Single molecule imaging of cholesterol-dependent cytolysin assembly

10.1101/2021.05.26.445776

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Tracking Perfingolysin O assembly and pore formation. We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway of the Cholesterol-Dependent Cytolysin, Perfringolysin O. This enables quantification of the stoichiometry of PFO complexes during assembly with millisecond temporal resolution and 20 nanometre spatial precision. Our results support a model of overall stepwise irreversible assembly, dominated by monomer addition, but with infrequent assembly from larger partial complexes. Furthermore, our results suggest a dominant proportion of inserted, but non-conductive intermediates in assembly.