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DissectIFT Report Summary

Project ID: 335623
Funded under: FP7-IDEAS-ERC
Country: Germany

Mid-Term Report Summary - DISSECTIFT (In vitro reconstitution and mechanistic dissection of Intraflagellar Transport in C.elegans sensory cilia)

Primary cilia are microtubule-based projections found on most eukaryotic cells. Even though the central importance of primary cilia to development and disease is now evident, molecular mechanisms underlying their assembly and function are far from being understood. The construction and maintenance of cilia relies on an ancient, universally conserved machinery termed IntraFlagellar Transport (IFT). IFT requires a multi-subunit, non-membranous protein complex assembled from more than 20 distinct subunits along with specialized motor proteins that are indispensable for the bi-directional transport on the axonemes. The main objective of our project is to provide a comprehensive mechanistic understanding of the IFT at the example of the C. elegans model system. Previous in vivo work argued for two kinetically distinct kinesin-2 motors (heterotrimeric KLP11/20/KAP and homodimeric Osm-3) to associate with the two distinct IFT complexes, termed IFTA (five subunits) and IFTB (over 15 subunits), respectively. To mechanistically dissect the coordinated transport by KLP11/20 and Osm-3 in vitro, we established an experimental strategy to specifically couple two motors using protein-double-stranded DNA hybrids. To follow differently fluorophore-labeled motor proteins, we built a state-of-the-art microscope set up that is designed to simultaneously track up to three fluorophores nanometer precision. By tracking one fluorescently-labeled head domain of the KLP11/20 at nanometer resolution, we unmasked how the motor adopted its nanometer-sized steps to its axonemal track. Our preliminary data on tracking two different fluorophores on the heterodimeric kinesin-2 motors showed that the setup is indeed capable of simultaneously resolving discrete displacements of the respective fluorophores with nanometer precision as well. This technological expertise along with our experimental capability to specifically assemble motor teams via motor-DNA hybrids will be instrumental to expose the molecular mechanisms of how the KLP11/20 and Osm-3 motors coordinate their actions with unprecedented details. To study the kinesin-2 motors within the context of their respective adaptors, we have recombinantly expressed the respective subunits of the IFTA and IFTB complexes and characterized their assembly properties. Our preliminary results suggest that the respective motors require pre-assembled subcomplexes to form transport-competent IFTA and IFTB complexes.

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