bioRxiv preprint doi: https://doi.org/10.1101/205690; this version posted March 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Quantifying nucleation in vivo reveals the physical basis of prion-like phase behavior 1,3 1,3 1,2,3 1,3 1,3 Tarique Khan , Tejbir S. Kandola , Jianzheng Wu , Shriram Venkatesan , Ellen Ketter , 1 1 1 1 1 Jeffrey J. Lange , Alejandro Rodríguez Gama , Andrew Box , Jay R. Unruh , Malcolm Cook , 1,2, and Randal Halfmann * 1 Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110 2 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA 3 These authors contributed equally *Corresponding author:
[email protected] Highlights ● Distributed Amphifluoric FRET (DAmFRET) quantifies nucleation in living cells ● DAmFRET rapidly distinguishes prion-like from non-prion phase transitions ● Nucleation barriers allow switch-like temporal control of protein activity ● Sequence-intrinsic features determine the concentration-dependence of nucleation barriers Summary 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 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.