Fig. 1. Macroscale physical characterization of MeV droplets in vitro.

(A) Schematic diagram of the structure of N and P; intrinsically disordered regions are presented as lines and folded domains are presented as boxes. (B) Fluorescence or DIC images of mixtures where phase separation events were observed: P₅₀N₅₂₅ (80 μM, 1% labeled) + P_1–507 (66 μM), P₅₀N₅₂₅ (80 μM, 1% labeled) + P_304–507 (200 μM), P₃₀₀N₅₂₅ + P_304–507 (290 μM), and N_1–525 + P_1–507 (coexpressed in E. coli). (C) The phase separation scaffold requires P₅₀N₅₂₅ and P_304–507 domains. Mixtures of different N and P truncation mutants were tested for the ability to trigger phase separation using DIC microscopy. “−” no phase separation events were observed; “+” phase separation events were observed; “NA” not tested. All mixtures were tested at several concentrations higher than 50 μM for each protein and several ratios including 1:1. Bottom panel represents coexpression of the full-length N and P proteins. (D) Picture of tubes with P₅₀N₅₂₅ and P_304–507. Sample with N and P mixed together becomes turbid. (E) Colocalization of P₅₀N₅₂₅-fluorescein (80 μM, 1% labeled) and P_304–507-Alexa Fluor 594 (200 μM, 0.5% labeled) droplets.