Structural Studies of the Intraflagellar Transport Complexes

Cilia and flagella are specialized organelles which are highly conserved from protists to mammals. They consist of a membrane-sheathed axoneme and nearly 700 associated proteins. These organelles play essential roles in cell signaling and have recently been associated with a plethora of human disorders. Cilia and flagella are devoid of ribosomes and membrane-bound vesicles; they are assembled and maintained by a process known as intraflagellar transport (IFT), which imports ciliary components from the cytoplasm and deploys them to target sites within the enclosed compartment of the organelle. IFT is carried out by two multi-protein complexes, IFT-A and IFT-B, which associate with motor molecules to move bidirectionally along the microtubules of the axoneme. IFT is responsible for both the transport of ciliary components and the removal of turnover products. While these complexes have been studied for several years, little is known about their architecture and how they are assembled. The lack of high-resolution structural information on these complexes has limited our understanding of how IFT functions at the molecular level. The proposed research focuses on the structural characterization of these protein complexes as a means to provide this missing information. We will use a combinatorial strategy in our structure determination, which will include crystallography and electron microscopy methods. Our structural studies will be complemented by structure-based mutagenesis and in vivo experiments to test our mechanistic hypotheses. The resulting structural information will enhance our understanding of how the two IFT complexes function and will provide hints as to how their malfunction leads to a broad spectrum of human disorders. This information may potentially guide the design of therapeutic drugs to provide much needed support for millions of patients worldwide.

The originally proposed project aimed to elucidate three-dimensional structural information of protein complexes essential for the assembly of a eukaryotic organelle called the cilium. Cilia are highly conserved in all ciliated cells and exist in most eukaryotes from protists to mammals. They are like antenna residing on the cell surface and playing essential roles in cell signaling. Defects in cilia have recently been associated with a plethora of human disorders, which are collectively called ciliopathies. Although the cilium consists of hundreds of proteins, it is devoid of ribosomes and membrane-bound vesicles. The cilium is assembled and maintained by a process known as intraflagellar transport (IFT), which imports ciliary components from the cytoplasm and deploys them to target sites within the enclosed compartment of the organelle. IFT is carried out by two multi-protein complexes, IFT-A and IFT-B, which associate with motor molecules to move bidirectionally along the microtubules of the axoneme. IFT is responsible for both the transport of ciliary components and the removal of turnover products. We had tried very hard to reconstitute the two IFT complexes by coexpressing their components in bacteria and/or insect cells. However, it turned out that the complexes were structurally dynamic and we could not obtain fully assembled complexes for structural studies. Therefore, we later turned to characterize a related cellular structure, the centriole, which anchors the cilium onto the plasma membrane. Besides its essential role in ciliogenesis, the centriole is also required for centrosome formation. Despite more than 100 years of microscopic observation, our understanding of centrioles is limited to low resolution. Extensive cell biological research on centriole duplication over the past decade has led to the identification of a core set of five centriolar proteins. However, there are no high-resolution structures available for most of these proteins, and the mechanistic basis of their contributions to centriole formation remains unclear. We have investigated how SAS-5 and SAS-6 participate in centriole assembly from the structural point of view as well as how ZYG-1, the master regulator of centriole assembly, is recruited to procentrioles. To the end, we have determined the crystal structure of the central coiled-coil domain of SAS-6 and further characterized its interaction with another SAS-5, characterized the oligomerization and potential function of SAS-5 in centriole assembly using various biophysical techniques, and determined the crystal structures of both the polo-like kinase ZYG-1 from nematodes and its homolog Plk4 from fruit flies. Our work has greatly advanced our understanding of centriole duplication at the atomic level and provides a new knowledge basis for future mechanistic studies of cell-cycle control and cilium biogenesis at the molecular level.