The cilium is an organelle that protrudes from the cell body and is responsible for the motility of unicellular organisms and of vertebrate cell types such as sperm cells. In addition, most vertebrate cells have primary non-motile cilia important for sensory reception and signalling. The assembly and function of cilia rely on intraflagellar transport (IFT), the bi-directional movement of macromolecules between the cell body and the cilium. As cilia do not contain ribosomes, IFT is required to move the approximately 600 different ciliary proteins from their site of synthesis in the cell body to their site of function in the cilium. IFT is powered by kinesin and dynein motors, which move cargoes along the microtubule-based axoneme of the cilium. The interaction between motors and cargoes is mediated by the IFT complex, a 1.6 MDa complex formed by 20 different proteins. Despite the importance of the IFT complex, very little is known about its architecture and how it is regulated. In this proposal, we want to address both aspects using a combination of structural and functional studies. The structural analysis of the IFT complex is daunting given its size and complexity. We are proceeding with the biochemical reconstitution of the core subcomplexes, which we plan to analyze using X-ray crystallography and electron microscopy. To date, we have solved the X-ray structure of a dimeric complex between an IFT GTPase and its binding factor, and have reconstituted one of the two core complexes (the 8-subunit IFT-B complex) in amounts and purity suitable for structural studies. While these studies are progressing, we plan to use similar approaches to tackle the other core complex (IFT-A) and the plethora of ciliary GTPases, with the ambitious goal of understanding the architecture and regulation of the the entire IFT complex. This will shed light on the molecular basis of ciliogenesis and the pathological consequences of its disruption.
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