While actin localizes to the nucleus from yeast to humans, the functions of nuclear actin remain poorly understood. Most studies on actin have focused on its roles in the cytoskeleton, where it forms networks of filaments that give cells their shape and allow them to move. However, actin also localizes to the nucleus. One nuclear site of actin is the nucleolus. The nucleolus is an essential nuclear organelle and mediates the formation of ribosomes which are required for all protein synthesis in the cell; thus, nucleolar function is required for life. The nucleolus also senses and responds to cellular stress, including high temperatures and starvation. Under stress conditions, the nucleolus stops non-essential processes to allow the cell to weather the stress, and when necessary, drives cell death. The objectives of this project are to define the nucleolar functions of actin and to show how these functions are controlled to maintain cell and tissue function, not only under normal conditions but in response to cellular stress. The Broader Impacts of this project include efforts to increase the diversity of students pursuing degrees and careers in biological sciences. One barrier to pursuing a scientific career is a lack of understanding about how scientific research is carried out. To help overcome this barrier, the project will develop a 6-week Course-based Undergraduate Research Experience (CURE) targeting ~80 underrepresented students in their first two years of undergraduate education, including those from communities of socioeconomic and educational disadvantage. Thus, this project will increase both the studentsâ€™ and societyâ€™s understanding of nuclear actin and how it is important for life.
To uncover the roles of nuclear actin in the nucleolus during tissue development, homeostasis, and response to cellular stress, this project uses the robust genetic system of Drosophila oogenesis. In this system, nuclear actin is critical for female fertility and is dynamically regulated during follicle development. Specifically, nuclear actin is enriched in the nucleolus. The nucleolus mediates many nuclear activities, including rDNA transcription, ribosome biogenesis, and chromatin organization. Inhibition of nucleolar functions disrupts its structure. Aberrant nucleolar morphology is seen when nuclear actin levels are altered, suggesting nuclear actin regulates nucleolar functions. Altering nuclear actin levels also perturbs chromatin organization, another function of the nucleolus. Additionally, both the nucleolus and nuclear actin respond to the same cellular stresses. Stress results in disruption of nucleolar activity and structure; it also increases nuclear actin levels driving nuclear actin rod formation. Further, during Drosophila oogenesis, nuclear actin rod formation results in one nuclear actin pool relocating from the nucleolus to the rods. Together these data lead to the projectâ€™s novel central hypothesis that: Nuclear actin dynamics regulate nucleolar structure and function to both maintain cellular homeostasis and respond to cellular stress, processes that are essential for follicle development. To test this hypothesis, the proposed study will use a combination of sophisticated genetics, advanced biochemistry, state-of-the-art microscopy, quantitative image analyses and unbiased approaches, including proteomics and a genetic screen. Together these investigations will 1. Define the form-specific roles of nuclear actin in regulating nucleolar structure and functions, including rDNA transcription, ribosome biogenesis, and chromatin organization; 2. Determine how nuclear actin contributes to the nucleolar stress response; and 3. Identify new regulators of nuclear actin. As the nucleolus has emerged as a key regulator of tissue homeostasis, uncovering the connections between the nucleolus and nuclear actin is critical for understanding both normal cell and tissue function, and how mis-regulation of nuclear actin contributes to cellular stress pathways and tissue dysfunction.
This Project is cofunded by the Division of Molecular and Cellular Biosciences and EPSCoR.
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.