Cilia and flagella are ancient, evolutionarily conserved organelles that project from cell surfaces to perform diverse biological roles, including whole-cell locomotion, movement of fluid, chemo-, mechano- and photosensation, and sexual reproduction. Consistent with their stringent evolutionary conservation defects in cilia or flagella are associated with a wide range of human diseases. Loss or dysfunction of nodal cilia perturbs left-right axis determination, whereas sensory cilia defects lead to polycystic liver and kidney disease. Likewise, dysfunction of ependymal cilia cause hydrocephalus, and defective transportation of proteins along the photoreceptor connecting cilium leads to retinal degeneration. Despite their profound importance, cilia and their roles in human physiology have only recently gained broader attention and the fact that most mammalian cells have the capacity to ciliate has been under-appreciated. Here we propose to perform a comprehensive analysis of the mammalian genome and proteome to identify and validate the fraction of human proteins involved in ciliary structure and function, and to determine their contribution to human genetic disease. First, we will use selective evolutionary conservation coupled with microarray analyses to differentiate between proteins necessary for the function of sensory cilia, motile cilia, or both. Second, focusing on proteins expressed in sensory cilia, we will interrogate their direct involvement in ciliary function by determining their subcellular localization in ciliated cells. Third, we will perform high-density mapping of consanguineous families with established ciliation disorders such as Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) and determine for each pedigree all regions in the genome that display homozygosity by descent. We will then intersect our computational and experimental ciliary proteome data with our genetic mapping information and identify novel BBS and NPH genes. Finally, to investigate the mechanism of dysfunction, we will suppress the mRNA message of novel disease genes in mammalian ciliated cells and model the consequences of loss of function mutations in ciliary morphology, intraflagellar transport and transcriptional regulation during the ciliary life cycle. These studies will further significantly our understanding of the function of the cilium, one of the least-studied cellular organelles, yet of major physiological importance, and provide novel tools to dissect the molecular basis of human genetic disease.
NIH ExPorter
NIH ExPorter