Plasma membrane changes during programmed cell deaths.pdf

Cell Research(2018)28:9- membrane changes during programmed cell deathsYingying Zhang1,Xin Chen1,Cyril Gueydan2,Jiahuai Han11State Key Laboratory of Cellular Stress Biology,Innovation Center for Cell Signaling Network,School of Life Sciences,Xiamen University,Xiamen,Fujian 361005,China;2Laboratoire de Biologie Molculaire du Gne,Facult des Sciences,Universit Libre de Bruxelles,1050 Brussels,BelgiumRuptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death,respectively.As such,apoptosis is usually considered a non-inflammatory process while necrosis triggers in-flammation.Recent studies on necroptosis and pyroptosis,two types of programmed necrosis,revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D(GSDMD)pores during pyroptosis.Importantly,the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores.Interestingly,it was found recently that secondary necrosis of apoptotic cells,a previously believed non-regulated form of cell lysis that occurs after apoptosis,can be programmed and executed by plasma membrane pore formation like that of pyroptosis.In addition,pyroptosis is associated with pyroptotic bodies,which have some similarities to apoptotic bodies.Therefore,different cell death programs induce distinctive reshuffling processes of the plasma membrane.Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity,not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response.In this review,we will discuss recent advances in the field of apoptosis,necroptosis and pyroptosis,with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their impli-cation in cell death-elicited immunogenicity.Keywords:programmed cell death;plasma membrane;mechanism;morphology;immunologyCell Research(2018)28:9-21.doi:10.1038/cr.2017.133;published online 27 October 2017Correspondence:Jiahuai HanE-mail:IntroductionPlasma membrane is central for homeostatic mainte-nance in mammalian cells.It is a direct barrier against extracellular environment;it harbors protein sensors and receptors transducing extracellular signals to elicit cellular responses;it contains transporters and channels involved in the trafficking of inorganic ions and small water-soluble organic molecules;and it participates in nutrient and macromolecule transport processes such as endocytosis and exocytosis.The loss of plasma mem-brane integrity would therefore undoubtedly put an end to cellular life.Cell death can result from activation of intrinsic cell death programs or passive disruption of membrane integ-rity by damaging environmental forces.Since all passively disrupted cells present a ruptured plasma membrane,which is a feature of the necrotic phenotype,necrotic cell death has long been defined as a form of uncon-trolled passive cell death 1.However,it is now clear,that besides apoptosis the best characterized type of programmed cell death with an intact plasma membrane,other intrinsic programs can lead to necrotic cell death 2.A very recent study has even shown that plasma mem-brane rupture in apoptotic cells undergoing secondary necrosis is also intrinsically programmed 3.While apoptosis is generally accepted as a non-inflam-matory process,the lytic nature of necrosis leads to the release of intracellular DAMPs(damage-associated mo-lecular patterns)and triggers inflammation 4.However,distinct programs such as necroptosis and pyroptosis or passive mechanical damages will induce different immunogenic effects 5,6.Furthermore,the idea that apoptosis is a non-inflammatory form of cell death may not be accurate since accumulating experimental data in-dicate that apoptosis can also be immunogenic due to the 10Plasma membrane changes during programmed cell deathsSPRINGER NATURE|Cell Research| of particular DAMPs 7.Secondary necrosis might only be partially responsible for DAMPs release during apoptosis since certain DAMPs are preferential-ly released by apoptotic rather than necrotic cells 6.Therefore,plasma membrane changes during apoptosis and programmed necrosis are clearly more complicated than previously expected.Apoptosis was an early focus in the field of cell death study.The mechanisms of nuclear condensation,DNA fragmentation,caspase activation,and phosphatidylser-ine flipping have been intensively studied 8.Although plasma membrane blebbing and apoptotic body for-mation are morphological characteristics of apoptosis,mechanistic information regarding those processes re-mains limited.Recent advances in necrosis study have drawn our attention to the plasma membrane.Transloca-tion of channel or pore proteins to the plasma membrane increases plasma membrane permeability and causes plasma membrane rupture in programmed necrosis 9-22.Plasma membrane pore formation is also responsible for the secondary necrosis of apoptotic cells 3.Due to the significant progress in the recent study of plasma mem-brane changes in different cell death programs,this arti-cle will first review updated information on necroptosis,pyroptosis,and apoptosis with an emphasis on the com-parison of the accompanying plasma membrane changes.We will then review and discuss how these plasma mem-brane changes in dying cells elicit immune responses.Apoptosis Apoptosis is the most well-studied cell death program,occurring in almost all tissues and being important for normal development and tissue homeostasis.It features morphological changes such as cell shrinkage,chromatin,and cytoplasmic condensation,nuclear fragmentation,breakage of cells and the subsequent formation of mem-brane-bound apoptotic bodies 23.While apoptosis can be induced by a variety of physiological or pathological stimuli and conditions,it is mediated by either the extrin-sic death receptor pathway or the intrinsic mitochondrial death pathway 24.The extrinsic pathway involves the recruitment and activation of procaspase-8,and activated caspase-8 then directly activates the effector caspases such as caspase-3 to initiate the execution process.The intrinsic apoptotic pathway is mediated by the cleavage of BID(BH3 interacting domain death agonist),a BCL-2 homology 3(BH3)-only protein.Truncated BID sub-sequently translocates to the mitochondria and activates the BCL-2 family members BAX(BCL-2-associated X protein)and BAK(BCL-2 antagonist/killer).Upon acti-vation,BAX and BAK induce mitochondrial outer mem-brane permeabilization and the release of proapoptotic mitochondrial contents into the cytoplasm,such as cyto-chrome c and other soluble mitochondrial intermembrane space proteins 25.Released cytochrome c promotes oligomerization of APAF-1(apoptotic peptide activating factor 1),an adaptor protein containing a caspase re-cruitment domain(CARD).Heptameric APAF-1 recruits procaspase-9 through the CARD-CARD interaction and forms the apoptosome,leading to proximity-induced ac-tivation of caspase-9,which in turn cleaves and activates effector caspases 26.Crosstalk between the extrinsic and intrinsic pathways could occur as both can use the same execution mechanism to elicit cell death.This common execution pathway is initiated by the cleavage of effector caspases,caspase-3/-6/-7 and results in DNA fragmentation,cytoskeletal reorganization,cytoplasmic condensation,and formation of apoptotic bodies 24,27,28.Events occurring at the plasma membrane of apoptotic cellsThe execution of apoptosis is orchestrated by the pro-teolytic cleavage of a wide range of cellular substrates by caspases,including cytoskeleton components(such as actin and catenin)and signaling elements 8.During the final step of apoptotic execution,modifications of the plasma membrane are undoubtedly finely tuned.Howev-er,little is known about how dying cells are dismantled.Morphologically,the plasma membrane will first under-go blebbing(formation of circular bulges),a transient stage which rapidly evolves toward bleb separation and generation of apoptotic bodies(Figure 1A).Mechanisms underlying these plasma membrane changes are partly described(Figure 2).Membrane blebbing Caspase-3 has been shown to be necessary for membrane blebbing as caspase-3-deficient cells fail to form membrane blebs 29,30.Rho-activat-ed serine/threonine kinase ROCK1 is a caspase-3 target 31,32.Activation of ROCK1 by caspase-3-mediated cleavage is Rho-independent and functions to regulate actin-myosin filament assembly,cell contractility,and membrane blebbing through phosphorylation of the my-osin light chain(MLC).ROCK1 plays no role in caspase activation,cytochrome c release,or phosphatidylserine externalization,but ROCK1-dependent membrane bleb-bing is required for the movement of DNA fragments to blebs and apoptotic bodies.Phosphorylation of MLC by ROCK1 promotes actomyosin contraction with con-sequential delamination of the plasma membrane from the cortical cytoskeleton membrane,leading to plasma membrane blebbing.Both the loss of the interaction with Yingying Zhang et al.11www.cell-|Cell Research|SPRINGER NATUREFigure 1 Morphological features of apoptosis,necroptosis,and pyroptosis and their linkages with immunogenicity.(A)Dying cells revealed by scanning electron microscopy.In RAW264.7 cells,apoptosis was induced by TNF+Smac mimetics;necro-ptosis was induced by TNF+Smac mimetics+zVAD;pyroptosis was induced by LPS priming followed by nigericin treatment.(B)Membrane blebbing followed by formation of apoptotic bodies is commonly observed in apoptosis.Under certain conditions,such as inhibition of PANX1 by trovafloxacin or further combined inhibition of actomyosin contraction by cytochalasin D or GSK 269962,apoptotic cells exhibit two apoptotic body-related morphological changes called apoptopodia and beads-on-a-string protrusions.These membrane-enveloped fragments can be immunogenic,non-immunogenic,or even immunosuppres-sive under different experimental settings.However,the regulated secondary necrosis of apoptotic cells mediated by DFNA5 can be highly inflammatory.In necroptosis,MLKL-mediated plasma membrane rupture leads to release of cellular contents and thus immunogenicity.Pyroptosis results from an inflammatory response induced by inflammasome activation,which is frequently observed in professional phagocytes and tightly associated with IL-1/IL-18 secretion.Whether GSDMD-mediated pyroptosis itself is immunogenic awaits further investigation.12Plasma membrane changes during programmed cell deathsSPRINGER NATURE|Cell Research| 2 Outlines of the signal transduction pathways leading to plasma membrane changes in apoptosis(including second-ary necrosis),necroptosis,and pyroptosis.(A)Apoptosis can be initiated by either intrinsic or extrinsic pathway.Caspase-3 activation resulting from either pathway cleaves ROCK1 to promote plasma membrane blebbing,followed by generation of apoptotic bodies.Caspase-3 can also cleave DFNA5 to generate the DFNA5 N-terminal fragment,which forms oligomers and translocates to the plasma membrane,leading to its rupture by the formation of non-selective pores and finally secondary necrosis.(B)In the necroptotic pathway,various external death ligands can initiate necrosome assembly.Once in the necro-some,RIP3 is autophosphorylated.Phosphorylated RIP3 recruits and phosphorylates MLKL,leading to MLKL oligomeriza-tion and translocation to the plasma membrane.MLKL oligomers execute necroptosis by generating cation channels,causing plasma membrane rupture.(C)Pyroptotic stimulation elicits inflammasome formation and subsequent caspase-1 activation.Activated caspase-1 cleaves GSDMD,generating the GSDMD N-terminal fragment,which oligomerizes and translocates to the plasma membrane and causes plasma membrane rupture via non-selective pore formation.cytoskeleton and the increase in hydrostatic pressure due to cell shrinkage constitute the physical bases driv-ing membrane blebbing.A few other proteins including p21-activated kinase(PAK)2 and LIM-kinase 1(LIMK1)were also reported to be activated by caspase to trigger cytoskeletal reorganization and membrane blebbing 31,33-36,but conflicting data exist.The flipping of phosphatidylserines,a feature of apoptosis,is ROCK1-independent but enriched in apop-totic blebs 31,32.Although considered as a hallmark of apoptosis and functioning as a eat-me signal to aid apoptotic cell recognition and clearance by phagocytes 37,phosphatidylserine flipping was reported to be inducible,reversible,and independent of cytochrome c release,caspase activation,and DNA fragmentation.It was also suggested that phosphatidylserine flipping may not affect the progression of the apoptotic program 38.Similarly,blebbing does not affect the development of apoptosis,and is also reported as being reversible 39,40.Apoptotic bodies Apoptotic bodies are non-uniform subcellular fragments released from apoptotic cells trapping cellular contents such as DNA,fragments of nucleus,and fragmented or intact organelles,the for-mation of which requires plasma membrane blebbing 31,41.Apoptotic bodies are 1-5 m in size,and are formed by defined cell types such as T lymphocytes and endothelial cells 37.However,not all apoptotic cells break up into apoptotic bodies.Indeed,apoptotic cells do not have to be disassembled into sub-particles to be efficiently cleared since evidence shows that in most cases,professional phagocytes tend to phagocytose their targets in their entirety.This is for example the case for macrophages and dendritic cells(DCs)that engulf entire apoptotic thymocytes or neutrophils 42,43.Non-pro-fessional fibroblasts,epithelial,or endothelial cells are also able to phagocytose entirely their dying brethren 44,45.Thus,the notion that apoptotic bodies form to help clear dead cells still needs to be verified.Cell rounding results from the detachment from the extracellular ma-trix,a process mediated by caspase-dependent disman-tling of cell-matrix focal adhesions and cell-cell adhesion Yingying Zhang et al.13www.cell-|Cell Research|SPRINGER NATUREcomplexes.This event represents the earliest step toward apoptotic body formation.Although the following mem-brane blebbing is known to be required for apoptotic body formation,the mechanism underlying the forma-tion of these apoptotic bodies is still largely unknown.Caspase-3-mediated activation of gelsolin is believed to mediate in part the morphological changes of apoptosis since the N terminal fragment of gelsolin yielded by caspase3-cleavage causes depolymerization of the ac-tin cytoskeleton in a calcium-independent manner 46.ROCK1-dependent expansion and retraction of the bleb is involved in debris packaging into the blebs lumen and finally apoptotic body formation 31,32.Pannexin 1(PANX1)channel is a four-pass trans-membrane channel reporte