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Several Usher syndrome (USH)-associated proteins are known to localize to the connecting cilium of photoreceptor cells. The unconventional myosin MYO7A (USH1B) was long accepted as the transport molecule responsible for the ciliary localization of USH proteins. However, based on the typical location of several of the USH proteins along the ciliary axoneme, the involvement of the main ciliary trafficking machinery, intraflagellar transport (IFT), seems apparent. The USH-associated scaffold protein PDZD7 is known to interact with SANS, Usherin, GPR98 and Whirlin, all of which can be found in the connecting cilium. Here, we report that PDZD7 provides the physical link of the USH-protein network to IFT-complex members. Tandem affinity purification (TAP) studies revealed a potential interaction between PDZD7 and several IFT molecules, including IFT25 and IFT27. TAP analyses of the other USH proteins were negative for IFT proteins, suggesting that this interaction is unique for PDZD7. In addition, a dedicated yeast two-hybrid screen of 200 (predicted) ciliary proteins revealed an interaction between PDZD7 and IFT57. The interaction between PDZD7 and selected IFT subunits was substantiated by co-immunoprecipitations. In accordance with these results, mRFP-tagged PDZD7, expressed in ciliated hTERT-RPE1 cells, localizes not only at the basal body, but also at the axoneme of a subset of cells. Pending further validation of the interaction between PDZD7 and IFT-B proteins, these first results suggest PDZD7 as a functional connection between USH-proteins and the IFT machinery. Future studies should reveal whether PDZD7 is involved in IFT of USH proteins in vivo.

Recent (affinity) proteomics studies have focused on the composition and dynamics of ciliary protein interaction networks. This has unveiled important knowledge about the highly ordered, interconnected but very dynamic nature of the cilium as a molecular machine in the cell. Disruption of members of functional modules of this machine leads to overlapping phenotypes. Detailed analyses of the binding repertoire, the biochemical properties and the biological functions of such modules have yielded the identification of new ciliopathy genes as well as new insights into the pathogenic mechanisms underlying ciliopathies. To gain better insights into the molecular disease mechanisms that underlie ciliopathies and to acquire knowledge about the general importance of the cilium for cellular homeostasis, we are conducting ciliary interactome studies and will combine this information with subcellular localization data, and functional data derived from gene knockdown in ciliated cells or knockout/mutant vertebrate models. This integrated dataset enables us to generate models of interacting functional modules associated with cell signalling cascades, developmental events or specific ciliary functions such as intraflagellar transport. To date, we have performed affinity proteomics for the majority of known ciliopathy-associated proteins and many other ciliary proteins. In addition, yeast two-hybrid experiments have been performed to study the physical interaction between ciliary proteins in more detail. Our current dataset shows high interconnectivity between many of the ciliopathy-associated proteins. These may be part of functional ciliary modules as many are associated with clinically overlapping phenotypes. In addition, new members of these modules are excellent novel candidate ciliopathy proteins.