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In vitro reconstitution and mechanistic dissection of Intraflagellar Transport in C.elegans sensory cilia

Objective

Cilia are microtubule-based protrusions of the plasma membrane found on many eukaryotic cells, including most cell types of the human body. Whereas the functions of motile cilia were immediately obvious, the role of the immotile or so-called primary cilia remained largely unrecognized for many decades. Once referred to as aberrant solitary cilia with no obvious function, these ancient structures now hold the promise of revealing no less than the secrets of multicellularity and development. Even though the importance of primary cilia 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. At the heart of IFT are the microtubule-associated motors, -kinesin and dynein-, that continuously ferry cargo in a bi-directional fashion needed for ciliary assembly and function. To pave the way towards a molecular understanding of this fascinating organelle, we propose to employ a bottom-up approach in which we stepwise reconstitute the IFT complex from recombinantly expressed subunits of the so far best understood primary cilium from C.elegans. The structural integrity and stability of the IFT complex will be characterized using multifaceted approaches such as chemical crosslinking or thermophoresis. To mechanistically dissect the kinesin-dependent transport in vitro, we will make use of enzymatic bulk and single-molecule assays. Collectively, these results will provide a quantitative understanding of the assembly and kinesin-dependent motility of the IFT machinery. Given that cells mobilize ~600 components to build their cilia, this experimental platform will significantly streamline future efforts to identify novel cargoes and the effects of putative regulators of the IFT machinery.

Field of science

  • /social sciences/social and economic geography/transport

Call for proposal

ERC-2013-StG
See other projects for this call

Funding Scheme

ERC-SG - ERC Starting Grant

Host institution

TECHNISCHE UNIVERSITAET MUENCHEN
Address
Arcisstrasse 21
80333 Muenchen
Germany
Activity type
Higher or Secondary Education Establishments
EU contribution
€ 1 497 740
Principal investigator
Zeynep ökten (Dr.)
Administrative Contact
Ulrike Ronchetti (Ms.)

Beneficiaries (1)

TECHNISCHE UNIVERSITAET MUENCHEN
Germany
EU contribution
€ 1 497 740
Address
Arcisstrasse 21
80333 Muenchen
Activity type
Higher or Secondary Education Establishments
Principal investigator
Zeynep ökten (Dr.)
Administrative Contact
Ulrike Ronchetti (Ms.)