Cellular Studies on the Oral-Facial-Digital Syndrome Type 1 Disease Protein
thesisposted on 10.01.2011, 12:58 by Carla Alexandra Mendes Lopes
Primary cilia were once considered vestigial structures with no obvious function. However, it is now clear that they have important roles in the control of cell proliferation and signalling during development. Indeed, defects in primary cilia are the major cause of many human diseases, collectively known as ciliopathies, that affect diverse organ systems. The growing list of ciliopathies includes oral-facial-digital syndrome type 1, a developmental disorder that features malformations of the face, oral cavity and digits, and is often associated with polycystic kidney disease. The aim of this thesis was to determine the cellular function of OFD1, the protein product of the gene mutated in this disease. Here, we show that OFD1, together with BBS4 and CEP290, proteins encoded by other ciliopathy disease genes, is primarily a component of centriolar satellites, PCM-1-containing particles that surround centrosomes and basal bodies. RNAi depletion experiments reveal that localization of all four of these proteins to centriolar satellites is mutually dependent. Intriguingly, upon satellite dispersal, either through depolymerization of the microtubule network or progression through mitosis, OFD1 and CEP290 remain associated with the centrosome, whereas BBS4 and PCM-1 do not. While others have shown that both CEP290 and BBS4 can physically interact with PCM-1, we show that OFD1 interacts via its fifth coiled-coil motif with the N-terminal coiled-coil domain of PCM-1, and that localization of OFD1 to satellites requires its N-terminal region encompassing the LisH motif. Furthermore, we also show that expression of C-terminal constructs of OFD1 causes mislocalization of PCM-1 and CEP290, while OFD1 and BBS4 functionally synergize in terms of determining organ laterality and body axis integrity in zebrafish. Together, these data highlight centriolar satellites as critical assembly points for proteins implicated in clinically distinct human diseases and that defects in one of these subunits affect the integrity of the whole complex. This work supports a model whereby centriolar satellites play a crucial role in regulating delivery of ciliopathy disease proteins to the basal body and primary cilium, thus extending our understanding as to why such different diseases show considerable phenotypic overlap.