Fig. 1: Schematic illustration of CC-dimer-based designed LLPS condensates in mammalian cells.

a Coiled-coil dimer-forming peptides as interaction modules between polypeptide chains. Each polypeptide chain comprises multiple repeats of two peptides that form two orthogonal CC dimers: (S1 and S2) and (S3 and S4). Each of the polypeptide chains has a different arrangement of CC-forming repeats, either interchangeable or clustered, connected by rigid (Single-Alpha Helix, SAH); or flexible (Gly-Ser, gs) linkers. Fluorescent proteins, either TagBFP (BFP) or mCitrine (mCit) are fused to the peptides to track condensate formation and localization in cells. Due to the use of two orthogonal weakly interacting CC pairs and their different arrangement both polypeptides cannot interact in a way to satisfy all the CC interaction domains, which means that one chain can interact with multiple other chains, leading to the formation of a polypeptide network and phase separation. b NIH-3T3 cells transfected with 100 ng of a single plasmid. In the first image plasmid encoding protein labeled with mCitrine containing interchangeable S1 and S3 modules and linked with SAH linker. In the second image, TagBFP fused to three repeats of S2 and three repeats of S4 with a gs linker. c NIH-3T3 cells transfected with 100 ng of both plasmids from b. Cit, BFP, and merged channels are presented. Scale bars, 10 μm.