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

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

Author information

1
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
2
Stowers Institute for Medical Research, Kansas City, MO 64110, USA; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
3
Stowers Institute for Medical Research, Kansas City, MO 64110, USA; Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA. Electronic address: rhn@stowers.org.

Abstract

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

PMID:
29979963
PMCID:
PMC6086602
[Available on 2019-07-05]
DOI:
10.1016/j.molcel.2018.06.016

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