Perforin activity at membranes leads to invaginations and vesicle formation.

The cytotoxic cell granule secretory pathway is essential for immune defence. How the pore-forming protein perforin (PFN) facilitates the cytosolic delivery of granule-associated proteases (granzymes) remains enigmatic. Here we show that PFN is able to induce invaginations and formation of complete internal vesicles in giant unilamellar vesicles. Formation of internal vesicles depends on native PFN and calcium and antibody labeling shows the localization of PFN at the invaginations. This vesiculation is recapitulated in large unilamellar vesicles and in this case PFN oligomers can be seen associated with the necks of the invaginations. Capacitance measurements show PFN is able to increase a planar lipid membrane surface area in the absence of pore formation, in agreement with the ability to induce invaginations. Finally, addition of PFN to Jurkat cells causes the formation of internal vesicles prior to pore formation. PFN is capable of triggering an endocytosis-like event in addition to pore formation, suggesting a new paradigm for its role in delivering apoptosis-inducing granzymes into target cells.

Electroformation of giant unilamellar vesicles from erythrocyte membranes under low-salt conditions.

Giant unilamellar vesicles (GUVs) are an attractive experimental model for studying various membrane-related phenomena. The procedure for GUV electroformation from erythrocyte ghosts under physiological conditions was introduced recently; however, it allows preparation of a limited number of GUVs. Here we describe an efficient, reliable, and simple method for electroformation of GUVs from native erythrocyte membranes at low salt concentration, which enables the formation of higher amounts of large, spherical GUVs. GUVs prepared according to the new procedure may not retain original lipid asymmetry; however, they preserved native proteins, lipids, and oligosaccharide heterogeneity and could be a suitable system for functional studies for which larger amounts of GUVs of complex composition are needed.

Human perforin employs different avenues to damage membranes.

Perforin (PFN) is a pore-forming protein produced by cytotoxic lymphocytes that aids in the clearance of tumor or virus-infected cells by a mechanism that involves the formation of transmembrane pores. The properties of PFN pores and the mechanism of their assembly remain unclear. Here, we studied pore characteristics by functional and structural methods to show that perforin forms pores more heterogeneous than anticipated. Planar lipid bilayer experiments indicate that perforin pores exhibit a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were very unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Furthermore, two different membrane-bound PFN conformations were observed, interpreted as pre-pore and pore states of the protein. The results collectively indicate that PFN forms heterogeneous pores through a multistep mechanism and provide a new paradigm for understanding the range of different effects of PFN and related membrane attack complex/perforin domain proteins observed in vivo and in vitro.

Human perforin permeabilizing activity, but not binding to lipid membranes, is affected by pH.

The various steps that perforin (PFN), a critical mediator of innate immune response, undertakes to form a transmembrane pore remains poorly understood. We have used surface plasmon resonance (SPR) to dissect mechanism of pore formation. The membrane association of PFN was calcium dependent irrespective of pH. However, PFN does not permeabilize large or giant unilamellar vesicles (GUV) at pH 5.5 even though the monomers bind to the membranes in the presence of calcium. It was possible to activate adsorbed PFN and to induce membrane permeabilization by simply raising pH to a physiological level (pH 7.4). These results were independently confirmed on GUV and Jurkat cells. The conformational state of PFN at either pH was further assessed with monoclonal antibodies Pf-80 and Pf-344. Pf-344 maps to a linear epitope within region 373-388 of epidermal growth factor (EGF)-like domain while the Pf-80 appears to recognize a conformational epitope. Pf-344 interacts with the EGF-like domain after PFN monomers undergo pore formation, the site recognized by Pf-80 is only accessible at acidic but not neutral pH. Thus, the Pf-80 mAb likely interacts with a region of the monomer that participates in oligomerization prior to insertion of the monomer into the lipid bilayer and thus may have therapeutic utility against PFN-mediated immunopathology.