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.     

The accidental protozoologist--my journey through the world of ciliates

The Past-President's Address has been the opportunity for the speaker to reminisce about the road traveled to get to this time in life. In this paper, I continue in that tradition. During my journey to the present day, I visited different laboratories, studying the genetics of mating and mating types in Paramecium, Tetrahymena, Blepharisma and Euplotes. I have met and worked with many distinguished scientists, including other Past-Presidents of the Society. I also became an active participant in the Society of Protozoologists. I hope the recounting of my trip will be both entertaining and enlightening.     

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.     

Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.     

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.     

The Paramecium circadian clock: synchrony of changes in motility, membrane potential, cyclic AMP and cyclic GMP

The behavior of a ciliate protozoan, Paramecium, is known to represent the electrical state of the cell membrane, and regulation of the membrane potential and ciliary motion are known to involve cAMP and cGMP. The present study shows the synchrony of circadian changes in motility, resting membrane potential and cyclic nucleotides in P. multimicronucleatum. Using an automated system for tracking isolated single microorganisms, the isolated Paramecium cells are confirmed to swim fast and straight during the day (and subjective day) and slowly, with frequent turning, at night (and subjective night). The resting membrane potential is more negative during the day than at night. cAMP and cGMP concentrations oscillate in a manner, such that both cAMP and cGMP are higher during the day (or subjective day) than at night (or subjective night). The ratio of cGMP to cAMP during the light and dark cycle (LD) fluctuates, paralleling the fluctuation of the resting membrane potential measured during the LD. These results suggest that the Paramecium will provide an excellent model to explore daily and circadian orchestration of second messengers mediating signals from ambient light/dark cycles and circadian pacemaker to ion channels and cilia, directly involved in daily and circadian cellular outputs of resting membrane potential and motility. Accepted: 23 January 1997     

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.