Perforin activity at membranes leads to invaginations and vesicle formation

Abstract

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. P athogen-infected and tumor cells are eliminated through granule-mediated apoptosis. Perforin (PFN), which is secreted together with proteases (granzymes) from cytotoxic T lymphocytes or natural killer cells, has a central role in this process, aiding the intracellular delivery of granzymes to initiate apoptosis of target cells (1, 2). Two models are currently available for the function of PFN. The first involves formation of stable plasma membrane pores that allow the direct transfer of granzymes into the target cell, which are later removed by membrane repair (1,3,4). This model is based primarily on visualization of pore-like structures on the membrane of the target cells (5-7). The other model, originally postulated by Podack and coworkers, proposes that the delivery process requires endocytosis of both PFN and granzyme, with the protease subsequently being released to the cytosol (4, 8).Mature PFN is a 533 residue protein consisting of three domains: an N-terminal membrane attack complex/perforin (MACPF) domain, an intervening EGF-like domain, and a Cterminal calcium-binding C2 domain, which is responsible for the initial Ca 2þ -dependent binding of PFN to membrane surfaces (9-11). The structures of PFN and other MACPF domain proteins (12-15) reveal they have folds related to those of the cholesteroldependent cytolysins (CDCs) of Gram-positive bacteria, such as pneumolysin from Streptococcus pneumoniae (16-19). These observations suggest that the effects of PFN and other MACPF proteins on membranes derive from a mechanism related to the CDCs, which involves membrane binding and oligomerization to large arc or ring structures. MACPF/CDC domains refold into membrane-inserted pore forms via an oligomeric prepore state; both prepore and pore states have been observed for PFN (15,20,21). It has been argued CDCs form pores as full ring-shaped oligomers and as arcs (17, 22); a recent study by us supports this view f...

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

“…POPC liposomes were prepared as reported. 7 Mica, glued to a metal disc, was freshly cleaved to obtain smooth surface and a Teflon chamber was stacked on the metal disc with vacuum grease. The LUV suspension was diluted in MilliQ water to a final concentration of 2 mg/ml in the presence of 2 mM Ca þ 2 and incubated on mica surface for 30 min at room temperature.…”

“…Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids. [6][7][8] In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and tumor cell plasma membranes was imaged by atomic force microscopy (AFM) and deep etch electron microscopy (DEEM), respectively. Further, the influence of an anti-human PFN mAb (pf-80) that rescues target cells from necrosis, 9 was examined.…”

Perforin: A Key Pore-Forming Protein for Immune Control of Viruses and Cancer

“…When ASM is released from the injured cell, it converts sphingomyelin in the outer leaflet of the plasma membrane to ceramide [38]. PFN also induces the formation of large endosome-like invaginations in artificial liposomes [73]. The idea that PFN activity at the plasma membrane leads to pores that are either too small or too rapidly removed to deliver Gzms directly to the cytosol, but that PFN pores formed in endosomal membranes are functional for Gzm delivery to the cytosol is consistent with a recent study of PFN pore conductance and cryo-electron microcroscopy in planar lipid bilayers and unilamellar vesicles of different lipid compositions and sizes [72].…”

Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack

“…Our recent structural studies have definitively shown that perforin monomers polymerize to form a range of pores, the majority having internal diameters of 120 to 170Å, which easily allows for trans-lumenal diffusion of granzyme molecules, typically ;50Å in diameter. 10 A lack of direct evidence for pores of this magnitude on the target cell plasma membrane previously led scientists to hypothesize a more complex, multistep route of granzyme delivery [11][12][13][14][15][16] : rather, perforin was proposed to perturb the target cell plasma membrane by forming very small or partial pores 17 to allow the cytosolic entry of calcium ions and trigger the coendocytosis of perforin and granzyme into an endosomal compartment. Within this compartment, perforin was suggested to finally reassemble into much larger pores, allowing "leakage" of granzymes into the cytosol.…”