Collaborative Research: Alpha-arrestins' impact on cellular physiology

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Eukaryotic cells are partitioned into membrane compartments, increasing reaction efficiencies and sequestering toxic intermediates. This organization necessitates communication between organelles to coordinate the cell’s metabolic program. Regulated nutrient transporter trafficking and exchange of nutrients at organelle contact sites maintain metabolite balance across membranes. This research focuses on a class of selective protein trafficking adaptors, known as the alpha-arrestins, and their role in maintaining nutrient balance and cell physiology. Conserved from yeast to man, alpha-arrestins are master regulators of protein trafficking, controlling membrane protein localization in response to cell signaling. In the absence of alpha-arrestins, nutrient transporters are retained at the cell surface, but studies have not yet defined how loss of alpha-arrestins impacts cell physiology. This project tests the global hypothesis that alpha-arrestins regulate nutrient transporters to maintain metabolite balance and mitochondrial function. This research will transform our understanding of alpha-arrestin function, defining their impact on metabolism and mitochondrial function. These research goals are intertwined with teaching and outreach objectives designed to train the next generation of scientists and broaden STEM participation. A course-based undergraduate research experience that uses alpha-arrestins as a paradigm to teach cell and molecular biology will be developed, providing 120 undergraduates with research experience. Graduate, undergraduate, and high school students will be mentored on independent research projects, and students from diverse backgrounds will be recruited to the research team. Research-driven lesson modules called ‘Pitt Kits’ will be developed in collaboration with high school teachers and deployed across Pennsylvanian public high schools. This project will advance our understanding of how alpha-arrestins act as sentinels to maintain nutrient balance and define how disruption of this process leads to organelle dysfunction. Preliminary data demonstrate that specific alpha-arrestins are required to maintain the balance of select amino acids in the cell. This is likely because each alpha-arrestin traffics a unique set of amino acid transporters. Amino acid imbalance is toxic and work from the Hughes lab has begun to define the underlying mechanisms for this toxicity, which had remained elusive for decades. In each case, excess amino acid leads to mitochondrial dysfunction, which appears to drive the cytotoxicity. Strikingly, initial studies from the O’Donnell lab show that in cells with dysfunctional alpha-arrestins, amino acid excess causes mitochondrial fragmentation and reduced mitochondrial activity. Mitochondria may act as bellwethers for amino acid levels as amino acid intermediates shuttle in and out of mitochondria, and this may sensitize this organelle to amino acid imbalance. During this project, the metabolic changes in cells where alpha-arrestins are impaired will be mapped and the nutrient transporters required for alteration in metabolite balance will be defined. Using high-content, confocal microscopy we will determine how alpha-arrestins regulate nutrient transporter trafficking and how this trafficking is linked to mitochondrial function. A machine-learning, automated quantification pipeline will help define transporter localization changes and mitochondrial form and function changes, ensuring that the data generated are rigorous and reproducible.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.