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.

Alpha-synuclein pore forming activity upon membrane association.

Alpha-synuclein is a natively unfolded protein widely expressed in neurons at the presynaptic level. It is linked to Parkinson's disease by two lines of evidence: amyloid fibrils of the protein accumulate in patients' brains and three genetic mutants cause autosomal dominant forms of the disease. The biological role of the protein and the mechanisms involved in the etiopathogenesis of Parkinson's disease are still unknown. Membrane binding causes the formation of an amphipathic alpha-helix, which lies on the surface without crossing the bilayer. Recent observations however reported that the application of a voltage induces a pore-like activity of alpha-synuclein. This study aims to characterize the pore forming activity of the protein starting from its monomeric form. In particular, experiments with planar lipid membranes allowed recording of conductance activity bursts with a defined and reproducible fingerprint. Additional experiments with deletion mutants and covalently bound alpha-synuclein dimers were performed to understand both pore assembly and stoichiometry. The information acquired allowed formulation of a model for pore formation at different conductance levels.