Structural and functional studies of cilia and Intraflagellar Transport by 3D cryo-EM
Cryo-transmission electron microscopy (cryo-EM) is a powerful technology that can be used to reveal the three-dimensional architecture and the assembly of macromolecular machines, organelles and cells. In cryo-EM the specimen is rapidly frozen in its native state allowing the best structure preservation of the biological object. We use cryo-EM and electron tomography to reveal the structural basis of flagella and cilia assembly and motility. Cilia and flagella play important roles in motility and sensory functions of eukaryotic cells ranging from protists to humans. Defects in ciliary assembly and function lead to a wide range of human diseases, called ciliopathies. The cilium/flagellum structure is based on a stereotyped assembly of microtubules, called the axoneme, which consists of more than 300 different polypeptides, organized in a variety of macromolecular complexes. The assembly takes place at the distal tip of the cilium and the transport of ciliary proteins from the cell body to the tip is mediated by the intraflagellar transport (IFT) machinery. IFT is the bidirectional movement of multiprotein complexes (named IFT particles) along the microtubules. IFT particles are formed by at least 20 IFT proteins and have been observed by EM to build linear arrays, called IFT trains. The movement of IFT trains and their cargo proteins along the microtubules is catalysed by dynein and kinesin motors. Using electron tomography we have shown that the IFT anterograde trains (cell body to tip) and the retrograde trains (tip to cell body) have a different morphology. Therefore the ciliary tip, site of dynamic growth and resorption of the axoneme, is also thought to be a main point of regulation and remodeling of IFT trains.
We currently study the assembly of the cilium by investigating the detailed 3D structure, the molecular arrangement, the protein composition, and the dynamics of the ciliary tip complex and of the IFT trains.