Institut de Biologie Intégrative de la Cellule

The centrosome is a tiny dense organelle located near the geometric centre of interphase cells, that duplicates only once per cell cycle. It is composed of two centrioles, the mother and the daughter, highly conserved eukaryotic microtubule-based organelles, surrounded by the pericentriolar material, responsible for the nucleation and the organisation of the microtubule networks. In cycling cells, as the principal microtubule organizing centre, the centrosome directs several fundamental processes, such as intracellular trafficking, cell polarity and division. In resting cells, the mother centriole turns into a basal body which docks at the plasma membrane and develops a non-motile sensory “primary” cilium. In some epithelial cells, a multiplication of basal bodies yields multiple motile cilia at the cell surface. By their dual function in motility and signal transduction, these “cell antennae” intervene in many physiological and developmental processes and their alterations in mammals result in a wide array of disorders, recognized as ciliopathies. More recently, loss of cilia has been described in many tumours so that ciliogenesis appears to be relevant to cancer.
In all cases, ciliogenesis is a multi-step process varying between both organisms and cellular types, but which involves key steps common to all systems: centriole/basal body duplication, basal body maturation, migration and anchoring at the cell surface, and eventually ciliary growth. Building such complex cell organelles requires, in addition to correct genomic expression, a set of appropriate molecular interactions in the 4D (space-time) space.

Basal bodies and cilia of Paramecium

Left : longitudinal view of a basal body and its cilium showing their continuity through a transition zone. Right : cross-sections at the levels marked by the black bars of (1) a basal body (BB) showing the nine-fold symetry of triplets of microtubules and of the cartwheel (cw), (2) a transition zone (TZ), and (3) a cilium showing the same nine-fold symetry of doublets of microtubules and the presence of a central pair of microtubules. Bar=200nm

Our interest have three main focuses:
1) Dissect molecularly and spatially the basal body anchoring process
2) Understand how defects in ciliogenesis may lead to tumour formation by studying the deubiquitinase CYLD, shown to play a role in basal body anchoring in mouse
3) The use of a ciliate microorganism - Paramecium tetraurelia - as a model for PCD (Primary ciliary dyskinesia) since Paramecium offers several technical facilities allowing rapid and efficient molecular and biochemical analyses of proteins involved in ciliary motility.

1)We are currently using a combination of biochemical, molecular and cell biology techniques with cryo-electron tomography in order to:
a) Identify interacting protein partners involved in the anchoring process using the proximity dependent biotin identification (BioID) technique and dissect their functional interactions. This will be dissected, first, in a unicellular multiciliated model, the ciliate Paramecium, which offers experimental facilities for post-genomic analysis such as RNAi, tagged-protein expression and cell biology analyses. It will provide a stepping-stone to our work in mammalian cells.
b) Gain insights into the structural mechanisms underlying the transition zone formation at nanometric resolution.

The depletion of the FOR20 protein in Paramecium impairs basal body anchoring

Left : view in confocal microscopy of a wild type (top) and a FOR20-depleted (bottom) paramecium stained with antibodies specific for basal bodies in green and for the cortical cytoskeleton (epiplasm) in red. Instead of being anchored at the surface, basal bodies stay in the cytoplasm in FOR20-depleted cells. Right : transmission electron microscopy view of a wild type (top) and a FOR20-depleted (bottom) paramecium. Basal bodies are anchored at the cell surface in wild-type cells, whereas they are found free in the cytoplasm or close to the cell surface but unanchored in FOR20-depleted cells.

2) Our recent work in mammalian cells has revealed an unexpected role of the deubiquitinase CYLD in basal body migration/anchoring. CYLD was originally identified as a tumour suppressor gene that is mutated in familial cylindromatosis, a genetic condition that predisposes patients to the development of skin appendages tumours. Most of the mutations of CYLD found in human cylindromas are predicted to cause carboxy-terminal truncations and catalytic inactivation of the deubiquitinating domain. Ubiquitination is a widespread mechanism for regulating many aspects of cell physiology, including protein degradation, DNA repair, receptor endocytosis, apoptosis…. The role of ubiquitination in basal body anchoring may be part of a more general function in ciliogenesis. In addition, we would like to understand how the ciliogenesis defects observed in the Cyld mouse mutant, mimicking the smallest truncation found in the human pathology, can be related to tumour formation.

A mutation in the CYLD gene impairs basal body anchoring

Left : double-staining analysis of basal bodies (in green) and cilia (in red) in ependymal cell cultures of wild type (top) and CyldΔ932 mutant (bottom) mice using specific antibodies 15 days after serum removal. Multiciliated cells show a large number of basal bodies irrespective of the presence or absence of cilia. Right : transmission electron microscopy image of trachea of wild type (top) and CyldΔ932 mutant (bottom) mice. In the wild type trachea, numerous regularly spaced anchored basal bodies are evident at the apical membrane. All of them show an axoneme. In the mutant trachea, the basal bodies stay in the cytoplasm and are not anchored at the apical membrane.

3) We set collaborations with clinical geneticists working on PCD in order to analyse functionally novel PCD candidate genes in our model, Paramecium.

C11orf70 Mutations Disrupting the Intraflagellar Transport-Dependent Assembly of Multiple Axonemal Dyneins Causing Primary Ciliary Dyskinesia.

A: Analysis of the swimming velocity of C11orf70 and control knockdown cells. B: Transmission electron micrographs of Paramecium cilia in cross-section showing normal 9+2 arrangement for the control knockdown (left) and absence of the outer (white arrows) and inner (gray arrows) dynein arms in the C11orf70 knockdown cells (right). Scale bar, 100 nm. C: Paramecia expressing the Paramecium C11orf70-GFP show a very high level of cytoplasmic GFP staining with protein also present in the cilia. The cell body was overexposed in order to allow the detection of cilia. Scale bars, 20 µm. D: Paramecium expressing either GFP alone (left), C11orf70-GFP (middle), or IFT46-GFP (right) were subject to deciliation after which the cilia were allowed to regrow for 15 min, in order to study the localization of the tagged proteins during reciliation. Direct GFP fluorescence (green, top) and polyclonal antibody staining against polyglutamylated tubulin (red, middle) are shown along with the merge (bottom). Paramecia expressing GFP are used as a negative control since the GFP protein enters the cilia passively (<27 kDA). The GFP staining is found distributed homogeneously along the entire length of the cilia. In contrast, IFT46-GFP accumulates at the ciliary tips during ciliary regrowth, as expected for an IFT-B family member. C11orf70-GFP is found at much higher levels in the cytoplasm than IFT46-GFP and is also present in the cilia. Upon ciliary regrowth, C11orf70- GFP behaves in a similar fashion to IFT46 GFP in cilia with an accumulation of both proteins observed at the ciliary tips. Scale bars, 10 µm. White boxes show higher magnification.

Keywords :

Centrosome, centriole, ciliogenesis, basal-body, transition zone, cryo-electron tomography, CYLD, ubiquitination, tumor suppressor, Primary ciliary dyskinesia