TY - JOUR T1 - Single molecule tracking the uncoupling of assembly and membrane insertion in Perfringolysin O JF - bioRxiv DO - 10.1101/2021.05.26.445776 SP - 2021.05.26.445776 AU - Michael J T Senior AU - Carina Monico AU - Eve E Weatherill AU - Robert J Gilbert AU - Alejandro P Heuck AU - Mark I Wallace Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/06/23/2021.05.26.445776.abstract N2 - 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 nanopore, 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.Competing Interest StatementThe authors have declared no competing interest.CDC(Cholesterol-Dependant Cytolysin)PFO(Perfringolysin O)MACPF(Membrane Attack Complex)DIB(Droplet Interface Bilayer)AFM(Atomic Force Mi-croscopy)TIRF(Total Internal Reflection)oSCR(Optical Single-Channel Recording) ER -