Figure 4.

The low-affinity interaction of TTP with DCP2 induces LLPS of TTP_Nin vitro. (A) Representative images of in vitro LLPS of the DCP2 proteins and TTP_N in the presence of a molecular crowding reagent, PEG8000. A range of protein concentrations from 250 to 10 μM were analysed. All DCP2 proteins undergo LLPS at high concentrations, while DCP2_ΔC, which has the strongest tendency to undergo phase separation, also does so at the lowest concentration investigated. TTP_N does not form phase-separated droplets in any of the tested conditions. Mixtures of TTP_N and DCP2_C-tail show phase separation at a lower concentration than DCP2_C-tail alone. Images were acquired by phase-contrast microscopy. (B) Representative images of in vitro phase separation of equimolar (90 μM) mixtures of Cy5-labelled TTP_N and XFD488-labelled DCP2 in the presence of a molecular crowding reagent, PEG8000. Overlap of Cy5 and XFD488 signals suggests co-localization of the two proteins. TTP_N partitions into phase-separated droplets in the presence of DCP2_C-tail and DCP2_ΔC, but not in the presence of DCP2_core. Brightfield images show additional phase-separated droplets in different focal planes that are not visible in the fluorescent images due to optical sectioning by the Zeiss Apotome. The imperfect overlap between the XFD488 and Cy5 fluorescent images stems from moving droplets and a time lag in image acquisition. Scale bars, 2 μm.