Defining the ciliary proteomes in vivo

PROJECT SUMMARY/ABSTRACT Cilia are complex and dynamic cellular structures crucial for normal development and tissue function. Genetic disruptions in ciliary proteins lead to a group of human diseases known as ciliopathies, which exhibit a broad spectrum of phenotypes including early gestational arrest, left-right body axis randomization, cystic kidney, liver, and pancreas abnormalities, hydrocephalus, neurological and cognitive disorders, blindness, anosmia, and obesity, etc. The manifestation and severity of ciliopathy disorders vary depending on the specific genetic mutation and its impact on ciliary function across different cell types and tissues. This variability has prompted the hypothesis that cilia exhibit distinct functions in various cell types, developmental stages, and environmental signaling contexts, as well as under pathological conditions and that this is dictated by alterations in the cilia proteome. Despite the clinical importance of cilia, there is a significant gap in our understanding of cilia functional roles and the signaling pathways they regulate in most cell types of the body. A key obstacle to advancing our knowledge of ciliary function is the lack of information on the ciliary proteome composition in different cell types and how it dynamically changes under varying conditions. This project aims to address this knowledge gap by developing a novel resource using the promiscuous ligase BirA*G3 fused with the transition zone protein B9d1 in mouse and cell-based models. The transition zone, located at the base of the cilium, serves as a crucial regulatory gate for controlling the entry and exit of proteins into and out of the cilium. The engineered cells and mouse lines will enable temporal and cell type-specific biotin labeling of proteins traversing the ciliary transition zone. Through the utilization of these innovative resources, we will assess the feasibility of this approach both in vivo and in vitro, with the goal of providing the research community with valuable tools to investigate the dynamics and diversity of the ciliary proteome. By enhancing our understanding of cilia function at the molecular level, this research has the potential to uncover novel insights into ciliopathy disorders and pave the way for the development of targeted therapeutic strategies.