TY - JOUR
T1 - Mechanistic Inferences From Analysis of Measurements of Protein Phase Transitions in Live Cells
AU - Posey, Ammon E.
AU - Ruff, Kiersten M.
AU - Lalmansingh, Jared M.
AU - Kandola, Tejbir S.
AU - Lange, Jeffrey J.
AU - Halfmann, Randal
AU - Pappu, Rohit V.
N1 - Funding Information:
This work was supported by grants from the US National Science Foundation (MCB1614766 and DMR 1729783 to RVP) and US National Institutes of Health (5R01NS056114 and 1R01NS089932 to RVP, and R01GM130927 to RH). We thank Furqan Dar for helpful discussions and Matthew King and Min Kyung Shinn for comments on the manuscript. A portion of this work was done to fulfill, in part, PhD thesis research requirements for T.S.K. as a student registered with the Open University, UK at the Stowers Institute for Medical Research Graduate School, USA. Original cytometric data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1594. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding Information:
This work was supported by grants from the US National Science Foundation (MCB1614766 and DMR 1729783 to RVP) and US National Institutes of Health (5R01NS056114 and 1R01NS089932 to RVP, and R01GM130927 to RH). We thank Furqan Dar for helpful discussions and Matthew King and Min Kyung Shinn for comments on the manuscript. A portion of this work was done to fulfill, in part, PhD thesis research requirements for T.S.K. as a student registered with the Open University, UK at the Stowers Institute for Medical Research Graduate School, USA. Original cytometric data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1594.
Publisher Copyright:
© 2021 The Author(s)
PY - 2021/6/11
Y1 - 2021/6/11
N2 - The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.
AB - The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.
KW - DAmFRET
KW - high-throughput
KW - machine learning
KW - nucleation
KW - prion
UR - http://www.scopus.com/inward/record.url?scp=85101119891&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2021.166848
DO - 10.1016/j.jmb.2021.166848
M3 - Article
C2 - 33539877
AN - SCOPUS:85101119891
SN - 0022-2836
VL - 433
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 12
M1 - 166848
ER -