Understanding the function of recycling endosomes

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My research program is focused on the role of membrane-bound recycling endosomes (REs) in protein trafficking and secretion. REs are specialized secretory compartments that were originally characterized for their ability to recycle cell surface receptors through endocytic and exocytic pathways. Recently, REs were found to have a role in intracellular protein trafficking and secretion beyond recycling receptors to cell surfaces. Studies have shown that REs are implicated in the secretion of proteins to the extracellular milieu. However, the function of REs in trafficking and secretion of proteins remains poorly understood. In this proposal, we aim to study the specific role of REs in contributing to the trafficking and release of cell-derived mediators using epithelial cells. Epithelial cells have robust protein trafficking systems that serve as perfect models for studying RE trafficking, and the secretion of proteins is well defined in these cells. They are also polarized cells, capable of directional release of proteins through apical and basolateral surfaces. Therefore, epithelial cells provide an ideal system for understanding protein trafficking through REs. Objectives The long-term objective is to determine the function of REs in protein trafficking and secretion in epithelial cells. There is very little known about the role of REs in mediator release from epithelial cells, and yet there is strong evidence for their existence in these cells. The specific short-term objectives are to: 1. Specifically label REs in epithelial cells and study their redistribution in response to specific stimuli (e.g. Toll-like, tyrosine kinase, and G-protein coupled receptors). 2. Determine mechanisms of RE-dependent secretory pathways in epithelial cells by application of gene deletion technology to effectively remove this compartment. 3. Characterize the role of rapidly and slowly recycled compartments of REs in mediator trafficking in epithelial cells. Specifically, we propose to resolve the requirements for REs in the release of proteins and mediators from epithelial cells. REs will be characterized for their role in mediator trafficking by the application of gene deletion technology. We will also delve more deeply into defining and characterizing RE subcompartments, known as rapidly and slowly recycled compartments of REs, in protein trafficking in epithelial cells. Using high resolution imaging, gene deletion technology (CRISPR and si/shRNA) and ultrastructural studies, we will map the fine distribution of RE-associated proteins during epithelial cell secretion of mediators. Outcomes My research program will reveal novel membrane trafficking compartments required for mediator trafficking in epithelial cells, important cells in the maintenance of barrier function. Understanding the function of REs in epithelial cells will lead to the elucidation of novel membrane trafficking pathways for REs that serve an essential function in protein secretion.