Anomalies of autogamy in Paramecium tetraurelia temperature sensitive mutant induced by heat treatment.

The run of autogamy in temperature sensitive mutant ts401, carrying also the mutation of trichocysts nd3a, at permissive (26 degrees C) and non-permissive temperature (35 degrees C) was studied and compared with this process in wild d4-2 stock of Paramecium tetraurelia. The effect of heat treatment on programmed nuclear and cortical events was investigated using cytological silver impregnation Fernandez-Galiano method and immunofluorescence technique with the application of two anti-alpha-tubulin antibodies. The appearance of cells with some large macronuclear fragments and an excess number of micronuclei suggests that nuclear differentiation is inhibited at restrictive temperature and that macronuclear regeneration takes place in this thermosensitive mutant. In such cells impaired oral reorganization was observed. After slight refeeding at the late autogamy, cells stopped at first postautogamous division were induced by heat treatment. The most striking feature of these abnormal divisions was the lack of a basal body duplication wave, which suggests that production of gamma-tubulin, structural protein being the essential component of the microtubule organizing centres, is disturbed at high temperature.

Fine oral filaments in Paramecium: a biochemical and immunological analysis.

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.

Nuclear and cortical regulation in doublets of Paramecium: II. When and how do two cortical domains reorganize to one?

Homopolar doublets with twofold rotational symmetry were generated in Paramecium tetraurelia and in P. undecaurelia by electrofusion or by arrested conjugation. These doublets underwent a complex cortical reorganization over time, which led to their reversion to singlets. This reorganization involved a reduction in number of ciliary rows, a progressive inactivation and loss of one oral meridian, and a reduction and eventual disappearance of one cortical surface (semicell) situated between the two oral meridians. The intermediate steps of this reorganization included some processes that resemble those previously described in regulating doublets of other ciliates, and others that are peculiar to members of the "P. aurelia" species-group and some of its close relatives. The former included a disappearance of one cortical landmark (a contractile vacuole meridian) and transient appearance of another (a third cytoproct) within the narrower semicell. The latter included a reorganization of the paratene zone and the associated invariant (non-duplicating) region to occupy the entire narrower semicell and a redistribution of zones of most active basal-body proliferation within the opposite, wider semicell. The final steps of reorganization involved anterior displacement, invagination, and resorption of one of the two oral apparatuses and eventual disappearance of the associated oral meridian. An oral meridian deprived of its oral apparatus, either by spontaneous resorption or microsurgical removal, could persist for some time in "incomplete doublets" before regulating to the singlet condition. The phylogenetically widespread events encountered in the regulation of doublets to singlets suggest that Paramecium shares some of the global regulatory properties that are likely to be ancestral in ciliates. The more specific events are probably associated with the complex cytoskeletal architecture of this organism and with the frequent occurrence of autogamy that was described in the preceding study (Prajer et al. 1999).

Tubulin polyglycylation: a morphogenetic marker in ciliates.

The occurrence of the tubulin post-translational modification, polyglycylation, in stable microtubular structures was investigated during morphogenesis in two ciliates, Paramecium and Frontonia atra, belonging to the Epiplasmata group. This analysis was carried out by means of immunofluorescence and post-embedding immunoelectron microscopy using two monoclonal antibodies, TAP 952 and AXO 49, respectively recognizing mono- and polyglycylated sites in alpha- and beta-tubulin. In the course of cell division, the TAP 952 epitope is detected in all microtubular structures including the newly assembled ones, such as cortical and oral basal bodies and cilia. In contrast, the AXO 49 epitope is only present in 'old' microtubular structures such as parental cortical and oral basal bodies and cilia. Our observations show that, in ciliates: 1) this tubulin post-translational modification takes place early in the course of morphogenesis; and 2) the lengthening of the polyglycine chains occurs after a great delay following addition of the first glycine residues on the tubulin glycylation sites, and following microtubule assembly. Thus, a sequential mechanism of polyglycylation is shown to take place in the tubulin molecule and during morphogenesis in Paramecium and Frontonia atra. Accordingly, polyglycylation, through a time-dependent polyglycine chain elongation process, appears to be a morphogenetic marker in ciliates.

DEFPARAM: a program package for aligning elliptical sections of biological objects containing an n-fold symmetry.

A program package has been developed to align automatically images of biological objects containing an n-fold symmetry, and to remove the distortions induced on their circular shape by the microtomy. It uses an original procedure based on correlation techniques and replaces usual manual processing. Examples of direct averaging of transverse sections of biological objects are given to illustrate the program's capabilities.

A scaffold for basal body patterning revealed by a monoclonal antibody in the hypotrich ciliate Paraurostyla weissei.

In the hypotrich ciliate Paraurostyla weissei, the infraciliature consists of basal bodies on which cilia are anchored together with associated dense material and microtubular rootlets. This is renewed at each morphogenesis, which occurs both in the fully differentiated cell and in a transient dedifferentiated stage, the zygocyst. In both situations, the cell must reconstitute its typical ciliature by properly patterning its basal bodies. Insights into these morphogenetic processes were obtained through an immunocytochemical study using an antipericentriolar material antibody displaying remarkable properties. In Paraurostyla, this antibody decorates the electron-dense material associated with the basal bodies in the interphase cell (as it does with centrosomes on metazoan cells) but, during morphogenesis, the antibody decorates a transient system of tracks which appear prior to basal body patterning and along which basal bodies will later be distributed. In all cases, tracks are initiated close to parental organelles and then elongate to form a system linking the anlagen together during their migration. During zygocyst morphogenesis, they extend along three main cellular meridians. As this antibody decorates an antigen associated with early steps of morphogenesis, it visualizes a proteinaceous system upstream to basal body patterning and provides a structural continuity between parental and newly assembled basal body systems.

Isolation and characterization of monoclonal antibodies to cytoskeletal and membrane proteins of the Paramecium cortex.

Monoclonal antibodies have been generated against whole cortical fragments of Paramecium to provide tools for the analysis of cortex morphogenesis as well as for the biochemical dissection of this complex membrane-cytoskeleton structure. Of 80 positive hybridomas in ELISA tests, 17 monoclonal were characterized by immunoblotting, immunofluorescence tests on sectioned as well as on Triton-permeabilized cells (including confocal microscopy), and by EM immunocytochemistry on permeabilized cells. Five classes of monoclonals were obtained directed, respectively, against the epiplasm (or elements tightly associated with it), the trichocysts, the microtubules, the surface membranes, and poorly defined intracellular antigens. Of these, the newest and most promising appear to be a set of monoclonals decorating both intensely and sharply some specific parts of the epiplasm (outer periphery, outer central part or core). These antibodies therefore provide the first demonstration of a molecular heterogeneity of composition at the level of individual epiplasmic scales in Paramecium. In addition, they offer powerful tools to follow the biogenesis of these structures during cell division. Finally, they have allowed the identification of a number of previously uncharacterized protein components of the cortex.

Rearrangement of the cytoskeleton and nuclear transfer in Tetrahymena thermophila cells fused by electric field.

This paper reports on electrofusion of Tetrahymena thermophila and on the reorganization of the cytoskeleton in fused cells. Important factors influencing the fusion yield are the number of electric pulses, the strength of alternate current field and cell density. The process of cell fusion consists of a mutual intermingling of cell membranes following their deformation at the contact zone, followed by the formation of cytoplasmic bridges and simultaneous disruption of portions of the submembranous cytoskeleton (epiplasm) and alveolar sacs. The course of further changes in cell organization depends on the polarity of fused cells. Homopolar fusion partners integrate by gradual translocation of portions of cortical cytoskeletal elements. In contrast, cortical integration of heteropolar fused cells is limited. Cytoskeletal integration is particularly promoted if the cells are incubated in non-growing conditions. Cortical integration leads to a high frequency of micronuclear transfer when a micronucleate strain is used as a donor and an amicronucleate strain is used as a recipient in the fusion experiments.

Induction of cybrid strains of Tetrahymena thermophila by electrofusion.

In this paper, the electrofusion of the ciliated protozoan, Tetrahymena thermophila, is described. Deciliated cells were brought into close membrane contact by dielectrophoresis in a weakly conductive medium. Then cell fusion was induced by application of repeated electric pulses. Up to 20 prestarved, logarithmic or stationary phase cells of the same or different mating types may form a single giant cell. The polykaryons are fully able to regenerate cilia and became motile. After an initial process of partial integration the polykaryons yield viable clones by separation of components. Cytoplasmic exchange between fused components occurs before separation. Cytoplasmically inherited chloramphenicol resistance was transmitted from one strain to another by electrofusion and drug-resistant cybrid strains were generated.

Subunit arrangement of Escherichia coli F1-ATPase studied by scanning transmission electron microscopy.

The shape and the arrangement of subunits in Escherichia coli F1-ATPase (ECF1) lacking the delta subunit have been explored with a high performance scanning transmission electron microscope. In tilting experiments, the ECF1 molecule appeared as a flat cylinder whose width (approx. 120 A) was about twice its height. The symmetry of front view projections of ECF1 has been investigated by computer analysis. In a population taken at random from the data bank, one third of the particles showed five-fold radial symmetry components, one third six-fold radial symmetry components and the last third no typical symmetry. The six-fold radial symmetry was consistent with a hexagonal arrangement of six large peripheric masses, which probably correspond to the three alpha and the three beta subunits of ECF1. The five-fold radial symmetry was tentatively explained by a fusion of two juxtaposed peripheric subunits. Lateral projections showed a zig-zag organization of the large masses, suggesting that the large alpha and beta subunits are located on two levels, with some degree of intercalation between the subunits of the two levels.

A STEM approach to the study of the proteinaceous architecture of a ribosome.

We used a high-resolution STEM to attempt to recognize the spatial localization of the macromolecular components of ribosomes isolated from Tetrahymena pyriformis. Images obtained either by the annular dark field detector or by the ratio-contrast mode (Z-contrast) show a ribosome as constituted of rounded dark areas, the number and size of which are compatible with those of the protein molecules recognized by biochemistry. Images of different ribosomes, when similar orientations are observed, exhibit a roughly constant pattern of the dark areas. A set of images of a tilted ribosome allows to recognize that some masses are in fact two more superimposed components. We propose that the dark areas represent essentially the proteinaceous architecture of the ribosome.

The mitoribosomes of a chloramphenicol-resistant cytoplasmic mutant of Tetrahymnea pyriformis differ from those of the wild strain.

The spontaneous CAP-resistant mutant, STR1, has been isolated from the sensitive St-strain of Tetrahymena pyriformis (Curgy et al., Biologie Cellulaire 37, 51-60, 1980; Perasso et al., Biologie Cellulaire 37, 45-50, 1980). The goal of the present work is to disclose if the resistance character is due to a modification in the mitoribosomes and if the CAP-treatment induces changes in their abundance and in their physico-chemical properties.The results show that the resistance character of the mutant is due to a reduced affinity of its mitoribosomes for CAP. This difference can be explained by modifications of at least one protein which is probably coded for by the mitochondrial genome.The mitoribosomes from CAP-treated sensitive cells tend to dissociate into their subunits and the electrophoretic pattern of their proteins suggests that at least two mitoribosomal proteins are necessary to bound the two subunits together. These proteins are probably translated in mitochondria.Finally, the CAP-treatment induces a decrease of the abundance of mitoribosomes in the sensitive cells whereas it induced an increase in the resistant cells. The latter change can be regarded as a regulatory mechanism owing to which a loss of efficiency of the mitoribosomes is compensated by their enlarged abundance.

[Ultrastructural characteristics of the cortex and of the membranelles of the ciliate Espejoia mucicola (Oligohymenophora, Hymenostomata, Tetrahymenina)]. / Caractéristiques ultrastructurales du cortex et des membranelles du cilié Espejoia mucicola (oligohymenophora, hymenostomata, tetrahymenina)

It was shown in a detailed study of the cortex and buccal organelles of Espejoia that, at the ultrastructural level, the general plan of organization of this ciliate conforms to that of Tetrahymenina. Specific variations seen in the cortex and membranelles bring out the very pronounced affinities of Espejoia to the genus Glaucoma and other related ciliates. In view of the foregoing and on the basis of reccent findings on morphogenesis, we confirm the taxonomic position of Espejoia mucicola among Tetrahymenina in the family Glaucomidae.