DAmFRET measures saturating concentrations and toxicities of protein phase transitions in vivo
Tayla Miller, Jianzheng Wu, Shriram Venkatesan, Alejandro Rodríguez Gama, Randal Halfmann
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 fluorescence 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.