The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

The raw Single-Molecule TIRF microscopy data was collected on microscopes using Micromanager (https://micro-manager.org/) and saved in TIFF format conforming to the OME standard (https://www.openmicroscopy.org/). 

Single-Molecule Localizations intensity traces were from the raw data using Picasso (https://github.com/jungmannlab/picasso) and titration data traces were generated using JIM (https://github.com/lilbutsa/JIM-Immobilized-Microscopy-Suite).

Analysis of traces was performed using Matlab (https://au.mathworks.com/products/matlab.html). All scripts for this analysis are included in the dataset. 

Raw tiff data can be opened using Micromanager, ImageJ, or any other image analysis program.