Fig. 3

MyD88 polymerises in a concentration-dependent manner and can be self-seeded. a Schematic diagram of the principle of two-colour seeding experiments testing the self-replication propensity of full-length MyD88 filaments. Full-length MyD88 is expressed in an mCherry-tagged version above its supercritical concentration to create filaments, which are gently spun and washed, then sonicated to increase the number of fragments. These “seeds” are then mixed in a sample expressing GFP-tagged full-length MyD88 at sub-critical concentrations. b Example of fluorescence time trace for MyD88 at 10 nM concentration. Unseeded sample demonstrating a monomeric time trace profile (above) with the seeded sample (below) showing polymerisation of GFP-MyD88 upon the addition of MyD88 “seeds”. c The B parameter (brightness) correlating with number of oligomers detected in typical time-traces as a function of protein concentration (nM), with and without “seeds” introduced. The subcritical, supercritical and “meta-stable” zones are labelled. Values are from approx. 50 repeated dilution experiments with the various protein concentrations and corresponding brightness values obtained plotted