The engineering of cellular decision-making ? such as entry into the cell cycle or stem cell differentiation ? is critical to our ability to regenerate functional cells and tissues. The development of these technologies is limited by the extent to which one can manipulate the endogenous cellular machinery. Membraneless organelles are naturally-occurring assemblies of intrinsically disordered proteins (IDPs) that form protein-rich, fluid-like phases in cells. With insightful engineering, these organelles can provide a means for directing cell physiology through the compartmentalization and release of regulatory molecules. Recently, we developed a novel platform for the assembly of membraneless organelles endowed with a myriad of novel properties and control motifs. Our methods are based on engineering intrinsically-disordered arginine-glycine rich RGG domains that are sufficient to drives phase separation. The premise of our application is the development of numerous, state-of-the-art IDP materials for assembly of new, designer membraneless organelles. We have developed methods to dynamically modulate the multivalency of RGG sequences and introduced protein-interaction motifs to allow sequestration of designated cargo. We propose to develop strategies for controlling the rapid release and sequestration of cargo proteins in response to optical and thermal stimuli. Further, we propose the develop optical and enzymatic controllers for organelle assembly and disassembly. Importantly, we have already demonstrated the expression of the sequences is sufficient to generate membraneless organelle in yeast and mammalian cell lines. Here we propose a novel, innovative paradigm in synthetic biology for controlling cellular responses through the manipulation of membraneless organelles. These organelles will be designed to sequester factors that control cell cycle commitment or cell fate decisions on cue, in response to light or biochemical stimulus. The ultimate goal of the proposed work is to control the dynamic progression through the cell cycle and cell fate decision- making in hematopoietic stem cells for regenerative medicine.
In aim 1, we will expand the toolbox of self- assembling IDPs by engineering optical and thermal switches within disordered protein materials for dynamic gating of organelle assembly, composition and release.
In aim 2, we will use membranless organelles for cell decision-making, rapidly sequestering and releasing target proteins to dynamically control cell proliferation and stem cell fate. Harnessing the power of compartmentalization, our designer organelles promise a tunable biochemical niche and a generalizable strategy for precisely engineering cell systems capable of responding to specific stimuli with predictable outcomes.

Public Health Relevance

The engineering of cell behaviors ? such as entry into the cell cycle or commitment to differentiation ? is critical to our ability to regenerate functional cells and tissues. A major limitation to this goal is the absence of appropriate materials that can direct rapid and reversible changes in cell function. Our development of synthetic membraneless organelles that can be tailored to rapidly sequester or release regulatory factors promises a significant advance in the field of cellular engineering for application in regenerative medicine.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Research Project (R01)
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Cellular and Molecular Technologies Study Section (CMT)
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Russell, Luisa Marie
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University of Pennsylvania
Anatomy/Cell Biology
Schools of Medicine
United States
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