HIGH RESOLUTION STRUCTURAL STUDY OF THE IN SITU AND PURIFIED INTRAFLAGELLAR TRANSPORT (IFT) COMPLEX: KEY MOLECULAR MACHINERY ESSENTIAL FOR CILIOGENESIS AND THE CELLULAR BASIS FOR PKD (POLYCYSTIC KIDNEY DISEASE)

Cilia and flagella are hair-like filaments located on the surface of many types of cells, including the one-celled green alga Chlamydomonas and kidney tubule cells. They can function as both motile and sensory organelles checking the external environment. Several heritable disorders have been recently shown to be ciliary diseases; the most common is the polycystic kidney disease (PKD), whose autosomal dominant variant affects 12.5 million people worldwide. The disease is characterized by uncontrolled division of kidney tubule epithelial cells, leading to formation of large cysts and kidney failure. The key genes for PKD have been shown to code for polycystins, calcium channels located on the ciliary membrane; these respond to the cilium bending caused by urine flux and in turn regulate the cell cycle, thus preventing cyst formation. PKD can result from defects in the polycystin proteins themselves or from an inability to form normal cilia. Analysis of mouse mutant models for PKD has revealed that targeting of polycystins to the ciliary membrane is dependent on a process called IntraFlagellar transport (IFT); it conveys ciliary components from the cell body, where they are synthesized, to their definitive site in the cilium, and recycles disposed components back to the cell body. IFT involves multi-protein complexes, the IFT particles, that carry ciliary components and are moved by molecular motors. IFT particles play a central role in cilia assembly and function, and hence in the pathogenesis of ciliary diseases. A good amount of molecular information is now available on IFT proteins. On the contrary, a high resolution ultrastructural analysis of IFT particles is still missing. Since a full understanding of IFT machinery is fundamental to establish the causes of PKD as well as of other ciliary-dependent diseases, our project is aimed to fill this gap and to obtain a high resolution 3D model for both in situ and isolated IFT particles.