Project Summary Cilia are ubiquitous and important microtubule-based organelles involved in sensation, developmental signaling, fluid flow and cell motility whose dysregulation leads to a range of human disease states including cancers and ciliopathies. The non-motile primary cilium functions as a signaling antenna. It has a distinct and tightly regulated protein and lipid composition while remaining contiguous with the plasma membrane of the cell. Proper cilium function depends on compartmentalization both for spatial separation of ciliary activities and high concentration of signaling components. While there has been extensive characterization of the proteins and lipids that compose the cilium, ciliary membrane, and the diffusion barrier at its base, much less is known about the composition and function of membrane regions proximal to the cilium that support proper signaling. In many cell types, the mature primary cilium sits in a microns-deep membrane invagination called the “ciliary pocket” that is an important but under-characterized hub of endocytic activity and signaling. The ultrastructure of the ciliary pocket is highly conserved and is distinct from both the ciliary and plasma membranes. It has been difficult to clearly define the ciliary pocket as a distinct compartment and determine its full contributions to ciliary signaling and function. This is in part due to the technical challenge of imaging this diffraction limited region and lack of tools to specifically perturb the pocket’s structure and composition. In this proposal I will address this gap through an interconnected set of aims, leveraging innovative technologies including proximity-based labeling and super-resolution microscopy as well as functional signaling assays to: 1) define the molecular composition of the ciliary pocket, 2) determine how ciliary pocket structure is established and maintained to support signaling, and 3) investigate extracellular roles of the ciliary pocket in signaling. In summary, I propose to establish a high-resolution map of the architecture and spatial and temporal dynamics of the mammalian ciliary pocket and define its role in supporting ciliary signal transduction. Defects in ciliary structure lead to aberrant signaling in a variety of disease states. The proposed research will expand our understanding of how the architecture of the periciliary membrane supports proper function of the primary cilium and has the potential to contribute new insights into the molecular basis of human diseases such as ciliopathies and cancer.