Francine Iftode

Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission⋆

Abstract One main difference between basal bodies and centrioles resides in the expression of their polarity: centrioles display a structural nine‐fold radial symmetry, whereas basal bodies express a circumferential polarity, thanks to their asymmetric set of rootlets. The origin of this polarity during organelle duplication still remains under debate: is it intrinsic to the nine‐fold structure itself (i.e. the nine microtubular triplets are not equivalent) or imposed by its immediate environment at time of assembly? We have reinvestigated this problem using the Ciliate Paramecium, in which the pattern of basal body duplication is well known. In this cell, all basal bodies produced within ciliary rows appear immediately anterior to parental ones. Observations on cells fixed with the tannic acid protocol suggest that, to be competent for basal body assembly, parental basal bodies have to be individually associated with a complete set of rootlets (monokinetid structure). During pro‐basal body assembly, full microtubular triplets were detected according to a random circumferential sequence; during the whole process, the new basal body and its associated rootlets maintained structural relations with the parental monokinetid structure by way of specific links. These results strongly suggest that basal body and associated rootlets (kinetid) polarity is driven by its immediate environment and provide a basis for the structural heredity property observed by Sonneborn some decades ago.

Development of surface pattern during division in Paramecium: III. Study of stomatogenesis in the wild type using antitubulin antibodies and confocal microscopy

Summary Stomatogenesis, during the vegetative division of Paramecium, is a complex morphogenetic process involving massive proliferation of basal bodies and their progressive patterning to generate a new oral apparatus in the immediate vicinity of the old one. This new oral apparatus will be inherited by the posterior daughter cell while the old one remains in the anterior daughter cell. We have carried out a detailed description of the whole process, using new antibodies and confocal microscopy, allowing visualization of all basal bodies and a number of other cytoskeletal elements, thus completing our previous description of morphogenesis. The following inferences could be made: 1) The new oral structures are exclusively formed from the anarchic field by a two step wave of basal body duplication. These two steps strictly parallel, slightly in advance, those we have previously detailed at the level of the “somatic” cortex, suggesting *) that a common set of biochemical cascades is involved, **) that the OA may be triggering these cascades. 2) The initial proliferation of basal bodies occurs in the immediate vicinity of the parental “paroral kinety” which itself remains invariant and appears to dictate the progressive patterning of the oral anlage (as can be inferred from the staining intensity, distance and alignment of the basal bodies of the anarchic field with respect to the paroral kinety). This suggests that the concept of cytotaxis or structural guidance can be extended to the genesis of an elaborate set of basal bodies. 3) The number of basal bodies within the anarchic field is determined during this formation of the new oral apparatus. Thus, the size and possibly part of the pattern of the new OA is determined in one generation, with storage of morphogenetic potentialities used in the next generation. In this process, a slight cortical rotation around the cell axis is effected at each division. 4) The whole morphogenetic assemblage up to the level of the whole cell displays an overall left-right asymmetry which is progressively built up upon the basic asymmetry of the basal body.

The surface pattern of Paramecium tetraurelia in interphase: an electron microscopic study of basal body variability, connections with associated ribbons and their epiplasmic environment

Summary In Paramecium tetraurelia, thousands of basal bodies with their three associated rootlets are anchored into the submembranous skeleton, the epiplasm, which is itself segmented into cortical units. In this study, we redescribe more precisely (after Pitelka [31], Hufnagel [19] and Allen [1]) the architecture of these structures after detergent treatment and tannic acid contrast reinforcement. Although this technique leads to loss of membranes and of some labile cytoskeletal elements, it allows to better underline how the three rootlets are linked together and to the basal body. The postciliary microtubules and ciliary rootlets (Kd fibers) are bound together by a “fingered node”, and the transverse microtubules to the previous fibers by dense links. The anterior basal body of the pairs is linked to the ciliary rootlet by a newly identified architecture in this species, the “bone-like node” [1]. A specific ciliary domain can be defined in the epiplasm of each cortical unit: epiplasmic rings encircling the basal bodies are closely apposed on the thicker part of the epiplasm and can include some apertures along the basal bodies towards the cilia. The thickness of the epiplasm seems to vary according to the presumptive elongation direction of the cortical units. Each basal body associated rootlet is specifically linked to the epiplasm itself. Because of their unexpected set of linkage organization, these rootlets may ensure the ciliary cohesiveness in the interphase cell as well as the transmission of the ciliary polarity during division as it was previously discussed [19]. We also show for the first time that there is a variability of the basal body architecture at the cellular level: its height, and the number of cartwheel elements change depending on its location on the cortex and its morphogenetical history; the longest basal bodies with 5 cartwheel elements are found in territories which result from hyperduplication and become invariant, i.e. do not undergo duplication during the next divisions, as is the case along the sutures and in the left oral and cortical areas [22]. These results are discussed in terms of morphogenetical behavior.

Nuclear and cortical regulation in doublets of Paramecium: I. Interactions between macronucleus and contractile vacuoles and their implication on the frequency of autogamy

Summary The macronucleus of ciliates is a huge “bag” of DNA whose mode of segregation during cell division is still not understood (no chromosome condensation, no spindle, no centrosomes or functional equivalents). We unexpectedly obtained some new hints on this process through the analysis of doublets of Paramecium . We had previously noted [32] that autogamy occurred at a seemingly higher frequency in regulating lines. In the present work, we studied this process in P. tetraurelia and in P. undecaurelia which has a long, more convenient, interautogamous interval. We confirmed a highly significant decrease in duration of the interautogamous interval in both species and tried to determine its causes. The process of regulation which concerns the return of doublet state to the singlet one was previously described in part [24. 61, 35]. We observed its course through successive vegetative and sexual cycles. We first described precisely the contractile vacuole (CV) systems, and their number, extension and relations with the nuclear division events, in normal cells and in tam mutant cells of P. tetraurelia which are known to show abnormal nuclear division [6, 58]. We deduced that cortical and nuclear events are correlated during division by means of the contractile vacuole system (immunocytology and TEM observations). In regulating doublets, the enlarged cell volume leads to an increase in the number of CVs on two meridians M1, M2 and to the changes of their positioning and extension. CVM2 disappears and the number of CVs decreases when the angle between both oral apparatuses decreases from 180° towards 90°. Our results strongly suggest that abnormal number, size and position of macronuclei in doublets are related to number, spreading and positioning of the CVs. During division of doublets, the macronuclei appear to be “quartered” between both dorsal surfaces bearing the CVMs, and this leads to gross asymmetries in the distribution of the macronuclear DNA to the two daughter doublets [8]. Abnormal numbers of micronuclei and macronuclear anlagen are observed along with the evolution of the regulation process and, in turn, appear to lead to numerous anomalies at autogamy, a process which also requires correct intracellular positioning of meiotic and postmeiotic nuclei. From such observations, we deduced that contractile vacuoles, through their microtubular rootlets, exert a major function in ensuring proper macronuclear segregation during amitosis, such as the role played by “atractophores” in other protists during extranuclear pleuromitosis [57].

Nuclear and Cortical Regulation in Doublets of Paramecium: II. When and How do Two Cortical Domains Reorganize to One?

Abstract 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).

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 programs capabilities.

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

SummaryThe 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.