WORKPACKAGE 2

Generation and characterization of in vitro models to study the role of BBS, OFD1 and NPHP in the formation and function of the primary cilium

OBJECTIVES
1. This WP will selectively modulate the expression of members of the BBS/OFD1/NPHP family in cell lines, and analyze established ciliary functions in these modified mammalian cells.
2. Mutational analysis in combination with protein modelling will be used to identify ciliary targeting motifs in BBS/OFD1/NPHP proteins.
3. To understand the dynamics of cargo recruitment to the cilium, the posttranslational modifications of selected BBS/OFD1/NPHP proteins will be analyzed in vitro and in vivo.
4. Genetic screens in C. elegans will define the components of the secretory pathway required to transport BBS/OFD1/NPHP proteins to the cilium/centrosome complex.
5. The effects of modulated BBS/OFD1/NPHP expression will be examined in canonical signalling cascades.

LIST OF PARTICIPANTS

3	UKLFR	Gerd Walz, Heymut Omran
4	CMNS	Alberto Luini
2	UCL	Philip Beales
1	FTELE.IGM	Brunella Franco

LIST OF DELIVERABLES
2.1 Generation of cell lines with selective depletion and selective over-expression of BBS/OFD1/NPHP. (Month 18).
2.2 Structural and functional characterization of the modified cell lines according to three criteria (ciliary integrity, calcium signalling, trafficking of ciliary marker proteins). (Month 24).
2.3 Definition of protein motifs and/or structural requirements for BBS/OFD1/NPHP to traffic to the cilium. (Month 36).
2.4 Identification of components of the secretory pathway required to transfer cargo proteins from the trans-Golgi network to the cilium. (Month 36).

LIST OF MILESTONES
M2.1 Establishment of cellular in vitro systems to induce or delete the expression of BBS/OFD1/NPHP proteins. (Month 36).
M2.2 Determination on how BBS/OFD1/NPHP are targeted to the CCC compartment, defining the underlying mechanisms of recruitment, vesicular transport, and the importance of posttranslational modifications. (Month 36).