CAREER: Regulation of clathrin-independent endocytosis by modulators of Rho GTPase function

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The cell surface is an important structure that defines the boundary between the inside and outside of the cell and contains proteins that help the cell to interact with and respond to its environment. Proteins are delivered to the surface using a process called exocytosis and are removed when a cell internalizes pieces of its surface in a process known as endocytosis. Balancing these two events allows the cell to control which proteins are on the surface at any given time and makes it possible to add new material or remove damaged proteins that may be harmful. This project combines cell biology, molecular biology and genetic approaches to study poorly understood pathways for endocytosis, termed clathrin-independent endocytosis (CIE), that are present in many cell types. Through this research, this project will provide cutting-edge research and training opportunities at many levels of education, including undergraduate and graduate trainees as well as high-school students. A major goal is to give young scientists the opportunity to see how the things they learn in the classroom can be applied to make new and exciting discoveries in the field of membrane biology.This research project uses budding yeast, a simple yet extremely powerful genetic model organism that has provided insight into many basic cellular functions that are conserved through evolution, including exocytosis and endocytosis. Although yeast was originally thought to use only clathrin-mediated endocytosis, studies in a mutant strain where this pathway was blocked revealed the existence of a new clathrin-independent pathway. This second pathway requires numerous proteins including Rho1, which regulates polymerization of the actin cytoskeleton and coordinates repair of the yeast cell wall. Rho1 undergoes cycles of activation and inactivation, and its activity can be restricted to specific sites within a cell. The relationship between Rho1 activity and location, and how these relate to its role in endocytosis, are poorly understood. Thus, this project will achieve several goals: (1) define the ability of proteins that regulate localized Rho1 activation and inactivation to regulate clathrin-independent endocytosis; (2) examine relationships between protein complexes that regulate exocytosis and endocytosis; (3) identify cargos that are internalized by clathrin-independent endocytosis under high osmolarity conditions that are known to facilitate this pathway; (4) examine the role of the osmotic stress response pathway in clathrin-independent endocytosis; and (5) identify genes that promote cargo internalization in mutant yeast strains lacking both clathrin-mediated endocytosis and the known clathrin-independent pathway to determine if additional pathways exist. Overall, the results of this research will provide new insights into the molecular machinery that controls clathrin-independent endocytosis, an important but poorly understood process in all eukaryotic cells. Using a simple model organism such as yeast makes it possible to rapidly identify the key components of these pathways, which in turn will make it possible to better understand related pathways in other organisms, including humans.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.