Cilia have sparked keen interest because of their proven importance in many human genetic diseases (collectively termed ciliopathies and including Joubert syndrome (JS)) and in vertebrate/mouse development;appreciation of their central role in aspects of cell signaling is growing, as well. Primary, immotile cilia are microtubule-based projections found on virtually every eukaryotic cell. We focus on the small GTPase, ARL13B, because its absence leads to defects in (1) cilia structure, (2) movement and localization of specific proteins to and (3) within cilia, and (4) patients with mutations in ARL13 have Joubert syndrome (JS), which involves renal, ocular, and brain anomalies. This application has four specific aims, in which we propose to (1) define the biochemical properties of ARL13B as a GTPase and identify and characterize novel effectors, (2) identify, purify, and characterize ARL13B GTPase activating proteins (GAPs), (3) determine the level of complexity in downstream ARL13B signaling and identify one or more effectors, (4) determine the tissue-specific consequences of JS causing ARL13B mutations in mice. We will use the complementary expertise of the two PIs to develop models for ARL13B signaling at cilia, with direct relevance to disease processes. We will pair a novel genetic deletion system in mouse embryo fibroblasts with detailed biochemical studies and modeling of ARL13B as a regulatory GTPase to develop molecular models for ARL13B's actions in cells and animals. Together, these approaches will allow us to identify and rigorously test downstream effectors and GTPase activating proteins (GAPs) of ARL13B actions in the development of the pathways affected by JS. Finally, by defining the in vivo, tissue specific function of the JS causative ARL13B mutations, we will both model the disease and identify the mechanism underlying tissue-specific Arl13b function and patient phenotypic variability. Together these aims will provide a solid experimental basis for establishing models of Arl13b actions, with clear implications for many aspects of cilia function, cell signaling, mammalian development, and human disease.
Primary cilia are found on virtually every cell type and are required for cell signaling, vital to development in vertebrates, with defects in cilia resulting i a host of human diseases called ciliopathies, which include Joubert syndrome (JS) and Bardet-Biedl syndrome. The focus of this multi-PI application is the small GTPase, Arl13b, which is enriched in cilia and mutated in a number of Joubert patients. Using biochemical, cell culture and in vivo models, we will test our central hypothesis that Arl13b regulates a number of processes central to signaling from the primary cilia via distinct pathways to better understand its mechanisms of action and dysfunction that lead to Joubert syndrome.