Pore-forming proteins (PFPs) are a large and structurally heterogeneous group of proteins produced by a wide range of organisms to permeabilize target cellular membranes. They play a fundamental role as virulence factors and as executors of endogenous machineries of regulated cell death in eukaryotic cells. The general mode of action of these proteins involves: binding to membranes, membrane insertion, and final pore assembly. This is a complex process often involving different structures with different functionalities. Despite these similarities, the detailed mechanism of pore formation differs from protein to protein. It remains unclear if different pore structures define different biological functions. Although pore opening is usually lethal, cell death is not the unique consequence of pore formation. Intracellular processes activated downstream pore formation include cytoskeletal alterations, assembly of the inflammasome, activation of the NF-ĸB pathway, secretion of pro-inflammatory cytokines, and membrane repair mechanism. Therefore, understanding how it is regulated holds therapeutic potential to control different biological processes such as infection, host defense, immunity and cell death.

This Methods Collection aims at providing a comprehensive protocol resource for the characterization of the molecular mechanism of action and the intracellular consequences of PFPs. We will cover biochemical and biophysical approaches, together with state-of-the-art techniques that provide information about pore structures and function at a single entity level.