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.

The conserved centrosomal protein FOR20 is required for assembly of the transition zone and basal body docking at the cell surface.

Within the FOP family of centrosomal proteins, the conserved FOR20 protein has been implicated in the control of primary cilium assembly in human cells. To ascertain its role in ciliogenesis, we have investigated the function of its ortholog, PtFOR20p, in the multiciliated unicellular organism Paramecium. Using combined functional and cytological analyses, we found that PtFOR20p specifically localises at basal bodies and is required to build the transition zone, a prerequisite to their maturation and docking at the cell surface and hence to ciliogenesis.

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.

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.

Multiple-axis tomography: applications to basal bodies from Paramecium tetraurelia.

Background Information: Transmission electron tomography is becoming a powerful tool for studying subcellular components of cells. Classical approaches for electron tomography consist of recording images along a single-tilt axis. This approach is being improved by dual-axis reconstructions and/or high-tilt devices (tilt angle>+/-60 degrees) on microscopes to compensate part of the information loss due to the 'missing wedge' phenomena.

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.

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.

Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold.

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as delta-, epsilon-, or eta-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of epsilon-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules.