Fig. 6: Design of chemical regulation of CC-LLPS.
From: Programmable de novo designed coiled coil-mediated phase separation in mammalian cells
a One polypeptide chain of a condensate-forming pair BFP-(S2)3(S4)3-gs, was separated into two chains, each with 3 CC domains BFP-(S2)3-gs and (S4)3-gs. FKBP and FRB domains were fused to each chain to trigger heterodimerization by the addition of rapamycin (RAPA). b NIH-3T3 cells transfected with the single long chain of the pair and two chemically regulated shorter chains. DMSO was added as control and below after the addition of 1 μM rapamycin, at 0 and after 30 minutes. c Time series after 1 μM rapamycin addition. Merge of mCitrine and BFP channel. d Single polypeptide chain was split into two condensation-incompetent chains and each of them connected to either FKBP or FRB domain. HEK293T cells were transfected with both parts. Condensates were observed after the addition of rapamycin. e Time series after 1 μM rapamycin addition. Insets from Supplementary Movie 5.f TEVp site was incorporated in one of the pairs of condensate-forming proteins at a position that forms condensate incompetent pairs after cleavage. Rapamycin-regulated split TEVp protease was used. After the addition of rapamycin FKBP/FRB heterodimerize, activating split TEVp which cuts the TEVp site leading to the dissolution of condensates. HEK293T cells transfected with plasmids for both polypeptides and split TEVp. After the addition of rapamycin condensates dissolve in a matter of minutes. g Time series after 1 μM rapamycin addition. Scale bars, 10 μm.
