Identification of proteins that regulate intraflagellar transport

Cilia are specialized cellular extrusions involved in motility and sensation. The development, maintenance and function of cilia require an evolutionarily conserved transport system, called intraflagellar transport (IFT). IFT is used to transport structural components and signaling molecules from the base of the cilia to the tip (mediated by kinesin motors) and back (mediated by dynein motors). Several different kinesin complexes have been identified, including heterotrimeric kinesin 2 and the homodimeric kinesin KIF17 (in mammals) or OSM-3 (in C. elegans). We have recently identified a conserved MAP kinase, DYF-5, that is involved in the regulation of IFT in C. elegans. In animals that lack dyf-5 IFT particles are no longer transported by OSM-3 kinesin, but only by heterotrimeric kinesin 2, although OSM-3 is still present and moves along the microtubular axoneme. In contrast, in wild type animals IFT particles are transported by both kinesins together. In addition, mutation of dyf-5 affects the length of cilia. DYF-5 homologues can be found in many other organisms, including Chlamydomonas and Leishmania, where DYF-5 homologues have similar functions in regulating cilia length. Mammals have three DYF-5 homologues, called MAK, MOK and MRK, which form a small family of MAP kinases. We aim to unravel the mechanism by which DYF-5 and its homologues regulate IFT. In this proposal we will use a biochemical approach to find out how DYF-5 and its mammalian homologues regulate IFT by identifying proteins that function in the same complex as DYF-5. First, we will generate polyclonal antibodies against C. elegans DYF-5 and its mammalian homologues. We will use the antibodies to determine where these proteins are localized in C. elegans and in several mammalian kidney cell lines that can develop cilia in culture. We will continue our studies with the mammalian proteins that localize to the cilia; I will refer to this (or these) protein(s) as MAK. Second, we will generate biotinylation and GFP tagged DYF-5 and MAK constructs to express these tagged proteins in C. elegans sensory neurons (DYF-5), or mammalian cells (MAK). We will generate two variants, constructs encoding the wild type kinase and kinase dead (kd) constructs. In addition, we will generate constructs to express the protein-biotin ligase BirA in these cells. The tagged DYF-5 and MAK constructs together with the BirA construct will be used to generate transgenic C. elegans or to transfect mammalian cells, respectively. Third, lysates will be made using gentle extraction procedures to preserve protein-protein interactions and biotinylated proteins will be isolated using streptavidin beads in a one step purification procedure. Isolated proteins will be analyzed on gel and by Western blotting and identified by mass spectrometry. Fourth, we will select a set of approximately ten most likely interacting proteins based on literature data and control pull downs. For these proteins we will try to confirm the interactions with DYF-5 and its mammalian homologues and determine where they localize in C. elegans or mammalian cells. Finally, we will determine the effects of loss- or gain-of-function of the confirmed DYF-5/MAK complex proteins on the cilia of C. elegans and mammalian cells. We expect that our approach will identify novel proteins that are involved in the regulation of IFT. This knowledge will help us understand how the localization of specific proteins in cilia can be regulated and how cilia length is regulated. Within our Department there is ample experience with various C. elegans techniques (Jansen lab), IFT and other intracellular transport systems (Jansen and Akhmanova labs), Biotin tagging (Grosveld and Akhmanova labs) and mass spectomery (Demmers, Center for Biomics).