Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior

Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein’s self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity. [Display omitted] •Distributed amphifluoric FRET (DAmFRET) quantifies nucleation in living cells•DAmFRET rapidly distinguishes prion-like from non-prion phase transitions•Sequence-intrinsic features determine concentration dependence of nucleation barriers•Prion cross-seeding occurs by conformational templating, not by condensation Prion phenomena result from protein phase separations that are rate limited by nucleation. Here, Khan et al. introduce a method to quantify nucleation in living cells. By comparing diverse proteins with and without prion behavior, they show that the kinetic barrier to nucleation derives from structural order in the new phase.