Cellular stress may lead to harmful conditions that alter or prevent the completion of a normal routine; therefore, a dynamic response must be established to maintain balance of cellular functions under stressful conditions. This project investigates how cells adapt to a stressful environment to return to normal. The researchers involved in this project will be undergraduate and graduate students who will participate in independent projects to determine the mechanisms of how genetic information is stored and influenced by a specific protein under normal and stressful conditions. They will have the opportunity to continue their research in Canada at the Lunenfeld-Tanenbaum Research Institute (Toronto, Ontario). Their findings will be presented at local, national, and international conferences such as at William and Mary (Williamsburg), San Diego, and Greece. In addition, these students will develop and use a children friendly workshop to teach local summer camp students and their parents about the importance of cells keeping a balance, under toxic conditions. This research will also allow selected students at William and Mary and Hampton University to travel to a leading research facility, Cold Spring Harbor Laboratory (Long Island, NY), to engage and build networks with scientists highly active in various disciplines. Thus, the overall scope of this project allows undergraduates and Master's students to integrate education and research in the stress response field.

Cells have protective mechanisms to safeguard stalled mRNAs from harmful conditions. They form stress granules (SG). SG are large (100-200 nm) cytoplasmic RNA-protein complexes that transiently form under stress and disassemble upon return to normal conditions. When SG coalesce for too long, they become toxic, disrupting cellular homeostasis, highlighting the importance of understanding SG clearance. Investigators in this project previously reported that SG assembly was decreased by the signaling protein MK-STYX (MAP kinase serine/threonine/tyrosine binding protein; a unique catalytically inactive phosphatase), whereas the active mutant of MK-STYX induces SG. The mechanism by which MK-STYX decreases SG remains unclear. The current project explores the molecular mechanisms by which MK-STYX regulates SG by addressing the following questions: 1) Which domain of MK-STYX elicits a decrease in SG? 2) What are the effects of MK-STYX on HDAC6 and the motor protein dynein? 3) Does MK-STYX have a role in autophagy, which clears stress granules? Investigators have identified a phenotype that will allow substantial structural and functional assays of MK-STYX, including identification of the MK-STYX domains and the important protein interactions of MK-STYX that lead to a decrease in SG. Various fluorescent protein-tagged mutants of MK-STYX, such as truncated domains and the active mutant (catalytic activity restored), will be overexpressed in HeLa and HEK/293 cells stably expressing the SG nucleator G3BP1, and observed for SG, and analyzed further through proteomics. HDAC6 is a critical component of SG; its deacetylation activity is required for SG. Colocalization, immunoblotting for acetylation and ubiquitination (important for HDAC6 function), immunoprecipitation, and deacetylation activity assays will address whether MK-STYX affects the activity of HDAC6. Dynein is required in conjunction with HDAC6 for SG. Thus, it is important to determine the effects of MK-STYX on these proteins. Since autophagy and MK-STYX each negatively affect SG formation, it is important to determine the effects of MK-STYX on autophagy. A combination of TEM, immunoblotting (LC3-autophagsomes marker, ubiquitin, etc.), radioactive pulse chase analysis, tandem mRFP/mCherry-GFP colocalization microscopy, and autophagic sequestration assays (autophagy inhibitors) will be used, resulting in the development and integration of computational analysis. The long-term objective of this research is to understand how MK-STYX regulates signaling pathways.

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.

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College of William and Mary
United States
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