized in pre-meiotic DNA synthesis. [3H]dThd was utilized very little in pre-meiotic DNA synthesis even if the precursor was taken into the cell, suggesting that the activity of thymidine kinase is low in these cells.

Animals↗
PubMed1983

Calcium and microtubule sliding in ciliary axonemes isolated from Paramecium caudatum.

Microtubule sliding was induced in axonemes obtained from isolated cilia of Paramecium caudatum when they were exposed to a reactivating solution containing ATP after mild treatment with trypsin. Over a very wide range of concentrations (1 nM-4 mM), Ca2+ in the reactivating solution had no effect on the proportion of axonemes that disintegrated as the result of microtubule sliding. Also, the velocity of sliding, determined by cinematography, and the polarity of the direction of sliding-force generation, determined by electron microscopy with regards to the base-to-tip axis of the cilium, were not affected by Ca2+. The results indicate that the Ca sensitivity, which is responsible for the ciliary reversal response, was removed from the axoneme, possibly as the result of trypsin treatment. It is thus unlikely that Ca sensitivity is attributable to the basic sliding machinery that powers ciliary movement.

Animals↗
PubMed1983

Attachment of Paramecium to polystyrene surfaces. II. Induction of the attachment by hydrophobic reagents or immune immunoglobulin G.

Mating-reactive cells of Paramecium multimicronucleatum attach to a polystyrene surface (Falcon 10070 culture dishes) irrespective of extracellular ion concentration and swimming velocity. This attachment is induced in cells that are not mating-reactive by treatment with purified immunoglobulin G from antiserum, which blocks cells from mating or from antiserum prepared against the surface-localized immobilization antigens. Treatment with certain hydrophobic reagents such as phenethylamine, benzylamine, amphetamine or phenylethylamine also strongly induces attachment. Similar results are obtained with P. caudatum. Micronuclear activation, which normally occurs in the conjugation process, does not occur, however, in cells that are induced to attach to the polystyrene surface.

Adhesiveness↗
PubMed1983

Infection of macronuclear anlagen of Paramecium caudatum with the macronucleus-specific symbiont Holospora obtusa.

The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Paramecium caudatum, which is incorporated into the host cells via the food vacuoles and infects their macronucleus exclusively, but never the micronucleus. Since these two kinds of nuclei originate from a fertilization nucleus, it is assumed that the macronucleus acquires a property necessary for it to be recognized by the bacterium at a certain time during the nuclear differentiation process. We found that this property is acquired by four of the eight postzygotic nuclei as soon as the four nuclei differentiate morphologically into the macronuclear anlagen.

Animals↗
PubMed1984

Antibody-induced membrane fusion in Paramecium.

Immobilization of cells by specific immune serum involves crosslinking between immunoglobulin G (IgG) and the i-antigen in the cell membrane. Globular material is seen to accumulate at the ciliary tips by phase-contrast and fluorescence microscopy in a manner analogous to 'capping' in more typical eukaryotes. When immobilized cells of Paramecium are examined by scanning electron microscopy, the fused ciliary tips are seen to be distended, discoidal membranes. Transmission electron microscopy often reveals several ciliary axonemes enclosed within a single, enlarged membrane that is oriented with the ferritin-labelled second antibody directed against the i-antigen antibody on the outer surface only. Fixed cells or living cells treated with immune Fab do not show membrane changes, but do bind antibody. Membrane fusion occurs only if cells are alive and the i-antigen is directly or indirectly cross-linked by intact immune IgG.

Animals↗
PubMed1984

Ca2+-dependent regulation of beat frequency of cilia in Paramecium.

Triton-extracted models of Paramecium cells prepared in the presence of Mg2+ and EGTA showed Ca2+ sensitivity not only in the direction of beat but also in the beat frequency of cilia. The beat frequency increased over a range of concentration of Ca2+ from 2 X 10(-7) to 4 X 10(-7) M, in which the model swam forwards. The frequency increased also above 10(-6)M-Ca2+, in which the model swam backwards. The increase in frequency was inhibited by calmodulin antagonists. Intracellular injection of a Ca2+ buffer giving a free Ca2+ concentration of 2 X 10(-7) to 5 X 10(-7) M in intact cells induced an increase in the beat frequency. Therefore, it is very likely that the beat frequency of cilia is regulated by the intracellular concentration of Ca2+.

Adenosine Triphosphate↗
PubMed1984

Comparative freeze-fracture study of perialgal and digestive vacuoles in Paramecium bursaria.

In the endosymbiotic unit of Paramecium bursaria (Ciliata) and Chlorella sp. (Chlorophyceae) algae are enclosed individually in perialgal vacuoles, which do not show acid phosphatase activity and thus differ from digestive vacuoles. Both types of vacuoles have been studied by freeze-fracture. Perialgal vacuoles are nearly spherical; their membrane always fits tightly to the algal surface. The vacuole size and shape do not vary much. During division of the algal cell into four autospores the vacuole diameter only doubles. After autospore formation the vacuole invaginates around the algal daughter cells and divides. Newly formed perialgal vacuoles remain in intimate contact and exhibit characteristic attachment zones before final separation. The two fracture faces of perialgal vacuole membranes are homogeneously covered with intramembranous particles (IMPs) but rarely show signs of vesicles pinching off or fusing with the membrane, except during vacuole division. The P-faces bear more IMPs (3164 +/- 625 IMP/micron 2) than the E-faces (654 +/- 208 IMP/micron 2). The range of IMP density on both faces is enormous, suggesting that the membrane is not static. Membrane changes are supposed to occur simultaneously with the enlargement of the vacuole and to be caused by fusion with cytoplasmic vesicles, as the fractured necks on vacuole membranes may indicate. Digestive vacuoles in P. bursaria show significant variations in size, shape, membrane topography and IMP density, as well as signs of endocytic activity. Different vacuole populations are present in P. bursaria according to different feeding conditions: ciliates fed for a long time have small vacuoles with few IMPs (322 +/- 198 IMP/micron 2 on the E-faces, 1438 +/- 458 IMP/micron 2 on the P-faces), which are probably condensed digestive vacuoles, whereas organisms fed for a short time have larger vacuoles with highly particulate faces (680 +/- 282 IMP/micron 2 on the E-faces, 2701 +/- 503 IMP/micron 2 on the P-faces) and thus are supposed to be older vacuoles. The digestive vacuole membrane changes continuously. Compared to digestive vacuoles perialgal vacuoles are characterized by small size combined with high IMP density on the two fracture faces. Their IMP densities resemble those of old digestive vacuole membranes. However, it would be premature to conclude that membranes of perialgal and old digestive vacuoles are identical. Membranes of old digestive vacuoles are mainly derived from lysosomal material, which presumably does not contribute to the formation of perialgal vacuole membranes as is indicated by the small vacuole diameter; fusion with lysosomes would considerably enlarge it.(ABSTRACT TRUNCATED AT 400 WORDS)

Chlorella↗
PubMed1984

Destruction of immaturin activity in early mature mutants of Paramecium caudatum.

Cells of Paramecium in the sexually immature period of the clonal life cycle contain a protein called immaturin, which represses mating activity when injected into sexually mature cells. The amount of immaturin at various stages of the immaturity period was examined by injecting cytoplasm from immature cells of one wild-type and two early mature mutant clones into mature cells of wild-type. The proportion of immature cells brought about by the injection increased until about 10-15 fissions after conjugation of the donor cells in all three clones. But the percentage of immature cells induced by the injection decreased rapidly from the 25th to the 30th fission of both early mature donors, while the percentage was still high with a wild-type donor of the same fission age. When cytoplasm of the Emt A (early mature mutant) homozygote taken immediately before maturation (about 27 fissions) was injected into the wild-type immature cells of 45 fissions after conjugation, 13% of the recipient cells expressed mating reactivity. But when cytoplasm of Emt A cells 20 and 50 fissions after conjugation was injected into the same cells, no reactive cell appeared. The results suggest that cytoplasm of the early mature mutant immediately before maturation contains some material that accelerates maturation when injected into wild-type cells in the late stage of immaturity. On the other hand, the immature cytoplasm of wild-type cells about 25 fissions after conjugation showed no effect when injected into early mature cells immediately before maturation. The function of the early mature gene product was suggested to be the destruction of immaturin activity.

Animals↗
PubMed1985

Isolation and characterization of macronuclei of Paramecium caudatum infected with the macronucleus-specific bacterium Holospora obtusa.

Macronuclei from Paramecium caudatum infected with Holospora obtusa may be isolated on sucrose step gradients. Macronuclei containing primarily infectious forms can be separated from those bearing predominantly reproductive forms. RNA polymerase activity in infected macronuclei is greater by a factor of 5 than that in uninfected macronuclei. Proteinase activity is also significantly higher.

Animals↗
PubMed1985

Infection and maintenance of Holospora obtusa, a macronucleus-specific bacterium of the ciliate Paramecium caudatum.

The gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum, which invades the host cell via a food vacuole, infects its macronucleus and grows exclusively in the nucleus. From infection experiments, we showed that a property of the macronucleus that is necessary for it to be recognized and infected by H. obtusa is commonly provided by P. caudatum, P. multimicronucleatum and 14 species of the P. aurelia complex, but not by P. jenningsi, P. bursaria, P. trichium, P. duboscqui, Didinium nasutum, Blepharisma japonicum, Pseudourostyla levis, seven species of Euplotes or Tetrahymena thermophila. Furthermore, it was also shown that the bacteria that infect the macronuclei of P. multimicronucleatum and the P. aurelia species complex always disappear from the nuclei within 5 days and the infected bacteria are maintained stably in the host nuclei in only 13 out of 22 strains of P. caudatum. The results indicate that the species specificity of the habitat of H. obtusa is not simply a matter of its ability to penetrate the host nuclear membrane but depends on unknown factors that exist only in certain strains of P. caudatum.

Bacteriological Techniques↗
PubMed1985

Synchronous exocytosis in Paramecium cells. VI. Ultrastructural analysis of membrane resealing and retrieval.

After the synchronous induction of exocytosis of secretory organelles (trichocysts) in Paramecium tetraurelia cells the process of membrane resealing and retrieval could be followed under synchronous conditions. The characteristic aggregates of membrane intercalated particles (MIPs) contained within the freeze-fractured cell membrane (rings and rosettes) and trichocyst membranes (annulus MIPs), in addition to collar striations on the top of trichocyst membranes, served as endogenous ultrastructural markers. This allowed us to follow the re-arrangement of membrane constituents during and after exocytosis with high temporal and spatial precision. Membrane specificity is maintained to a considerable extent (approximately 99.5%), as judged from the rare occurrence of aberrant resealing (according to freeze-fracture data) and from the rather minute shift of glycocalyx components (according to electron staining experiments) during normal membrane resealing. Coated pits are not involved in membrane retrieval (155 ghosts analysed); since the membrane regions involved in exocytotic fusion are backed by apposed materials, probably proteins, this may restrain membrane constituents from intermixing. Another factor for maintaining membrane specificity is the fact that resealing of the exocytotic opening occurs much more rapidly than in most other systems. The retrieval operates with a half-life of 3 (strain 7S) to 9 min (K401); the involvement of cortical microtubules in the retrieval can be largely excluded, since only two microtubules (of unidentified origin) were seen to approach ghost structures in 4074 cases analysed during this period of intense ghost retrieval. Phalloidin microinjected at a dose that blocked all cytoplasmic streaming (before synchronous exocytosis was induced) did not abolish membrane resealing and retrieval, which, therefore, may be passive processes.

Animals↗
PubMed1985

Regulation of ciliary reversal in triton-extracted Paramecium by calcium and cyclic adenosine monophosphate.

A Triton-extracted model of Paramecium swims forwards when the Ca2+ concentration in the reactivation medium containing ATP is below 10(-6) M and swims backwards when Ca2+ concentration is above 10(-6) M. We found that cAMP (adenosine 3':5'-cyclic monophosphoric acid) inhibited Ca-induced backward swimming of the model and caused forward swimming even when the [Ca2+] was above 10(-6) M. This effect of cAMP was abolished by an inhibitor of cAMP-dependent protein kinase. In order to study the possible role of phosphorylation in the regulation of ciliary orientation, ATP in the reactivation medium was replaced by an ATP analogue, ARP gamma S (adenosine 5'-O-3-thiotriphosphate), which irreversibly thiophosphorylates proteins. In ATP gamma S medium, the model ceased both swimming and ciliary beating, but the orientation of cilia was dependent on [Ca2+]. At low [Ca2+], cilia were perpendicular to the cell surface and, with increase in [Ca2+], their orientation gradually changed towards the cell anterior. Such a change in ciliary orientation corresponds roughly to the change in the swimming direction observed in ATP medium. The ciliary orientation towards the anterior of the cell in ATP gamma S medium at high [Ca2+] was maintained when [Ca2+] was decreased. In contrast, in ATP medium, the swimming direction was reversibly changed with changes in [Ca2+]. These results suggest that the ciliary orientation is regulated not only by Ca2+ but also by cAMP, probably via protein phosphorylation.

Animals↗
PubMed1985

Intracellular positional control of survival or degeneration of nuclei during conjugation in Paramecium caudatum.

The survival or degeneration of nuclei produced after meiosis in Paramecium caudatum depends upon their position in the cytoplasm. The surviving nucleus lies in the paroral region, which is the region around the cytostome. In this study, this was confirmed by quantitative measurements of the location of surviving nuclei in both stained and living conjugating cells. Observation of nuclear behaviour in living cells shows that there is an active mechanism for localizing one of the four meiotic products into the paroral region after the second meiotic division. When this mechanism is inhibited by vinblastine, none of the four nuclei gets into the paroral region and all of them become pycnotic, before degenerating. The results show that migration into the paroral region is essential for survival of the nucleus and that microtubules are involved in this nuclear migration.

Animals↗
PubMed1986

Rectilinear particle arrays in freeze-fracture replicas of the surface membrane of Paramecium tetraurelia.

Freeze-fracture replicas of the plasma membrane of unfixed, uncryoprotected Paramecium tetraurelia bear large rectilinear arrays of 11 nm particles arranged in 7-11 parallel rows. The arrays are of sufficient size to leave impressions in replicas of the underlying outer alveolar membrane, and are apparent as parallel ridges in replicas of the surface coat of deep-etched cells. By noting the location of arrays in replicas of identified portions of the cortex of P. tetraurelia, it has been possible to map the distribution of arrays over the cell surface. The arrays are found primarily over the anterior surfaces of the cell, covering an area that extends from the preoral suture over the left adoral field and a large portion of the anterior dorsal surface. Freeze-fracture analyses of cells taken from a number of different stages of a culture cycle suggest that the particle arrays are not replicated as an integral part of the cortex during cell division, but are assembled and oriented in the membrane as the cells mature. The appearance of small intramembranous particle complexes in the plasma membrane of cells in logarithmic growth phase supports this hypothesis, possibly representing an assembly stage in the formation of the larger particle arrays. The facts that the particle arrays are apparent in replicas of the surface coat of cells, are found primarily at the anterior of the cell body, and have a highly specific orientation with respect to the cell surface, strongly suggest that they function as chemoreceptors in P. tetraurelia.

Animals↗
PubMed1987

Clonal lifespan of Paramecium tetraurelia: effect of selection on its extension and use of fissions for its determination.

Paramecia cells, like human diploid cells cultured in vitro, provide a useful model system for understanding the mechanism that limits division potential. The reported maxima of the clonal lifespan of Paramecium tetraurelia fall into two ranges: from 220 to 258 fissions and from 310 to 325 fissions. We found that neither the selection of vigorous lines nor the cryptic occurrence of autogamy offers a plausible explanation for the much longer lifespans in the latter range. We found the sporadic occurrence of very long clonal lifespans, such as 330 fissions, without selection and autogamy. Selection, which was evaluated by using different methods to maintain lines, had little effect on the extension of the maximal clonal lifespan, whereas it did have a marked effect on the extension of the mean clonal lifespan. Autogamy, which was checked with two closely linked marker genes, was frequent, but only during the period when lines were terminating. Statistical analysis on the mean clonal lifespans of two groups of subclones cultured at 25 and 20 degrees C or with rich and poor nutrition showed that the clonal lifespan that was fission-related under favourable conditions tended to dissociate from fissions under less favourable conditions. We discuss mechanisms that determine the clonal lifespan, programmed events contributing to the maximum clonal lifespan and random events contributing to the mean clonal lifespan.

Animals↗