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.
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.
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.
3) We set collaborations with clinical geneticists working on PCD in order to analyse functionally novel PCD candidate genes in our model, Paramecium.
Keywords :
Centrosome, centriole, ciliogenesis, basal-body, transition zone, cryo-electron tomography, CYLD, ubiquitination, tumor suppressor, Primary ciliary dyskinesia
Contact
TASSIN Anne-Marie [Senior Researcher - CNRS]
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