Defining the form-specific functions and regulation of nuclear actin within the nucleolus

From yeast to humans, actin – a protein best known for its role in forming cytoskeletal filaments that give cells their shape and allow them to move – also localizes to and functions inside the nucleus. This nuclear localization of actin was controversial initially, so the functions of nuclear actin are only just now being uncovered. This project will define functions of nuclear actin within the nucleolus, a subcompartment of the nucleus. The nucleolus is a non-membrane bound organelle that is the site of ribosomal RNA (rRNA) synthesis and, so, mediates ribosome formation; ribosomes produce proteins, the factors that mediate cellular functions. Thus, too little or too much nucleolar activity has deleterious outcomes, from cell dysfunction to cell or organism death. The project will use Drosophila, a robust genetic system, and the non-essential tissues of oogenesis (egg development) to advance understanding of how nuclear actin tightly controls nucleolar functions, providing insights into normal cellular function and development, as well as into how misregulation can contribute to diseases. This project will also train the next generation of biologists by developing undergraduate laboratory course modules that bring this research into the classroom and by mentoring trainees in the research lab. The functions of actin in both the cytoplasm and the nucleus depend on the form of actin – monomers, polymers, filaments, and networks of filaments. The objective of this proposal is to define the form-specific roles of nuclear actin in the nucleolus. Actin localizes to the nucleolus and regulates RNA polymerase I (RNAPI) activity across organisms. The team discovered that during Drosophila oogenesis, monomeric and polymeric nuclear actin localize to the nucleoli with distinct developmental patterns, suggesting that nuclear actin has stage-specific and form-specific roles in regulating nucleolar functions. Indeed, increasing nuclear actin results in increased ribosomal RNA (rRNA), abnormal nucleolar morphology, and increased protein translation. However, the roles of the different forms of nuclear actin in modulating the nucleolus remain unknown. The project will uncover how monomeric vs polymeric actin regulate RNAPI activity – from licensing of rRNA genes to functioning within the RNAPI complex – and the downstream effects on cellular function. It will also use two unbiased approaches – proteomics and a targeted genetic screen – to identify new regulators of nuclear actin and nucleolar activity. As the nucleolus is a key regulator of cellular homeostasis, uncovering the connections between the nucleolus and nuclear actin is critical for understanding both normal cell function, and how misregulation of nuclear actin contributes to cellular dysfunction. 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. NSF Award ID: 2545583 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Tina Tootle | Institution: University of Iowa, IOWA CITY, IA | Award Amount: $500,000 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2545583 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2545583.html