Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia.

The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.

Centrin deficiency in Paramecium affects the geometry of basal-body duplication.

BACKGROUND: Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication. RESULTS: We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies. CONCLUSIONS: Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.

Gamma-tubulin and MTOCs in Paramecium.

The characterization of the two Paramecium gamma-tubulin genes, gammaPT1 and gammaPT2, allowed us to raise Paramecium-specific antibodies, directed against their most divergent carboxy-terminal peptide and to analyze the localization and dynamics of gamma-tubulin throughout the cell cycle. As in other cell types, a large proportion of the protein was found to be cytosolic, but in contrast to the general situation, gamma-tubulin was found to be permanently associated to four types of sites: basal bodies, the micronuclear compartment--within which mitotic and meiotic spindles develop without membrane breakdown, the pores of the contractile vacuoles and the cytoproct which are cortical microtubular organelles fulfilling excretory functions. In addition, a transient site of gamma-tubulin and microtubule assembly was observed at the site of nuclear exchange during conjugation. This complexity accounts for the nucleation of most of the numerous and diverse microtubule arrays present in Paramecium. The sites and mode of nucleation of the microtubule bundles formed in the macronuclear compartment during division remain unclear. These observations lead us to discuss the relationships between microtubules, gamma-tubulin and MTOCs.

A Centrin3-dependent, transient, appendage of the mother basal body guides the positioning of the daughter basal body in Paramecium.

Basal bodies are tightly controlled not only for their time of duplication but also for their movements, which ensure proper division and morphogenesis. However, the mechanisms underlying these movements only begin to be explored. We describe here a novel basal body appendage in Paramecium, the anterior left filament (ALF), which develops transiently from the mother basal body before duplication and disassembles once the new basal body is docked at the surface. By comparing the ultrastructure of dividing wild type cells to that of cells defective in basal body duplication, either by depletion of conserved proteins required for basal body assembly, or by mutation, we showed 1) that assembly of the ALF requires PtCen3p, one of the two basal body specific centrins and 2) that absence of the ALF correlates with a failure of the newly assembled basal bodies to tilt up to their docking site at the surface. This correlation suggests that the function of the ALF consists in anchoring centrin-containing contractile fibers which pull up the new basal body toward its site of docking. The presence in T. thermophila of an ALF-like appendage suggests the conservation of an ancestral mechanism ensuring the coupling of basal body duplication and cell morphogenesis.

Functional diversification of centrins and cell morphological complexity.

In addition to their key role in the duplication of microtubule organising centres (MTOCs), centrins are major constituents of diverse MTOC-associated contractile arrays. A centrin partner, Sfi1p, has been characterised in yeast as a large protein carrying multiple centrin-binding sites, suggesting a model for centrin-mediated Ca2+-induced contractility and for the duplication of MTOCs. In vivo validation of this model has been obtained in Paramecium, which possesses an extended contractile array - the infraciliary lattice (ICL) - essentially composed of centrins and a huge Sfi1p-like protein, PtCenBP1p, which is essential for ICL assembly and contractility. The high molecular diversity revealed here by the proteomic analysis of the ICL, including ten subfamilies of centrins and two subfamilies of Sf1p-like proteins, led us to address the question of the functional redundancy, either between the centrin-binding proteins or between the centrin subfamilies. We show that all are essential for ICL biogenesis. The two centrin-binding protein subfamilies and nine of the centrin subfamilies are ICL specific and play a role in its molecular and supramolecular architecture. The tenth and most conserved centrin subfamily is present at three cortical locations (ICL, basal bodies and contractile vacuole pores) and might play a role in coordinating duplication and positioning of cortical organelles.

An Sfi1p-like centrin-binding protein mediates centrin-based Ca2+ -dependent contractility in Paramecium tetraurelia.

The previous characterization and structural analyses of Sfi1p, a Saccharomyces cerevisiae centrin-binding protein essential for spindle pole body duplication, have suggested molecular models to account for centrin-mediated, Ca2+-dependent contractility processes (S. Li, A. M. Sandercock, P. Conduit, C. V. Robinson, R. L. Williams, and J. V. Kilmartin, J. Cell Biol. 173:867-877, 2006). Such processes can be analyzed by using Paramecium tetraurelia, which harbors a large Ca2+ -dependent contractile cytoskeletal network, the infraciliary lattice (ICL). Previous biochemical and genetic studies have shown that the ICL is composed of diverse centrin isoforms and a high-molecular-mass centrin-associated protein, whose reduced size in the démaillé (dem1) mutant correlates with defective organization of the ICL. Using sequences derived from the high-molecular-mass protein to probe the Paramecium genome sequence, we characterized the PtCenBP1 gene, which encodes a 460-kDa protein. PtCenBP1p displays six almost perfect repeats of ca. 427 amino acids (aa) and harbors 89 potential centrin-binding sites with the consensus motif LLX11F/LX2WK/R, similar to the centrin-binding sites of ScSfi1p. The smaller (260-kDa) protein encoded by the dem1 mutant PtCenBP1 allele comprises only two repeats of 427 aa and 46 centrin-binding sites. By using RNA interference and green fluorescent protein fusion experiments, we showed that PtCenBP1p forms the backbone of the ICL and plays an essential role in its assembly and contractility. This study provides the first in vivo demonstration of the role of Sfi1p-like proteins in centrin-mediated Ca2+-dependent contractile processes.