Figure 4.

New technologies to measure and control protein condensation in vivo. (A) Yeast transcriptional reporting of aggregating protein (yTRAP) relies on tagging a protein of interest with a synthetic zinc finger (ZnF), which binds specifically to its cognate promoter. This yTRAP module contains an activation domain of VP16. In the soluble state, proteins diffuse freely and activate the reporter (in this case, mNeonGreen but could be easily interchanged or another reporter added for dual sensing of proteins). Upon aggregation, the signal is lost. The fluorescence can be measured sensitively with a CCD camera or flow cytometry. Three different strains of a yeast prion can be separated with this method. (B) DamFRET (distributed amphifluoric FRET). A protein contacting a prion-like domain (PrLD) is attached to mEOS3.1. Upon conversion of a subpopulation of mEOS1, two fluorophores can act as FRET pairs, if they are in close proximity. Therefore, the FRET signal acts as a proxy for concentration-dependent nucleation of prion-like proteins. Optodroplets rely on the photolyase homology region (PHR) domain of Cry2 protein of Arabidopsis thaliana. This domain self-associates upon exposure to blue light. The intrinsically disordered regions (IDRs) can be induced in vivo for supersaturation.