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

Abstract

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.

Figure 1

PFN treated cells that deliver GzmB lack signs of membrane damage and show minimal Ca²⁺ influx. (a) Calibration of Ca²⁺ influx by flow cytometry. Standardization performed as described in methods. Values plotted against free [Ca²⁺], (mean+S.D., n=4). (b) Minimal Ca²⁺ influx occurs in PFN-treated, PI negative cells. Fluo-3 and Fura Red loaded Jurkat cells were treated with indicated concentrations of PFN in the presence of PI and intracellular Ca²⁺ flux (nM) was measured in the PI negative subset using the Ca²⁺ calibration curve from (a); (n=4; mean+S.D.). (c) GzmB but not PFN is cleared rapidly from the cell surface. Jurkat cells were treated with PFN (7.6 nM, in presence of 0.2 mM Ca²⁺) or GzmB (1.92 nM) for 30 min on ice, washed, chased at 37 °C for the times indicated and stained. Isotype control subtracted mean fluorescence intensity (MFI, geometric mean) of GzmB or PFN binding on PI negative cells was plotted against the time of chase at 37 °C (MFI+S.D., n=3)

Figure 2

Anti-PFN mAb, pf-80, can be detected on target cell surface complexed with PFN. (a) pf-80 mAb can be detected on target cell plasma membranes complexed with PFN. Jurkat were treated with PFN (0–2.3 nM) in absence or presence of pf-80 (0.25 fold molar excess) or an isotype control antibody (1 fold molar excess), washed and stained with an Alexa 488 tagged anti mouse secondary antibody. Fluorescence signal in PI negative cells was analyzed, (n=3,M+S.D.). (b) pf-80 does not interfere with binding of PFN. PFN (0–2.3 nM) was either co-incubated with pf-80 (0.25 to 1 fold molar excess; simultaneous incubation) and Jurkat or pre-incubated with pf-80 alone for 15 min at room temperature followed by introduction of cells. After 10 min, cells were washed and stained with Alexa 488 tagged anti-mouse secondary antibody (n=3; mean+S.D.). (c) Membrane bound PFN:pf-80 complexes are not eluted by EGTA. PFN (0 to 1.7 nM) was pre-incubated with pf-80 (1 and 3 fold molar excess) for 15 min followed by introduction of cells. After 10 min, cells were washed in presence or absence of EGTA (5 mM), stained with Alexa 488 anti-mouse antibody and fluorescent signal in PI negative cells analyzed. (n=3, mean+S.D.)

Figure 3

Anti-PFN mAb, pf-80 blocks PFN mediated necrosis but enhances PS flip-flop and GzmB delivery. (a) pf-80 mAb blocks PFN mediated necrosis but enhances PS flip-flop. Cells were treated with PFN (1.97 nM)±pf-80 mAb in varying molar ratios. PI was present throughout and measured necrotic events defined as PI Hi events (top panel). PS flip-flop (bottom panel), was measured under identical conditions, shown as % events in the lower right quadrant of the FACS dot plot. Isotype matched Ab served as a control (representative figure from one of three studies). (b) pf-80 mAb augments PFN-mediated GzmB delivery. Jurkat cells were treated with PFN alone (1.97 nM), GzmB alone (1 μg/ml) and PFN plus GzmB±0.25 molar excess of pf-80 mAb or isotype control for 1 h at 37 °C. PI and caspase 3–7 activity were analyzed at 1 h. Values in mid-right gate show caspase 3–7 activation and a representative figure from one of three studies is shown. (c) Summary. PFN concentrations ranged from 0 to 1.97 nM for the necrosis and PS flip-flop experiments (inset) and from 0 to 2.1 nM for caspase 3–7 activation. GzmB was at 1 μg/ml (n=3, mean+S.D.)

Figure 4

The anti-PFN mAb, pf-80, augments the number of incomplete pores/arcs in PFN treated POPC bilayers. (a–f) Imaging of structures formed by PFN on planar bilayers using AFM. (a) PFN (15.2 nM) was added (15 min) at RT to POPC planar bilayer adhered to mica and imaged in tapping mode. Rings were usually 22 nm in diameter; arcs are also shown (arrowheads); (b) A field with lower protein density is shown here height profile extends 12 nm above the bilayer. (c) A magnified image from a small region of (a) demonstrates uniform sized pores (22 nm) and smaller rings. Images are from two out of seventeen different fields (7 experiments). (d–f) Mica adhered POPC planar bilayer was treated with PFN alone (15.2 nM). (g–i) Imaging of structures formed by PFN in presence of pf-80. PFN (15.2 nM) added to bilayer with 0.25x (g) or 0.5x (h) molar excess of pf-80 mAb (3.8 and 7.6 nM) for 15 min at RT. The height of structures, primarily arcs, is in the range 10–12 nm. i. A magnified image depicting arcs is shown where pf-80 was used at a 1:0.5 molar ratio. Images are representative for one of 10 fields from two experiments. (j) Summary: the number of rings and arcs for PFN:pf-80 molar ratios from 1:0–1:1 with PFN constant at 15.2 nM (mean+S.D.) are shown. Isotype matched antibody used as described in Figure 1. The number of experiments (n) for each condition, the number of fields and the total number of events analyzed are indicated. A paired t-test (2-tailed with two sample unequal variance) was utilized to measure statistical significance

Figure 5

Effects of pf-80 mAb on single channel conductance values mediated by PFN. (a) Conductance distribution of PFN channel. Planar lipid membranes (PLM) consisting of POPC/cholesterol (1/1 mol) were treated with PFN as indicated in the absence of mAb pf-80. (b) Conductance distribution of PFN channel in the presence of mAb pf-80. PLM were treated with PFN as indicated in presence of pf-80 at 1:0.25 molar ratio. Representative current traces are shown on the left. (c) Summary. Discrete versus continuous increase in conductance is shown. PFN was used at 15. 2 nM and pf-80 at 3.8 nM (ratio of 1:0.25) and 7.6 nM (ratio of 1:0.5)

Figure 6

The anti-PFN mAb, pf-80, augments the number of incomplete pores/arcs on PFN treated T24 bladder carcinoma cells. (a and b): Imaging of structures formed by PFN on T24 using DEEM. (a) PFN (3 nM) was added (4 min) at 37 °C to 2% paraformaldehyde fixed T24 cells. Cells were washed, fixed in 2% Glutaraldehyde, frozen and deep etched followed by production of platinum replicas and imaging. Rings (arrows) ranged in external diameter from 19 to 34 nm. A small proportion of Arcs were also observed (arrowheads); (b) Fixed T24 cells were treated with pf-80 (9 nM) followed by PFN (3 nM). Rings (arrows) and Arcs (arrowheads) are shown. Scale bar=100 nm. (c) Summary: the number of rings and arcs for PFN alone or PFN:pf-80 at 1:3 molar ratio with PFN constant at 3 nM (mean+S.D.) are shown; Representative of 5-8 fields. A paired t-test (2-tailed with two sample unequal variance) was utilized to measure statistical significance