Real-time visualization of perforin nanopore assembly

Perforin is a key protein of the vertebrate imm une system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10-20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptopic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we have carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane- bound prepore intermediate state, typically cons isting of up to ~8 loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subseque ntly recruit additional prepore oligomers to grow the pore size