Role of WDR35 in the formation of functional cilia

Cilia are microtubule-based organelles present on the surface of almost all mammalian cells that play key sensory and sometimes motile functions. Defects in cilia structure or function lead to a group of human diseases called the ciliopathies. In order to function, cilia must maintain a distinct protein and membrane composition from the surrounding plasma membrane and cytosol, highly enriched in signaling receptors and effectors. How this compartmentalization occurs remains unclear. There is no known protein synthesising machinery in cilia and the transition zone at the base of cilium forms the diffusion barrier, which does not allow free exchange between the cytoplasm to cilioplasm. Biogenesis of the cilium (ciliogenesis) requires many carriers and adaptors to facilitate passage of cargo across the transition zone. Once inside the cilia intraflagellar transport (IFT) proteins move the cargo along microtubules ‘railways’ of the axoneme. IFTs are known to assemble in two protein complexes, IFTA and IFTB protein complexes. IFT-B complex known to be made of 16 different proteins mediate anterograde transport with the help of kinesin motors, and IFT-A complex made of 6 different proteins help in retrograde transport powered by dynein motors. However, the exact mechanism of transport of cargo to cilia and entry across the diffusion barrier is not well understood, and the functions of each IFT protein remain unclear. In this thesis, I describe the critical role of one of the IFT-A complex protein WDR35/IFT121 in the formation of functional cilia by transporting structural elements of cilia via a vesicular mediated pathway. Null mutations in the IFT-A component WDR35/ IFT121 are embryonic lethal in both mouse models and human ciliopathies (Mill et al., 2011). Small, unstable WDR35 mutant cilia are formed but fail to become enriched in diverse classes of integral and membrane-associated proteins (Caparrós-Martín JA et al., 2015; Fu W et al., 2016). To elucidate its role in the entry of membrane proteins to cilia, I present live and fixed cell imaging experiments to visualize the dynamics of membrane protein localization at the periciliary base. I also performed interaction studies by immunoprecipitation and mass spectrometry to define the molecular mechanism by which IFT proteins establish functional cilia. There are few reports of some IFTs having similarity to COPI, II and clathrin vesicles (Jékely G and Arendt D, 2006; Taschner M et al., 2012; Dam TJPV et al., 2013). To further explore its role, I performed deep sequence searches and homology modeling of the entire IFTA complex. I found that three of its core components and WDR35 have structural homology to COPI complex proteins α and ß’. To test whether WDR35 could function as a coatomer, I performed transmission electron microscopy tomography on cilia mutants. I found that in contrast to the electron-dense vesicles observed around WT cilia, Wdr35-/- cilia had a ten-fold increase in the number of vesicles all lacking this outer electron density. This suggests WDR35 may be involved in coating cilia-bound vesicles in order to transfer cargo into cilia, functioning similar to COPI complex proteins which selectively transports cargo between the endoplasmic reticulum and Golgi

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß′ COPI coatomer subunits and demonstrate an accumulation of ‘coat-less’ vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation