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

Abstract

Protein 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 D istributed Am phifluoric 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.

A First Glimpse of Nucleation of Phase Transitions in Living Cells

“…Given these complexities, the ability to study prion formation in living cells seems like a mirage. And yet, here we are: in this issue, Khan et al (2018) report the development and deployment of a novel method known as Distributed Amphifluoric FRET (DAmFRET) that affords a first glimpse of the processes that initiate the formation of prion-like supramolecular assemblies within living cells. To achieve this, Khan et al turned budding yeast into a veritable test tube for high-throughput experiments that enable comparative assessments of ''prion-ness'' across a range of proteins.…”