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

Lopez, Jamie A.; Susanto, Olivia; Jenkins, Misty R.; Lukoyanova, Natalya; Sutton, Vivien R.; Law, Ruby H. P.; Johnston, Angus P. R.; Bird, Catherina H.; Bird, Phillip I.; Whisstock, James C.; Trapani, Joseph A.; Saibil, Helen R.; Voskoboinik, Ilia

doi:10.1182/blood-2012-07-446146

Cited by 212 publications

(242 citation statements)

References 41 publications

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“…Very low concentrations of PI (1.0-1.5 mM) are typically used in cell death assays to detect massive plasma membrane rupture during secondary necrosis. By adding significantly more PI (100 mM) to the medium, PI uptake can be detected entering the target cell specifically at the site of NK synapse upon granule delivery 17 ( Figure 3a). Using these parameters, the initiation phase of GzmB-mediated apoptosis (mediated by WT or GzmA À / À NKs) was 4.9 ± 0.2 or 3.3 ± 0.1 min, respectively, whereas the initiation phase of GzmB À / À NK-mediated athetosis was significantly longer (16.9 ± 1.02 min; Po0.0001, Figure 3b).…”

Section: Resultsmentioning

confidence: 99%

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

et al. 2013

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Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.

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Section: Resultsmentioning

confidence: 99%

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

et al. 2013

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“…2 This structure has seemed the obvious candidate for providing granzyme passage into a target cell cytosol. Perplexingly, however, PFN efficiently induces translocation of GzmB in targets that exclude even low molecular weight cationic fluorophores, 5,6,17,18 suggesting that cylindrical pores are somehow dispensable for granzyme delivery leaving another form of PFN responsible.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2014

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Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets. Cell Death and Differentiation ( The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation. 1 Ring-shaped transmembrane PFN pores hereafter called 'cylindrical pores', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis. [2][3][4] In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal, 5 that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding. 6 This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. 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 t...

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“…31 Furthermore, Propidium Iodide (PI) rapidly diffuses through the perforin pore upon delivery of the lethal hit, when added to the assay medium. 32 These events can be monitored by imaging the cells in real time using time-lapse microscopy 32 and allows accurate quantification of the proportion of NK-tumor cell interactions that trigger calcium influx and result in a successful lethal hit.…”

Section: Dnam-1 Ligands Promote Nk Cell Degranulation and Aml Cell Lysismentioning

confidence: 99%

“…2 £ 10 4 NK cells were labeled with Fluo-4 for 20 min (1 mM Fluo-4 and 0.02% [w/v] Pluronic F-127 carrier at 37 C/10% CO 2 ), then added to adherent targets, in media containing 100 mM PI (PtdIns). 32 Chamber slides were mounted on a heated stage within a temperature-controlled chamber maintained at 37 C, and constant CO 2 concentrations (5 or 7%) were infused using a gas incubation system with active gas mixer ("The Brick;" Ibidi). Optical sections were acquired through sequential scans of Fluo-4 (excitation 488 nm; 5-mm pinhole), PI (excitation 561 nm; 1.38-mm pinhole), or Brightfield/DIC on a TCS SP5 confocal microscope (Leica Microsystems, Deerfield, IL) using a 40£ (NA 0.85) air objective and Leica LAS AF software.…”

Section: Time-lapse Microscopymentioning

confidence: 99%

Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

et al. 2016

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Acute myeloid leukemia (AML) is associated with poor natural killer (NK) cell function through aberrant expression of NK-cell-activating receptors and their ligands on tumor cells. These alterations are thought to promote formation of inhibitory NK-target cell synapses, in which killer cell degranulation is attenuated. Allogeneic stem cell transplantation can be effective in treating AML, through restoration of NK cell lytic activity. Similarly, agents that augment NK-cell-activating signals within the immunological synapse may provide some therapeutic benefit. However, the receptor-ligand interactions that critically dictate NK cell function in AML remain undefined. Here, we demonstrate that CD112/CD155 expression is required for DNAM-1-dependent killing of AML cells. Indeed, the low, or absent, expression of CD112/CD155 on multiple AML cell lines resulted in failure to stimulate optimal NK cell function. Importantly, isolated clones with low CD112/155 expression were resistant to NK cell killing while those expressing abundant levels of CD112/155 were highly susceptible. Attenuated NK cell killing in the absence of CD112/CD155 originated from decreased NK-target cell conjugation. Furthermore, we reveal by time-lapse microscopy, a significant increase in NK cell 'failed killing' in the absence of DNAM-1 ligands. Consequently, NK cells preferentially lysed ligandexpressing cells within heterogeneous populations, driving clonal selection of CD112/CD155-negative blasts upon NK cell attack. Taken together, we identify reduced CD155 expression as a major NK cell escape mechanism in AML and an opportunity for targeted immunotherapy.

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

Abstract: Key Points Granzymes diffuse through perforin pores on the target cell plasma membrane.

Cited by 212 publications

References 41 publications

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

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

Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

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