Biophysics of Phase Separation in Protein-RNA Systems

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The project will develop and use novel tools to study how cellular materials are organized to facilitate chemical and biological functions that support life, while integrating broader impacts in teaching, training and outreach. The biology of humans and other organisms is critically dependent on interactions and chemistry of a host of cellular molecules. A key mechanism that underlies these correct interactions in a huge background of other molecules is the selective organization of molecules into different compartments. Indeed, cellular compartmentalization by a surrounding membrane has been studied in detail for decades. In contrast, another type of cellular compartment that lacks a membrane is common but less well-studied. These membraneless or "droplet" organelles are spontaneously formed by a phase transition (referred to as liquid-liquid phase separation, a process similar to oil-in-water droplet formation). In this project, novel methods to address unfilled gaps in our understanding of the physical principles underlying this process will be developed. The work is expected to result in new tools, insights and predictive principles that are broadly applicable in cell biology. The project will also integrate broader impacts in teaching, training and outreach, and aim to broaden participation in science. The work will be used as an excellent training ground for students in performing interdisciplinary research in a cutting-edge and timely area of research. The multidisciplinary nature of the research will also facilitate enhancement of graduate student curriculum, as well as outreach and communication of the research area to the broader community.Several membraneless organelles in cells contain proteins and RNA, both cellular macromolecules that are involved in a wide range of cellular processes. Understanding how molecular-level structure and interactions of these molecules link to the biophysics of droplet formation and membraneless organelles continues to pose challenges. The project will develop and use a combination of novel single-molecule and ensemble methods to study a series of important questions in this area. Single-molecule methods can provide access to critical information that is normally hidden by averaging over a large number of molecules. A method will be developed to probe size and conformational distributions in droplets smaller than typically detectable currently, by use of multicolor single-molecule detection. Another method will be developed to probe intermolecular interactions and conformational properties using single-molecule and chemical biology tools. These new tools and existing methods will be used to probe the determinants of complex phase transitions, non-equilibrium substructure formation and conformational patterning of protein-RNA droplets. The project is expected to reveal new insight in these aspects of cellular phase transitions and their functional consequenceThis 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.