DAmFRET measures saturating concentrations and toxicities of protein phase transitions in vivo

Abstract

Protein phase transitions broadly govern protein function and dysfunction. However, analyzing the consequences of specific phase transitions in cells is hindered by the low throughput and limited resolution of fluorescence microscopy, and this problem is compounded for proteins with complex phase behavior such as those implicated in age-associated neurodegenerative diseases. As one solution to this problem, we incorporated an orthogonally fluorescent proxy of total protein expression to adjust for effective cell volume differences in a flow cytometric assay for protein self-association Distributed Amphifluoric FRET (DAmFRET), thereby allowing the intracellular saturating concentrations of different proteins to be precisely compared in single experiments. We further found that the effective cell volume decreased in cells experiencing proteotoxicity, which provided a simple way to assign toxicity to specific phases of ectopically expressed proteins.

Protein supersaturation powers innate immune signaling

“…volume. 57 Most of the continuous DFDs (61 of 66) did not exhibit self-seeding (Figure 1E, Table S1) . As a proxy for saturating concentration, for each self-seeded DFD, we obtained the concentration at which half of cells exhibited AmFRET.…”

“…With these questions in mind, we systematically assessed the intrinsic capacity of every human DFD to 1) supersaturate in living cells, and 2) template the nucleation of other DFDs. We achieved this goal using a combination of high throughput fluorescence microscopy and a powerful cytometry method, Distributed Amphifluoric FRET (DAmFRET) ( 32, 41 ), to evaluate sequence-encoded nucleation barriers to self-assembly in the fully orthogonal biological context of budding yeast cells. This effort uncovered 18 protein modules with prion-like behavior, and further identified DFD interactions that regulate their nucleation.…”

“…In seventeen cases the cells acquired FRET discontinuously, switching semi-stochastically to a discrete high FRET state over a range of concentrations. We previously showed that discontinuous transitions result from large sequence-encoded nucleation barriers that are characteristic for prion-like self-assembly, whereas continuous transitions instead reflect phase separation through dynamic low-affinity interactions ( 24, 32, 41, 42 ).…”