Identification of novel ciliogenesis factors

Cilia are finger-like cellular projections ranging from a few microns to several millimeters in length that perform critical sensory and motile functions in eukaryotic cells. Best known in their role as photoreceptors in the eye and as the flagellum of sperm, cilia are nearly ubiquitous in the human body. Defects in cilia assembly and function are consequently associated with a wide array of human developmental and adult disorders, collectively known as ciliopathies. Despite intensive research the causative mutation in many ciliopathy cases remains unknown, suggesting additional ciliogenesis factors remain to be identified. The goal of this stand-alone project is to identify and characterize such factors using a combination of bioinformatics and reverse genetics employing the fruitfly Drosophila melanogaster and the nematode worm C. elegans as experimental models. Starting point of this work is the observation that cilia are not universally present in eukaryotes, but have been lost multiple times over the course of evolution. The resulting presence and absence pattern in the fully sequenced genomes of diverse eukaryotes can be used to derive a phylogenetic signature for cilia genes. Novel genes that present this signature are excellent candidates for additional players in cilia assembly and function. To evaluate these genes, we will perform high-throughput assays in the fruitfly Drosophila and the nematode worm C. elegans to identify genes which present cilia phenotypes. Promising candidates will then be further characterized using an array of sensitive light and electron microscopy-based assays developed in the lab. Of particular interest is a small set of uncharacterized genes that is found in all ciliated species and is likely to play a fundamental role in cilia assembly. The approaches briefly outlined above aim to capitalize on the advantages of Drosophila melanogaster and C. elegans as experimental models to further our understanding of cilia assembly and function, fundamental cellular activities which are of key relevance to human development and disease. An important part of this work will be to apply our findings to vertebrate and clinical models, in collaboration with experts in the field. -1-

Cilia are finger-like cellular projections ranging from a few microns to several millimeters in length that perform critical sensory and motile functions in eukaryotic cells. Best known in their role as photoreceptors in the eye and as the flagellum of sperm, cilia are nearly ubiquitous in the human body. Defects in cilium assembly and function are associated with a wide array of human developmental and adult disorders, collectively known as ciliopathies. Despite intensive research the causative mutation in many ciliopathy cases remains unknown, suggesting additional ciliogenesis factors remain to be identified. The goal of this stand-alone project was to identify and characterize such factors and to better understand the molecular mechanisms underlying cilium assembly and function more generally, using the fruitfly Drosophila melanogaster and the nematode worm C. elegans as experimental models. Starting point for this work was the observation that cilia are not universally present in eukaryotes, but have been lost multiple times in different branches of the tree of life over the course of evolution. The resulting presence and absence pattern in the fully sequenced genomes of diverse eukaryotes can be used to derive a phylogenetic signature for ciliary genes. Novel genes that present the same signature are naturally excellent candidates for additional players in cilium assembly and function. This approach led us to identify a single cluster of 386 genes including most known ciliary components, but also 152 genes that have so far not been functionally characterized. To evaluate these genes, we performed high-throughput assays in the fruitfly Drosophila and the nematode worm C. elegans to identify those genes which present ciliary phenotypes. Remarkably, novel genes scored in our assays at a rate similar to known ciliary genes within the cluster, suggesting that most if not all 386 genes are cilium-related. In our downstream characterization we focused on a handful of particularly promising candidates, which we placed within the cilium assembly pathway using an array of sensitive light and electron microscopy-based assays developed in the lab. Beyond this main body of work, this standalone project also included a characterization of the unusual features of the ciliary base in C. elegans, which helped shed light on the mechanisms underlying the assembly of a related cellular organelle, the centrosome, as well as the establishment of a tool to characterize the molecular interactions involved in assembly of cellular structures whch should be widely applicable also to other cellular contexts and experimental models. Finally, we set out to characterize the role of genes involved in human ciliopathies in collaboration with clinicians from the UK and France, in work that is still ongoing.