Cilia and Flagella, Molecular Motors, Sensory Transduction, Proteomics, Molecular Basis for Diseases Involving Cilia and Flagella

Our research is concerned with the biology of cilia and flagella, including the non-motile primary cilia that are present on most cells in our bodies and function as cell antennae, receiving signals from the environment and transmitting these to the cell body. Our findings have important implications for human development and male infertility, and for diseases of the lung, kidney, and eye, all of which contain cilia. Such diseases are known as "ciliopathies."

In many of these studies we are using the unicellular Chlamydomonas, a model flagellated organism amenable to biochemical, genetic, and molecular genetic approaches. We recently completed a proteomic analysis of the Chlamydomonas flagellum. This has resulted in a virtual "gold mine" of data that has and will continue to form the basis for many exciting projects. Because the proteins of cilia and flagella have been highly conserved throughout evolution, the human homologues of most of these proteins are readily identified. This opens the door to understanding the functions of many previously uncharacterized ciliary proteins. We currently are investigating the functions of several proteins whose homologues in humans or mice are known to cause disease, including blindness (Leber congenital amaurosis), cystic kidney disease, hydrocephalus, and syndromic ciliopathies such as Bardet-Biedl syndrome and primary ciliary dyskinesia. Typically, we explore the functions of these proteins in Chlamydomonas and then in the mouse to be sure that what we learn from Chlamydomonas is applicable to mammals.

In addition, many members of this laboratory are participating in a large-scale project to generate and identify insertional Chlamydomonas mutants for all the genes encoding flagellar proteins. Chlamydomonas cells are transformed with a selectable marker that integrates at random into the genome, disrupting any gene at the site of insertion. Using PCR, we can then readily determine the genomic sequence flanking the insert, and thus identify the mutated gene. The mutant can then be characterized structurally and biochemically to understand the function of the mutated gene.

Finally, we are studying a process called "intraflagellar transport" (IFT), which involves the active movement of multi-subunit protein particles from the base to the tip of the cilium or flagellum, and back to the base again (Fig. 1). These particles carry cargo necessary for assembly and maintenance of the cilium or flagellum, and also transport signals from the cilium or flagellum to the cell body and vice versa (Fig. 2). We are characterizing the motors responsible for this transport, the individual polypeptides that make up the IFT particles, and the proteins and protein complexes that interact with the IFT particle and generally function as cargo adaptors. These studies are providing new insights into a process that is essential for the assembly of almost all cilia and flagella.

Figure 1. The intraflagellar transport (IFT) machinery. During IFT, linear arrays of IFT particles (yellow) are transported towards the 'plus' (distal) ends of the flagellar outer doublet microtubules (blue) by kinesin-II (pink), and towards the 'minus' (proximal) ends of the microtubules by cytoplasmic dynein 1b (green). The IFT particles, which are composed of at least 19 different proteins, are believed to be carrying precursors that are necessary for the assembly of the flagellar axoneme. The IFT particles are linked to the flagellar membrane (grey lines), and there is evidence that their cargo also includes membrane proteins.

Figure 2. IFT and targeting of proteins to the flagellar compartment. Flagellar membrane proteins are carried by vesicles from the Golgi apparatus to the base of the flagellum, where they fuse with the plasma membrane of the cell. In this figure, proteins destined for the flagellar membrane are sorted into specific vesicles that are then targeted to the base of the flagellum. This sorting and targeting appears to be aided by one or more IFT-particle proteins that cycle from the base of the flagellum back through the endomembrane system, where they become associated with the proteins that are destined for the flagellar membrane. Once the vesicle is exocytosed, the IFT-particle proteins, with attached flagellar membrane proteins, become incorporated into IFT particles and are moved through the flagellar pore (involving outer doublet-membrane links in the flagellar transition zone) into the flagellar compartment.