Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum

Microtubule dynamics in Paramecium caudatum were investigated with an anti-alpha-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-alpha-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.     

Die stoffwechselphysiologischen Benziehungen Zwischen Paramecium bursaria Ehrbg. und Chlorella spec. in der Paramecium bursaria-Symbiose

Zusammenfassung Der endosymbiontische Verband von Paramecium bursaria Ehrbg. mit Chlorella spec. (grünes Paramecium) wurde physiologisch und cytologisch untersucht. Ein Vergleich der Eigenschaften der Symbiosecinheit mit denen der getrennt kultivierten Symbiosepartner ergab die folgenden Merkmale und Unterschiede: 1. Der symbiontische Verband hat bis zu einer Beleuchtungsstärke von 6000 lux eine stärkere Photosyntheseleistung als die aus ihm isolierte und in Massenkultur in einem definierten Medium kultivierte Alge. Algenfreie P. bursaria zeigen nur eine minimale Fähigkeit zur CO₂-Fixierung. 2. Der Kompensationspunkt der Photosynthese liegt beim algenhaltigen Paramecium bei ca. 4000–5000 lux, derjenige der getrennt kultivierten Alge bei ca. 200–400 lux. 3. Die Symbioseeinheit hat im Dunkeln im Vergleich mit algenfreien P. bursaria einen niedrigeren, im Vergleich mit der frei kultivierten Alge jedoch einen höheren Sauerstoffbedarf. 4. Das grüne Paramecium nimmt weniger Kohlenhydrate aus dem Medium auf als algenfreie Paramecien, hat aber eine höhere Aufnahmeleistung als die isoliert gezogenen Algen. 5. Im Symbioseverband besitzt die symbiontische Alge im Licht eine kompakte Lagerung der photosynthetischen Membranen und eine massive Stärkeablagerung. Die Vergiftung der Photosynthese durch 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff (DCMU) oder die Kultur im Dunkeln führt in algenhaltigen Paramecien zu einer aufgelockerten Lagerung der Thylakoide und einer Verringerung der Stärkeablagerung. Die Algen-population unterliegt im symbiontischen Verband einem komplexen Regulationsmechanismus, bei dem u. a. der intracelluläre Kohlenhydratspiegel eine Rolle spielt. Die geschilderten Ergebnisse werden im Zusammenhang mit der Ökologie des grünen P. bursaria diskutiert.

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The metabolic interactions between Paramecium bursaria Ehrbg. and Chlorella spec. in the Paramecium bursaria-symbiosisII. Symbiosis-specific properties of the physiology and the cytology of the symbiotic unit and their regulation

The endosymbiotic association of Paramecium bursaria Ehrbg. with Chlorella spec. (green Paramechim) was studied both physiologically and cytologically. Comparison of the properties of the symbiotic unit with those of the symbiotic partiners which bad been isolated from it revealed the following features and differences: 1. Up to 6000 lux the photosynthetic capacity of the symbiotic unit is higher than that of the isolated symbiotic algae grown independently in mass culture under defined conditions. Alga-free. Paramecium bursaria (colourless Paramecium) show a very low rate of CO₂-fixation. 2. The green Paramecium has a higher compensationpoint of photosynthesis (4000–5000 lux) than the isolated alga (200–400 lux). 3. Green paramecia consume less oxygen in darkness than colourless organisms but more than the isolated algae. 4. The uptake of carbohydrates from the culture medium by green paramecia is lower than the uptake by alga-free P. bursaria but higher than the one of the isolated algae. 5. Symbiotic algae within the intact symbiotic unit show tightly packed photosynthetic membranes and an intense deposition of starch. In the presence of 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) or in darkness the arrangement of thylakoids is less compact and the deposition of starch is reduced. The growth and the number of the symbiotic algae in situ is regulated by a complex mechanism to which the intracellular level of carbohydrates belongs. The results are discussed in connection with ecological aspects of the Paramecium bursaria-endosymbiosis.

     

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

The temperature-sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley-Liss, Inc.     

Ionic mechanisms of depolarizing and hyperpolarizing quinine receptor potentials in Paramecium caudatum

The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca²⁺ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K⁺ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca²⁺ channels, and the hyperpolarizing quinine receptor potential by an activation of K⁺ channels. Accepted: 3 October 1997     

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum

Summary. Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria. Correspondence and reprints: Department of Cytology and Histology, Faculty of Biology and Soil Sciences, Saint Petersburg State University, Universitetskaya naberezhnaya 7/9, 199034 Saint Petersburg, Russia.     

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.     

Studies on the Macronuclei of Doublet Paramecium tetraurelia: Distribution of Macronuclei and DNA at Fission*

SYNOPSIS. Doublet Paramecium tetraurelia would be expected to contain 2 macronuclei if their nuclear complement were strictly analogous to that of singlets. However, most doublets are unimacronucleate. It is shown in this study that dimacronucleate cells are present only in young clones. Unimacronucleate cells arise either through abnormalities in the determination and distribution of macronuclear anlagen during the first cell cycle after conjugation, or from dimacronucleate cells through abnormal division and segregation of macronuclei during the fission process. When a change in the number of macronuclei occurs through abnormalities in the division and segregation of daughter macronuclei, the daughter cells produced typically have DNA contents more similar than those expected from either random segregation of daughter macronuclei, or from the normal segregation pattern in ciliates in which changes in the number of macronuclei in progeny cells do not occur. This suggests that part of the regulation process of macronuclear DNA content in Paramecium may occur through control of the segregation pattern of daughter macronuclei.     

Quantitative Changes in Periplasmic Proteins of the Macronucleus-Specific Bacterium Holospora obtusa in the Infection Process of the Ciliate Paramecium caudatum

ABSTRACT. The Gram-negative bacterium Holospora obrusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum. The infectious form of this bacterium infects the host macronucleus through digestive vacuoles and differentiates into the reproductive form two days after the infection in the nucleus. The monoclonal antibodies IF-3–1 and IF-3–2 reacted with 39 and 1S kDa periplasmic proteins, respectively, that were specific for the infectious form of H. obrusa. Because the antigens were not detected in the reproductive form of the bacterium, it appears that expression of the proteins decreases during or soon after the infection. Using these antibodies, quantitative changes in the antigens in the early infection process were examined by immunoblotting and immunogold electron microscopy. Immunoblotting showed that the amounts of both antigens were reduced within 1 h after the bacteria were engulfed into the digestive vacuoles of the paramecia, but that the amounts of IF-3–2 antigens declined earlier than the IF-3–1 antigen. Immunogold labeling showed that the level of IF-3–2 antigens became very low in the bacteria in the host digestive vacuoles, whereas there was no similar decrease in amount of IF-3–1 antigens. Possible functions of the antigens are discussed. The IF-3–1 antigens decrease in concentration in parallel with the decrease in the periplasmic region.     

Fine Structure of the Contractile Vacuole Pore in Paramecium*

The pore through which a Paramecium contractile vacuole communicates with the external environment is a 1.2 μm long and 1 μm diameter cylindrical orifice in the pellicle. During diastole, the vacuole:pore junction is closed by a substantial diaphragm which parts to the side at systole. The diaphragm is composed of inner and outer membranes continuous with the vacuole and pore membranes, respectively, and an intervening cytoplasmic layer containing filaments and irregular membranous tubules and vesicles. Microtubules, organized into 2 sets, are an important component of the pore apparatus. One set of ～ 16 microtubules forms an annulus around the pore. These microtubules are organized into a right-handed helix with a pitch of 0.5-0.6 μm, and thus complete slightly more than 2 turns in their course from the level of the diaphragm to the pore outer lip. They appear to be embedded in a layer of dense material immediately adjacent to the pore membrane. The other set consists of 5 or more bands of 10–20 microtubules which radiate in a slight left-handed helix from an insertion at the pore out over the vacuole surface to the ampullae.