Centriole and cilia: organization and dynamics – CCOD

The structure of cell components, as well as their dynamic, takes place in a 3D environment. De facto, we have to answer the structural questions using methods which allow us to obtain 3D volumes of the cell components at the highest resolution possible. To achieve it, we use cryo electron microscopes where the sample kept at -178°C close to native state. Images taken from these microscopes and treated with appropriate software, lead to 3D structural information of the cell components. To determine the localization of components from centrioles, cilia and flagella in the 3D volume, we have to use technology to label the proteins of interest in a specific manner. We develop state-of-the-art technology to label proteins using probes capable of interacting with heavy atoms visible at the electron microscope.

Our project started in 2012, January 1st. Currently we have successfully frozen the first samples. Reconstructed 3D volumes allow us to say that the native structure of the sample has been conserved. 3D volumes of flagella from two different strains of Trypanosoma are currently being analyzed.

Confidential data

Since the project started six months ago and the discipline is highly competitive, no result can be publicly broadcasted.

Centrioles (in centrosome) and basal bodies (in cilia and flagella) are similar systems that play essential roles in cell division and motility. The deregulation of the centriole duplication contributes to tumor formation, and for basal bodies leads to the perturbation of intraflagellar transport (IFT), the mechanism responsible for assembly and function of cilia and flagella. This ultimately can lead to ciliopathies, and hence it is interesting to understand how they function.

We propose to use cryo-electron tomography, which provides 3D information of biological structures close to their native state at nanometric resolution, together with cellular and molecular biology methods, to provide new insights into the centriolar organization and the IFT dynamics. For this purpose, isolated centrioles from Drosophila melanogaster embryos and flagella from Trypanosoma brucei as well as human isolated centrosomes will be analyzed. Three topics will be addressed in this study: (1) how does the central tube of the centrosomal cartwheel relate with the symmetry preservation during centriolar duplication; (2) how do the protofilaments arrange in microtubules B and C comparing to microtubule A in centrioles and basal bodies; and (3) how do anterograde and retrograde IFT particles organize within the flagellum. This study will contribute to the understanding of basic mechanisms that regulate centrosome and cilia assembly from a structural point of view.