Like factories, cells organize processes by placing them in distinct rooms (organelles). The project will create a model system to understand how cells utilize compartments without distinct walls. Design rules for the formation and function of these compartments will be established. The rules will be tested by constructing an membraneless organelle that houses enzymes that direct a series of reactions. The project will also focus efforts to foster a diverse STEM workforce through student recruiting, training, and teaching at all levels. Graduate and undergraduate students will be recruited. Outreach activities on enzymes will be developed for middle school students and summer research experiences will be provided to local high school students.
Membraneless organelles (M-organelles) provide spatial and temporal organization through selective phase separation. They are important in cell signaling, transcriptional control, and stress response. However, a robust model system that replicates their function does not yet exist. This project will develop a model system based on electrostatically driven liquid-liquid phase separation of enzymes. A model enzyme cascade of glucose oxidase and horseradish peroxidase will be incorporated into these synthetic M-organelles. Protein engineering of the enzymes will be used to determine the design rules for multiple protein partitioning. A library of fluorescent fusion proteins will be biosynthesized and their incorporation in the synthetic M-organelles will be quantified by fluorescence microscopy. The development of these design principles will enable control over the relative ratio of the sequenced enzymes in the synthetic M-organelles. Importantly, the impact of the synthetic M-organelle microenvironment on the efficiency of the enzyme cascade will be evaluated. The spatial distribution of the enzymes, substrates, and product will be monitored and used to determine detailed reaction kinetics. These combined efforts will help to establish rules for the formation and function of M-organelles.
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.
