Rapidly Inducible De Novo Synthesis of Hydrogels in Living Cells

Abstract

We report a design, synthesis and characterization of hydrogels inside living cells which will serve as a biomolecular sift. We achieve this by rapid induction of a sol-gel phase transition. A hydrogel is defined as a non-fluid, cross-linked polymer network. Inducing polymerization of soluble multivalent molecules could thus lead to in situ hydrogel formation. To probe a spatiotemporally dynamic cellular processes, the phase transition must take place at a specific subcellular location in a rapidly inducible manner. To achieve this, we utilized a chemically inducible dimerization technique, through which a pair of protein domains bind to each other only in the presence of the corresponding chemical dimerizer. Our choice of protein pairs are FKBP (FK506 binding protein) and FRB (FKBP-rapamycin biding domain) which dimerize in the presence of rapamycin. For the in-cell hydrogel formation, we induced polymerization of the multivalent FKBP (FKBPX) and FRB (FRBX) molecules spaced with a long peptide linker by adding a chemical dimerizer. By targeting the FRBX to a particular cellular location and FKBPX to the cytosol, we were able to rapidly produce a hydrogel in a spatio-temporally controlled manner. Subsequently, we performed biophysical characterization of the synthetic gels both in vitro and in vivo and found that the gels allow passive diffusion of most of the proteins but not larger molecular complexes or cellular organelles. The present study is highly unique in its quality execution of rapidly inducing de novo synthesis of hydrogels in living cells.