PT  - JOURNAL ARTICLE
AU  - Senior, Michael J T
AU  - Monico, Carina
AU  - Weatherill, Eve E
AU  - Gilbert, Robert J
AU  - Heuck, Alejandro P
AU  - Wallace, Mark I
TI  - Single molecule tracking the uncoupling of assembly and membrane insertion in Perfringolysin O
AID  - 10.1101/2021.05.26.445776
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2021.05.26.445776
4099  - http://biorxiv.org/content/early/2022/06/23/2021.05.26.445776.short
4100  - http://biorxiv.org/content/early/2022/06/23/2021.05.26.445776.full
AB  - 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)