Eukaryotic cilia and flagella are specialized organelles that are highly conserved from protists to mammals. These organelles consist of the membrane-sheathed axoneme, which is an extension of the mother centriole, and at least 360 associated proteins. Cilia have attracted much attention in recent years because of their role in the transduction of extracellular signals and their association with an expanding number of human disorders. Such disorders include respiratory distress syndrome, male sterility, polycystic kidney disease, retinal degeneration, and Bardet-Biedl syndrome.

Cilium assembly is initiated by the docking and fusion of mother centriole to the apical membrane of the cell. Cilia are assembled and maintained through intraflagellar transport (IFT). This process is carried out by two distinct protein complexes, IFT complex A and B, which contain at least six and sixteen subunits, respectively. These complexes transport ciliary cargos within cilia and flagella by interactions with the microtubule-associated motor proteins kinesin-II and dynein.

Whereas the complexes IFT A/B have been studied for some years, little is known about their architecture and assembly. The lack of high-resolution structural information on these complexes has now become a limiting step in gaining an understanding of their function at the molecular level. We are currently developing protocols for producing soluble and stable protein complexes by reconstituting in vitro or co-expressing all components in bacterial or baculovirus-insect cell expression system. Our goal is to elucidate at the atomic level the assembly mechanisms of the protein complexes for cargo transport to and within the cilium.

We mainly use X-ray crystallography to visualize these proteins and their complexes. Other biophysical techniques such as dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation will as well be employed to study the architecture and assembly of the protein complexes. Large assemblies will also be examined by cryo-electron microscopy. Our structural studies will be complemented by site-directed mutagenesis and in vitro/vivo experiments to test our mechanistic hypotheses. The available new structures will enhance our understanding of how these complexes function and provide hints as to how their malfunction leads to human diseases.