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. 2016 Sep;26(9):1007-20.
doi: 10.1038/cr.2016.100. Epub 2016 Aug 30.

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis

Xin Chen  1 , Wan-Ting He  1 , Lichen Hu  1 , Jingxian Li  1 , Yuan Fang  1 , Xin Wang  1 , Xiaozheng Xu  1 , Zhuo Wang  1 , Kai Huang  1 , Jiahuai Han  1
Affiliations

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis

Xin Chen et al. Cell Res. 2016 Sep.

Abstract

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis. VSports手机版.

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Figures

Figure 1
Figure 1
Pyroptotic and necroptotic cells have distinct morphological features. (A-C) Viabilities of RAW-asc cells treated with DMSO or TS (TNF and smac mimetic, A), or TSZ (TNF, smac mimetic and the caspase inhibitor z-VAD, B), or primed with LPS for 4 h followed by nigericin (Nig) treatment or no treatment (C) for different periods of times as indicated. Apoptotic cells were identified by annexin V staining. Necroptosis and pyroptosis were determined by propidium iodide (PI) staining. Results shown are mean ± SD from three independent experiments. The final concentrations of 10 ng/ml TNF, 100 nM smac mimetic, 20 μM z-VAD, 1 μg/ml LPS and 10 μM Nig were used here and in subsequent experiments unless otherwise stated. (D-F) Representative time-lapse images of RAW-asc cells treated as in A-C. Cell morphology was visualized by wide-field light microscopy (upper panel) and cell membrane integrity was monitored by PI uptake (lower panel). (G) Representative scanning electronic microscopy (SEM) images of RAW-asc cells treated as in A-C. Arrowhead indicates explosion of necroptotic cells and arrow points to bubbling of pyroptotic cells. Scale bar, 5 μm (D-F) and 2 μm (G).
Figure 2
Figure 2
GSDMD-N translocates to plasma membrane in pyroptotic cells. (A) Deconvolution microscopy of MLKL-HA-expressing RAW-asc cells treated with DMSO or TSZ for 5 h. After treatment, cells were immunostained for HA and counterstained with Hoechst and PI. PI positive indicates dead cell. (B) Plasma membrane marker Hras-RFP was expressed and detected in MLKL-HA-expressing RAW-asc cells. Cells were treated and analyzed as in A. (C) Deconvolution microscopy of HA-GSDMD-reconstituted Gsdmd−/− RAW-asc cells primed with LPS for 4 h and subsequently treated with or without Nig for 2 h. (D) The same as in C except that plasma membrane marker Hras-RFP was expressed and detected in HA-GSDMD-reconstituted RAW-asc cells. (E) Gsdmd−/− RAW-asc cells were reconstituted with GSDMD-Flag, then treated and analyzed as in C. (F) Quantification of cells in C with uniformly diffused GSDMD (UD), plasma membrane enriched GSDMD plus PI-positive (PM, PI+) and plasma membrane enriched GSDMD plus PI-negative (PM, PI−). (G) Purified recombinant GST-GSDMD-N and GST-GSDMD-C were incubated with a general lipid strip, respectively. Protein-lipid binding assay was performed as described in the Material and Methods. (H) Raw-asc cells were primed with LPS for 4 h and then pretreated with 3MA (10 mM), YM201636 (80 μM), SF1670 (2 μM) or PITenin-7 (20 μM) for 1 h. Cells were then treated with Nig for 2 h and cell survival was determined by PI staining. Results shown are mean ± SD representative of three independent experiments. Scale bar, 5 μm.
Figure 3
Figure 3
Forced interaction of GSDMD-N leads to pyroptosis. (A) GSDMD-N-HBD*-HA or GSDMD-C-HBD*-HA was expressed in CHO cells. These cells were treated with DMSO or 1 μM 4-OHT for 1 h. Cell survival was determined by PI staining. Results shown are mean ± SD from two independent experiments. The same concentration of 4-OHT was used in subsequent experiments unless otherwise stated. (B) Representative time-lapse imaging of GSDMD-N-HBD*-HA-expressing CHO cells treated with 4-OHT. Cell morphology was detected by wide-field light microscopy (upper panel) and cell membrane integrity was monitored by PI uptake (lower panel). Arrow indicates bubbling of pyroptotic cells. (C) Representative SEM images of GSDMD-N-HBD*-HA-expressing CHO cells treated with DMSO or 4-OHT for 1 h. Arrow indicates bubbling of pyroptotic cells. (D) Deconvolution microscopy of GSDMD-N-HBD*-HA-expressing CHO cells treated with DMSO or 4-OHT for 1 h. PI was used to detect dead cells. (E) The same as D, except that plasma membrane marker Hras-RFP was expressed and detected in GSDMD-N-HBD*-HA-expressing CHO cells. (F) Quantification of cells in D with uniformly diffused GSDMD-N-HBD*-HA (UD), plasma membrane enriched GSDMD-N-HBD*-HA plus PI-positive (PM, PI+) and plasma membrane enriched GSDMD-N-HBD*-HA plus PI-negative (PM, PI−). Scale bar, 5 μm (B, D and E) and 2 μm (C).
Figure 4
Figure 4
GSDMD-N but not GSDMD-C forms oligomer during pyroptosis. (A) Gsdmd−/− RAW-asc cells were reconstituted with Flag-GSDMD and GSDMD-Flag respectively, and then primed with LPS for 4 h followed by Nig stimulation for 0 min, 30 min, 60 min and 120 min. The culture supernatants together with their corresponding cell extracts were resolved by 4% - 12% gradient SDS-PAGE. Immunoblotting of Flag was performed. (B) The same cells were treated as in A. The culture supernatants together with their corresponding cell extracts were analyzed after DSS crosslinking. (C) Flag-GSDMD-reconstituted cells were primed with LPS followed by Nig stimulation or no stimulation. Lysates of these cells were fractionated by gel-filtration chromatography (Superose 6, fractions sizes at top) and followed by immunoblot analysis.
Figure 5
Figure 5
N-terminal five amino acids are indispensable for GSDMD-N to form oligomers and trigger pyroptosis. (A) Sequence alignment of N-terminal 20 amino acids of human GSDMA, GSDMB, GSDMC, GSDMD and mouse GSDMD (upper panel), and schematic representation of N-terminal deletions of GSDMD-N fused with HBD* (lower panel). (B) Viabilities of CHO cells expressing different GSDMD-N-HBD*-HA deletion mutants after stimulation with DMSO or 4-OHT for 0.5 h. Data represented the mean ± sd of three independent experiments. (C) Immunoblot analysis of lysates of CHO cells in B resolved by 4% - 12% gradient SDS-PAGE under non-reducing condition. (D) Deconvolution microscopy of CHO cells expressing different GSDMD-N-HBD*-HA deletion mutants treated with 4-OHT for 1 h. After treatment, cells were immunostained for HA and counterstained with Hoechst and PI. PI positive indicates dead cell. Scale bar, 5 μm.
Figure 6
Figure 6
GSDMD-N non-selectively increase plasma membrane permeability. (A-C) Intracellular ion concentration were monitored in GSDMD-N-HBD*-HA-expressing CHO cells treated with 4-OHT. Sodium indicator (ANG-2, A), calcium indicator (Fluo-4, B) and potassium indicator (APG-2, C) were used together with PI. For each ion indicator, a series of representative time-lapse images were shown in the left panel and relative intensity change of ion indicator and PI fluorescence in one representative cell was graphed over time in the right panel. (D) GSDMD-N-HBD*-HA-expressing CHO cells were treated with 4-OHT for 0.5 h in PBS (CTRL) or PBS containing 30 mM PEG400, PEG1450, PEG4000 or PEG8000. Cells were stained by PI and analyzed under microscope. (E) GSDMD-N-HBD*-HA-expressing CHO cells were treated with 4-OHT in PBS or in PEG4000 PBS for 0.5 h, or were treated with 4-OHT in PEG4000 PBS for 0.5 h and then were changed into PBS for another 0.5 h. Cells were stained with PI and analyzed under microscope. Results shown are mean ± SD from two independent experiments. Scale bar, 10 μm.
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