Single molecule tracking the uncoupling of assembly and membrane insertion in Perfringolysin O

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore-formation of the archetypical cholesterol-dependent cytolysin, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 millisecond temporal resolution and 20 nanometre spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes.