Dynamic formation/disassembly of membraneless organelle model systems by post-translational modification: Mechanisms and consequences

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Living cells contain different subcellular compartments that perform a range of functions necessary for survival. Some of these compartments, such as the nucleus and mitochondria, are separated from the rest of the cell by membranes. Others, termed membraneless organelles, lack any obvious physical boundary from the rest of the cell. It was recently discovered that many membraneless organelles are actually liquid droplets formed by intracellular phase separation. This project will investigate the consequences of droplet formation on biochemical reactions and how cells could take advantage of droplet-forming phase transitions to regulate biochemical pathways. The findings will uncover new paradigms for intracellular organization and enable new classes of artificial microscale bioreactors that incorporate such organization. The project will directly impact students at the graduate and undergraduate levels, who will perform this research at the intersection of chemistry, biology, physics and materials science. By involving K-12 teachers it will also reach high school and middle school students. Small teams of teachers will work in the Principal Investigator's laboratory each summer developing grade-level appropriate hands-on content in emulsion science. Back in their classrooms, implementation of this new content will engage middle and high school students with current scientific progress and its connection to their everyday lives. Emulsions where droplets of one phase are suspended in another liquid phase are common in consumer products such as salad dressings or sunscreens, and can be used to illustrate fundamental principles in multiple disciplines including chemistry, mathematics, physics, biology, and food science. The Principal Investigator and graduate students will also aid in development of chemistry content for a K-12 education approach being developed by colleagues in the Education department to facilitate high-level science comprehension.Liquid-liquid phase coexistence, which causes biomolecule-rich aqueous droplets termed coacervates to form in the cytoplasm or nucleoplasm of eukaryotic cells, has only recently been realized as a pervasive organizational motif for membraneless organelles, and is not yet well understood. This project will provide new insight into physicochemical driving forces underlying intracellular organization by liquid-liquid phase coexistence, as well as potential consequences of coexisting peptide-rich phases for biochemical reactions. Non-uniform solute distribution between coexisting phase compartments (coacervate droplets) will impact the rates of biochemical reactions, providing a mechanism to control reaction rates via droplet formation and dissolution. Post-translational modifications, specifically phosphorylation and methylation events in intrinsically disordered regions of key proteins, are thought to be a major mechanism for regulating the formation and dissolution of membraneless organelles. This project will use serine phosphorylation and arginine methylation to control phase separation in peptide-based model systems, as a means of regulating the rates of enzymatic reactions. This project has three research objectives: (1) Evaluate mechanisms for reaction control by droplet formation/dissolution. (2) Develop biomimetic peptide coacervate system that responds to arginine methylation. (3) Evaluate the hypothesis that spatial and temporal occurrence of distinct droplet phases can be controlled by different post-translational modifications, and provides a means of selectively modulating enzymatic reactions.