Experimental Model Systems for Intracellular Compartmentalization: Dynamic Formation/Disassembly of Model Organelles in Artificial Cells

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Technical Description: This proposal is based on the idea that it may be possible to realize key structural and functional aspects of subcellular organization due to the physicochemical phenomena that arise in solutions that are crowded with macromolecules "like the cytoplasm and nucleoplasm are" even in the absence of specific biomolecular interactions. Of particular interest is aqueous phase separation, which commonly occurs in polymer solutions and offers a means of compartmentalization. This work will evaluate two hypotheses: (1) that phase separation in the crowded intracellular environment could be largely responsible for the existence and properties of non-membranous organelles, and (2) that these structures could in turn serve as templates for membrane assembly. Experimental models of subcellular cytoplasmic and nuclear compartments will be developed based on crowded solutions of RNA, polyamines, proteins, and neutral polymers. Protein phosphorylation will be used to drive formation and disassembly of microcompartments that will serve as model organelles. Three research objectives are proposed: (1) Dynamic assembly/disassembly of model non-membranous organelles based on aqueous phase compartments that form in response to protein phosphorylation state. (2) Templated membrane formation around these model organelles. (3) Primitive model for mitosis/cell cycle in cell-sized lipid vesicles that contain the crowded solution and phase compartment-based model organelles. Nontechnical Description: Intracellular organization is a hallmark of living cells, with both membrane-bounded (e.g., nucleus) and non-membranous organelles (e.g., nucleolus, P-granules) performing key cellular functions. The central questions driving these investigations are: What role do relatively nonspecific chemical and physical effects play in subcellular organization and the associated functions of biological cells, and how does complexity arise from a small number of simple molecular components? The investigators hypothesize that, despite the deliberate simplicity of the proposed model cells, they will be able to mimic complex biological processes and specific biochemical interactions, such as the reversible formation and dissolution of RNA and protein-rich compartments, the formation of interior membranes around pre-existing protein-rich compartments, and finally to model cell division in a very primitive mimic of the mitotic cell. Broader Impacts. Two graduate students will be trained at the interface of molecular and cell biology, chemistry, biophysics and materials science. Undergraduate students will be recruited to work on this project during the academic year for course credit and in the summer through the various on-site REU programs, with a target of one student during the academic year and one or two students during the summer. Support for one K-12 teacher each summer is included in the budget. The investigators will team with the existing Research Experiences for Teachers program in the PSU MRSEC. Teachers will perform experiments and develop curriculum materials to bring back to their own classrooms the following school year. Real-world examples go beyond the intracellular organelles that motivate the intellectual merit of this proposal to also include, e.g., food science, drug delivery, and environmental remediation.This award is being funded by the Systems and Synthetic Biology Cluster in MCB/BIO and co-funded by the Chemistry of Life Processes Program in CHE/MPS.