Quantifying nucleationin vivoreveals the physical basis of prion-like phase behavior

Abstract

SummaryProtein self-assemblies modulate protein activities over biological time scales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these time scales 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.HighlightsDistributed Amphifluoric FRET (DAmFRET) quantifies nucleation in living cellsDAmFRET rapidly distinguishes prion-like from non-prion phase transitionsNucleation barriers allow switch-like temporal control of protein activitySequence-intrinsic features determine the concentration-dependence of nucleation barriers