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

Mol Cell. 2018 Jul 5;71(1):155-168.e7. doi: 10.1016/j.molcel.2018.06.016.


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.

Keywords: DAmFRET; amyloid; low-complexity sequences; nucleation barrier; phase transition; photoswitchable; prions; protein aggregation; proteotoxicity; supersaturation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Flow Cytometry
  • Peptide Termination Factors / genetics
  • Peptide Termination Factors / metabolism*
  • Prion Proteins / genetics
  • Prion Proteins / metabolism*
  • Protein Aggregates*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*


  • Peptide Termination Factors
  • Prion Proteins
  • Protein Aggregates
  • SUP35 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins