Structural analysis of Intraflagellar Transport

Cilia/Flagella (interchangeable terms) are complex structures emanating from the surface of a wide variety of eukaryotic cells. They consist of a microtubule-based axoneme, which is built onto a centriole-derived basal body and is surrounded by the ciliary membrane. Cilia are involved in a large number of biological processes, including cell motility (e.g. in protozoans or sperm cells), signal reception (e.g. in the rod and cone cells of the retina) or movement of fluids and objects across the cell surface (e.g. in the fallopian tubes or in the respiratory tract). Defective assembly and maintenance of cilia is linked to a variety of human diseases, commonly referred to as "ciliopathies", with phenotypes including blindness, deafness, obesity, sterility, cyst formation, but also severe developmental defects. The developmental abnormalities were recently explained by the finding that primary cilia (non-motile cilia found on the surface of almost all mammalian cells) serve an important function in various signal transduction pathways (including Wnt and Shh signaling), mainly by specific localization of receptors and downstream molecules to the ciliary compartment. The central process in ciliary assembly is called Intraflagellar Transport (IFT), a transport mechanism which moves building blocks from the cell body to the tip of the cilium (anterograde transport) and brings turnover products back to the cell body (retrograde transport). The key platform for IFT is the highly conserved IFT complex, which is found in organisms as diverse as single-celled green algae and humans and consists of about 20 proteins forming two distinct sub-complexes responsible for binding to both the cargo proteins as well as the motors that provide the force for movement. Since the discovery that cilia play an important role in signal transduction pathways, a lot of effort has been put into biochemical analysis of the IFT complex. Nevertheless, our current knowledge about IFT, especially concerning the overall architecture of the IFT complex and the regulation of the different steps in the transport process, remains largely obscure. Most notably, only the crystal structure of one of the smallest IFT subunits (human IFT25) is currently known, and extensive structural analysis of other subunits as well as bigger sub- complexes will certainly be essential for a deeper mechanistic understanding of IFT. The proposed work aims at gaining a more detailed understanding of the architecture of the IFT complex in a number of different ways. Subunits and sub-complexes (from the model organism Chlamydomonas reinhardtii) will be produced recombinantly to determine their three-dimensional structure. Furthermore, detailed analysis of protein-protein interactions (by systematic pull-down and co-expression studies) and identification of the responsible domains in the various IFT proteins will increase our understanding of the overall IFT complex architecture. Electron microscopy will be used to visualize larger assemblies of recombinant IFT proteins (from 300 kDa upwards). Lastly, the gained knowledge from the structural studies will be tested in vivo in cells of Chlamydomonas reinhardtii. The combination of these approaches will shed light on the molecular basis of the IFT process and help to understand the defects caused by certain mutations of IFT proteins in human patients.