MINIREVIEW Death-associated protein kinase (DAPK) and signal transduction: blebbing in programmed cell death Miia Bovellan 1,2, *, Marco Fritzsche 1,3, *, Craig Stevens 4 and Guillaume Charras 1,2 1 London Centre for Nanotechnology, University College London, UK 2 Department of Cell and Developmental Biology, University College London, UK 3 Department of Physics, University College London, UK 4 Institute of Genetics and Molecular Medicine, Edinburgh University, UK Introduction Blebs are balloon-like protrusions of the cell mem- brane that appear and disappear on a minute time- scale. The bleb lifecycle can be subdivided into three steps: nucleation, expansion and retraction. Blebs form when the actomyosin cortex of the cell contracts and increases the pressure inside the cell, leading to either detachment of the membrane from the cortex or cortex rupture [1–3]. Expansion lasts  10–30 s [4], during which time cytosol flows from the cell body into the bleb. During this time the bleb is devoid of filamentous actin when examined by light microscopy. As expan- sion slows, an actin cortex reforms under the mem- brane of the bleb. Retraction lasts  2 min [1] and is driven by the activity of myosin [4]. Blebbing has been observed in a variety of cellular phenomena, including cell spreading [5,6], viral infection [7], cell movement, cytokinesis and cell death. During cell motility, blebbing occurs in many cell types, including embryonic and cancer cells (reviewed in [8]). In particular, zebrafish germ cells have been shown unequivocally to move through blebbing in the presence of an extracellular chemoattractant gradient [9]. Some cancer cells solely utilize blebbing for motil- ity [10], whereas others can switch between lamellipo- dial motility and blebbing motility depending on extracellular cues [11]. Blebbing motility appears Keywords actin; blebs; cytoskeleton; myosin Correspondence G. Charras, London Centre for Nanotechnology, University College London, UK Fax: +44 207 679 0595 Tel: +44 207 679 2923 E-mail: g.charras@ucl.ac.uk *These authors contributed equally to this work (Received 11 March 2009, revised 20 August 2009, accepted 28 September 2009) doi:10.1111/j.1742-4658.2009.07412.x Death-associated protein kinase (DAPK) regulates many distinct signalling events, including apoptosis, autophagy and membrane blebbing. The role of DAPK in the blebbing process is only beginning to be understood and, in this review, we will first summarize what is known about the cytoskeletal proteins and signalling cascades that participate in bleb growth and retrac- tion and then highlight how DAPK integrates with these processes. Mem- brane blebs are quasispherical cellular protrusions that have a lifetime of approximately 2 min. During expansion, blebs are initially devoid of actin, although actomyosin contractions provide the motive force for growth. Once growth slows, an actin cortex reforms and actin-bundling and con- tractile proteins are recruited. Finally, myosin contraction powers bleb retraction into the cell body. Blebbing occurs in a variety of cell types, from cancerous cells to embryonic cells, and can be seen in cellular phe- nomena as diverse as cell spreading, movement, cytokinesis and cell death. Although the machinery that executes this is still undefined in detail, the conservation of blebbing phenomenon suggests a fundamental role in meta- zoans and DAPK offers a door to further dissect this fascinating process. Abbreviations DAPK, death-associated protein kinase; ERM, ezrin ⁄ radixin ⁄ moesin; MAP1B, microtubule-associated protein 1B; MLC, myosin light chain. 58 FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS particularly common among cells moving through gels and, in some cases, this can occur in the absence of integrin-mediated adhesion [10,11]. One major differ- ence between blebbing in apoptosis and in motility is that for cells to be able to move by blebbing, bleb formation needs to be polarized towards the direction in which the cell is moving. How this is achieved is presently unclear and may depend on the cell type [8]. During cytokinesis, blebbing takes place primarily at the poles of the dividing cell [12–16]. Its role is not well understood and may just be a consequence of increased cell contractility or a weakened membrane– cortex association during cell division. Intriguingly, the integrity of the actin cortex appears essential, as depo- lymerization of the actin cortex in the pole region inhibits the progression of cleavage furrow and eventu- ally cytokinesis [17]. In view of these results, one might speculate that blebbing may represent an effective way for the dividing cell to increase cortical surface area. Blebbing is probably the most striking phenomenon observed during cell death, whether necrotic or apop- totic. Apoptotic blebs appear indistinguishable from blebs in ‘healthy’ cells: their growth is dependent upon actomyosin contraction, their lifecycle is only a few minutes, and they retract. In contrast, necrotic blebs are larger and more transparent when examined by bright-field microscopy (G. Charras, unpublished observations). They form independently of actomyosin contraction [18], relying instead on an influx of ions and water flow into the cell [19,20], grow over a period of tens of minutes, and do not retract [19]. Bleb nucleation Bleb nucleation is the result of actomyosin contrac- tions. Indeed, inhibition of myosin contractility by treatment with the myosin-II ATPase blocker blebbist- atin impedes blebbing [2,21]. Two distinct mechanisms of bleb nucleation have been observed experimentally: delamination of the cell membrane from the actin cor- tex due to a transient increase in intracellular pressure [2] (Fig. 1A, left) or a rupture of the cellular actin cor- tex [3] (Fig. 1A, right). In the first scenario, myosin motor proteins contract the actomyosin cortex, giving rise to a localized compression of the cytoplasm. As the fluid phase of the cytoplasm (cytosol) cannot drain instantaneously, this gives rise to a localized increase in intracellular pressure, which, if large enough, can cause the membrane to tear from the actin cortex and nucleate a bleb [22,23]. Whether delamination is purely mechanical or facilitated by a biochemical mechanism is unknown. The exact location where a bleb is nucle- ated in a zone of elevated intracellular pressure could be determined by locally lower membrane–cortex adhe- sion energy. In particular, phosphatidylinositol 4,5-bis- phosphate has been proposed to play a primordial role in determining membrane–cortex adhesion, either through regulation of the ezrin ⁄ radixin ⁄ moesin (ERM) family of actin–membrane linker proteins or by being chelated by myristoylated alanine-rich C kinase sub- strate [24]. Delamination from the actin cortex is observed in filamin-deficient blebbing cells: the actin cortex appears intact during bleb expansion and no fracture of the actin cortex is apparent in light micros- copy images [2]. Filamin-deficient melanoma cells bleb constitutively because of decreased adhesion energy between the cortex and the cell membrane due to a lack of filamin [1,25,26]. Consistent with this, filamin rescued cells or cells expressing a constitutively active mutant of the ERM protein ezrin show a marked decrease in blebbing [4,25]. The second possible mecha- nism is that blebs result from rupture of the actin cortex. In this scenario, myosin contraction leads to fracture of the actin cortex and the cytoplasm flows into the bleb, something that has been observed experi- mentally in L929 cells [3]. Expansion of a bleb After nucleation, cytosol flows through the bleb neck to inflate the bleb (Fig. 1B). As the bleb expands, its surface area must increase, because the lipid membrane can only be stretched a small amount [27,28]. There are several mechanisms through which bleb expansion could proceed: tearing of the membrane from the actin cortex (Fig. 1B), unfolding of membrane wrinkles, or flow of lipids in the plane of the membrane (Fig. 1B). In the first scenario, if the expansion process is fast enough, the membrane tension becomes sufficient to break links between the membrane and the actin cor- tex, thereby making more surface area available and increasing the bleb neck diameter (Fig. 1B). This has been observed experimentally in filamin-deficient bleb- bing cells [23]. Second, excess membrane in cells can be stored in the form of folds and microvilli [28]. Therefore, an increase in bleb surface area could sim- ply be the result of unfolding of membrane wrinkles, but experimental data suggest that this alone is insuffi- cient to account for the observed growth of surface area [23]. In the third scenario, when expansion is slow, membrane tension increases moderately and causes membrane lipids to flow into the bleb through the bleb neck, thereby adding surface area. Lipid flows have been observed in cells during tether extraction [29,30], but have yet to be examined during bleb for- mation. Bleb expansion eventually ceases for one of M. Bovellan et al. Blebbing in programmed cell death FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS 59 two reasons: either the local pressure transient decreases below the threshold needed for expansion and the bleb reaches equilibrium, or reassembly of the actin cortex is sufficiently advanced to halt expansion. Reconstitution of an actin cortex An actin cortex starts to reform in the bleb once expansion slows down (Fig. 1C). The signal that trig- gers cortex reassembly is not known. One possibility is A B C D Fig. 1. Schematic diagram of the three phases of blebbing resulting from either a local detachment of the cortex from the membrane (left) or from a local fracture of the cortex (right). (A) High local intracellular pressure (black arrows) tears the membrane from the actin cortex (left) or the actin cortex ruptures and cytosol is expelled from the cell body (right). (B) Cytosol flows into the bleb and the resulting expansion is accommodated by tearing of the membrane from the actin cortex and by flow of lipids into the bleb membrane through the bleb neck. (C) As bleb expansion slows down, a new actin cortex reforms. (D) Recruitment of myosin to the new cortex is followed by bleb retraction, which starts forcing cytosol back into the cell body (black arrows). During this active process, the actin cortex and the membrane crumple. Blebbing in programmed cell death M. Bovellan et al. 60 FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS that no signal is needed, as constitutive turnover of the cortex could eventually reassemble a cortex under the bleb membrane. In dividing cells, the half-time of the actin cortex turnover is  45 s [31], which is comparable with the timescale for bleb expansion ( 30 s). Therefore, further experiments will be needed to examine this hypothesis in bleb cortex reassembly. How the cell knows that the membrane has delami- nated from the actin cortex is unclear. Factors, such as phosphatidylinositol 4,5-bisphosphate [24] or mechano- sensitive ion channels, could detect the detachment of the membrane from the cortex and start a signalling cascade, leading to cortex regrowth. The exact mecha- nism leading to the reassembly of an actin cortex under the bleb membrane is also unclear: F-actin could grow from the elongation of small seeds or be nucleated de novo. Indeed, very short actin filaments or actin seeds from the old cortex, undetectable by light microscopy, could persist under the bleb membrane during expansion and lead to cortex reassembly by rapid actin filament elongation. Second, an unknown actin nucleator might polymerize filaments de novo under the bleb membrane. Indeed, the most studied F-actin nucleators, the Arp2 ⁄ 3 complex and the formin Dia1, are not present in blebs of filamin-deficient blebbing cells [4]. However, the presence of regulators of actin nucleation, RhoA and RhoGEFs, at the bleb membrane and the ultrastructure of the actin cortex [4] suggest that if there is a nucleator needed for the reas- sembly of the actin cortex, it is probably a formin. In particular, it has recently been proposed that diapha- nous-related formin FHOD1 is the nucleator of actin cortex in blebs [32]. Reassembly of an actin cortex under the bleb mem- brane appears to result from the sequential recruitment of membrane–cortex linker proteins, actin, actin-bun- dling proteins and contractile proteins. Indeed, as bleb expansion slows, the ERM protein ezrin (and possibly moesin) is rapidly recruited to the bleb membrane [4] to link the forming actin cortex to the membrane [33]. Interestingly, ezrin is recruited to the membrane inde- pendently of actin [4]. Actin is recruited to blebs after ezrin, followed by recruitment of tropomyosin and the actin-bundling protein, a-actinin. Finally, myosin is recruited and is concentrated in a few distinct dots along the cortex [4]. When examined by scanning elec- tron microscopy in detergent-extracted cells, the newly reassembled actin cortex has a cage-like structure [4]. This ultrastructural organization is intriguing and raises a few interesting questions. First, it is not known whether the filaments in this cage-like structure are physically cross-linked, or whether they can slide past one another. Second, viewing the ultrastructural locali- zation and organization of myosin along the cage-like structure of the cortex should allow better understand- ing of how cross-linked actin gels contract. Bleb retraction The exact mechanism that causes bleb retraction is unknown. During retraction, the total amounts of actin polymers, a-actinin and tropomyosin do not appear to change significantly, indicating that net actin polymerization is downregulated once a continuous rim has been assembled, and that recruitment of the cross-linking proteins comes to a steady state. The mechanical work needed to force the cytosol back into the bleb and crumple the actin cytoskeleton is provided by myosin heads moving along the actin filaments (Fig. 1D) [4]. During this active process, two different forces resist the myosin contractions: the pressure resulting from forcing the cytosol back into the cell body and the restoration force from bending the actin network. Dynamic changes in the ultrastruc- ture of the actin network due to binding and unbind- ing of actin-bundling proteins may also play a role, but this has not yet been examined experimentally. Once retraction is complete, it is unclear whether the bleb cortex integrates into the cell cortex or whether it is immediately depolymerized and replaced by cortex. The role of death-associated protein kinase (DAPK) in blebbing, apoptosis and autophagy DAPK is a calcium ⁄ calmodulin-regulated, cytoskele- ton-associated serine ⁄ threonine kinase that functions as a positive mediator of apoptosis in response to vari- ous stimuli, including interferon-c, Fas and transform- ing growth factor-b [34]. In accordance with its pro-apoptotic activity, recent evidence suggests that DAPK functions as a tumour suppressor: DAPK expression is frequently lost in tumours and tumour cell lines due to promoter hypermethylation [35], it can inhibit tumour metastasis in vivo [36] and it can sup- press transformation in vitro [37]. In addition, DAPK can activate autophagy, which has recently been shown to be antitumorigenic [38–40]. Overexpression of DAPK can significantly induce membrane blebbing in various cell types [41–43], but relatively little is known about the genetic pathways by which DAPK regulates membrane blebbing, or whether these blebs are more akin to those observed during apoptosis, autophagy or cytokinesis. Phenotypically, blebs in apoptotic cell death resem- ble those of ‘healthy’ cells. Growth and retraction M. Bovellan et al. Blebbing in programmed cell death FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS 61 occur over similar timescales [44] and, in both auto- phagic and apoptotic cell death, bleb formation is dependent on contraction of the actomyosin cortex [44,45]. Indeed, treatment of serum-deprived cells with the caspase inhibitor z-VAD-FMK enables apoptotic cells to bleb for hours to days and depolymerization of the actin cortex inhibits this dynamic blebbing after prolonged treatment (> 10 min) [44]. As in healthy cells, myosin provides the motive force for bleb extru- sion: inhibitors of myosin phosphorylation inhibit blebbing during apoptotic cell death [44] and increased phosphorylation of myosin regulatory light chain is observed (apoptosis [44]; autophagic cell death [46]). In caspase-dependent apoptosis, caspases also destabilize the cytoskeleton through cleavage of a variety of cyto- skeletal proteins, either directly or indirectly through calpain [47]. After bleb expansion ceases, an F-actin cortex forms under the membrane of retracting apop- totic blebs [48]. However, an interesting contrast to healthy blebbing is that during the execution phase of apoptosis, the ERM family proteins dissociate from the cell membrane [49]. The presence of the other actin-binding proteins identified during reassembly of a contractile actin cortex under the membrane of blebs in filamin-deficient blebbing cells has not been exam- ined in blebs of cells undergoing cell death. Although the proteins involved in the execution of blebbing appear similar in apoptotic and autophagic cell death, the upstream signals that lead to membrane blebbing differ markedly. In particular, increased phos- phorylation of myosin light chain (MLC) results from different processes in apoptotic and autophagic cell death. During apoptosis, depending on the death stim- ulus, the upstream regulator of phosphorylation dif- fers. When apoptosis is provoked by tumour necrosis factor-a, cycloheximide, anti-Fas serum or calpain inhibitors, MLC phosphorylation occurs downstream of caspase-cleaved Rho kinase I [45,50]; whereas when cell death is the result of serum withdrawal, MLC phosphorylation is the result of MLC kinase activation [44]. In spite of different regulators upstream of MLC, it appears that RhoA activation plays a key role in apop- totic blebbing, as treatment of cells with C3 toxin inhib- its blebbing [44,45,50] and RhoA-GTP concentration increases in apoptotic cells [45]. During caspase-inde- pendent apoptosis of cells targeted by T lymphocyte cytotoxic granules, granzyme B cleaves Rho kinase II, making it constitutively active and leading to membrane blebbing [51]. In contrast, in DAPK-mediated cell death, blebbing is independent from Rho kinases or the Rho pathway, resulting instead from increased myosin contractility induced by phosphorylation of MLC at Thr18 and Ser19 by DAPK family proteins, such as DAPK and zipper (ZIP) kinase. In the case of DAPK, phosphory- lation of MLC at Thr18 and Ser19 can occur either directly [46,52] or indirectly through the induction of ZIP kinase activity [43] and this leads to the formation of actin stress fibres without the concomitant stimula- tion of focal adhesion assembly seen with other kinas- es, such as MLC kinase and Rho kinase [52]. One hypothesis is that this uncoordinated regulation of stress fibres and focal adhesions results in disruption of the cytoskeletal structure, leading to membrane blebbing and eventually to the activation of apoptosis [53]. In some cell types, overexpression of DAPK can lead to membrane blebbing and the appearance of autophagic vesicles [54], but little is known about how DAPK exerts its effects on autophagy and the induction of membrane blebbing may play a role. For example, microtubule-associated protein 1B (MAP1B) was recently identified as a DAPK-binding protein that functions as a positive cofactor for membrane blebbing [55]. Overexpression of DAPK together with MAP1B resulted in the disruption of microtubules, the induc- tion of membrane blebbing and concomitant autopha- gic vesicle formation. Intriguingly, blebbing could be inhibited by treatment with the autophagy inhibitor 3-methyladenine [55]. However, 3-methyladenine is a general inhibitor of phosphoinositide-3-kinase [56], and thus interferes with numerous cellular processes in addition to autophagy. Nevertheless, the concomitant membrane blebbing and autophagy observed in DAPK overexpressing cells suggests a degree of interplay between these processes. Interestingly, the kinase activ- ity of DAPK was required for MAP1B-stimulated membrane blebbing [55], suggesting that phosphoryla- tion of MAP1B or other substrates, such as MLC, may play an important role in DAPK-induced blebbing. In contrast, DAPK may also play a role in inhibit- ing cell blebbing through the regulation of cytoskeletal proteins such as tropomyosin, which plays a role in the formation and stabilization of stress fibres [57]. In endothelial cells, oxidative stress quickly activates extracellular signal-regulated kinase, resulting in the activation of DAPK and phosphorylation of tropomy- osin-1 by DAPK on Ser283 [58,59]. Overexpression of a Ser283Glu phosphorylated tropomyosin-1 mutant triggers the formation of stress fibres, whereas the expression of a nonphosphorylatable Ser283Ala tropo- myosin-1 mutant is not associated with stress fibres and leads to membrane blebbing in response to H(2)O(2) [59]. Furthermore, when DAPK expression Blebbing in programmed cell death M. Bovellan et al. 62 FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS was attenuated with siRNA, cells lost their stress fibres and underwent rapid membrane blebbing in response to oxidative stress, which could be rescued by overex- pression of a constitutively active mutant tropomyo- sin-1 [59], suggesting a role for DAPK in the inhibition of membrane blebbing through tropomyosin phosphorylation. However, DAPK is not the only kinase exerting its inhibitory effects on blebbing via tropomyosin. Indeed, DAPK is insensitive to the kinase inhibitor ML-7 and the treatment of cells with ML-7 and the subsequent exposure of cells to oxida- tive stress result in rapid membrane blebbing. In this situation, ML-7 treatment causes a decrease in tropo- myosin-1 Ser283 phosphorylation [58], despite the lack of effect on DAPK [52], suggesting that other kinases may also phosphorylate this site. It should also be noted that DAPK has other cyto- skeletal functions. For example, DAPK can induce apoptosis by suppressing integrin-mediated cell adhe- sion and survival signalling [53], and can inhibit the association of talin head domain with integrin to sup- press the integrin–Cdc42 polarity pathway [60]. These studies are intriguing and suggest additional mecha- nisms through which DAPK may regulate blebbing. However, further studies are required to determine whether these pathways are related. General conclusion Blebbing occurs as part of the normal cell growth pro- cess, and although blebbing is one of the characteristic hallmarks of programmed cell death, its exact contri- bution to cell death remains unclear. It has been suggested that vigorous blebbing may help mix intracellular content or deplete cellular DNA [47], or that blebbing may be a way of shedding membrane to attract macrophages to the site of cell death [61] or sig- nal extrusion by neighbouring cells [47,62]. However, neither of these hypotheses is fully satisfactory. First, blebbing does not appear to be essential to cell death, as staurosporine, a potent kinase inhibitor of blebbing, is often used as a pro-apoptotic treatment [44]. In view of this, one might hypothesize that if blebbing is needed for mixing intracellular content, death without blebbing may just be slower. Second, if blebs were a signal to neighbouring cells, their presence during autophagic cell death would appear counterintuitive. Nevertheless, the conservation of blebbing in all types of cell death probably points to an as yet unknown common role. Overexpression of DAPK leads to mem- brane blebbing in some settings, whereas in others the same phenotype is observed upon DAPK depletion. This may reflect differences in the input signal, or DAPK gene dosage may be an important factor. Inter- estingly, full-length DAPK tagged with green fluores- cent protein associates strongly with stress fibres and leads to large pseudopodial protrusions; whereas DAPK constructs lacking the cytoskeleton localization domain mediate profuse blebbing [46]. Whether the large pseudopodial protrusions resulting from full- length DAPK overexpression bear all the hallmarks of blebs merits further attention. Clearly, further studies are required to decipher the biological significance of membrane blebbing and to elucidate the mechanisms by which DAPK can regulate this fascinating process. Acknowledgements The authors gratefully acknowledge funding from the Human Frontier Science Program through a Young Investigator grant to GC and the UCL Comprehensive Biomedical Research Centre for generous funding of microscopy equipment. GC is a Royal Society univer- sity research fellow. References 1 Cunningham CC (1995) Actin polymerization and intra- cellular solvent flow in cell surface blebbing. J Cell Biol 129, 1589–1599. 2 Charras GT, Yarrow JC, Horton MA, Mahadevan L & Mitchison TJ (2005) Non-equilibration of hydrostatic pressure in blebbing cells. Nature 435, 365–369. 3 Paluch E, Piel M, Prost J, Bornens M & Sykes C (2005) Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments. Biophys J 89, 724–733. 4 Charras GT, Hu CK, Coughlin M & Mitchison TJ (2006) Reassembly of contractile actin cortex in cell blebs. J Cell Biol 175, 477–490. 5 Bereiter-Hahn J, Luck M, Miebach T, Stelzer HK & Voth M (1990) Spreading of trypsinized cells: cytoskele- tal dynamics and energy requirements. J Cell Sci 96, 171–188. 6 Pletjushkina OJ, Rajfur Z, Pomorski P, Oliver TN, Vasiliev JM & Jacobson KA (2001) Induction of cortical oscillations in spreading cells by depolymer- ization of microtubules. Cell Motil Cytoskeleton 48, 235–244. 7 Mercer J & Helenius A (2008) Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 320, 531–535. 8 Charras G & Paluch E (2008) Blebs lead the way: how to migrate without lamellipodia. Nat Rev Mol Cell Biol 9, 730–736. 9 Blaser H, Reichman-Fried M, Castanon I, Dumstrei K, Marlow FL, Kawakami K, Solnica-Krezel L, Heisen- berg CP & Raz E (2006) Migration of zebrafish primor- M. Bovellan et al. Blebbing in programmed cell death FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS 63 dial germ cells: a role for myosin contraction and cyto- plasmic flow. Dev Cell 11, 613–627. 10 Pinner S & Sahai E (2008) PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE. Nat Cell Biol 10, 127–137 (erratum appears in Nat Cell Biol 10, 370). 11 Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Brocker EB & Friedl P (2003) Compensation mechanism in tumor cell migra- tion: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160, 267–277. 12 Boss J (1955) Mitosis in cultures of newt tissues. IV. The cell surface in late anaphase and the movements of ribonucleoprotein. Exp Cell Res 8, 181–187. 13 Porter K, Prescott D & Frye J (1973) Changes in sur- face morphology of Chinese hamster ovary cells during the cell cycle. J Cell Biol 57, 815–836. 14 Fishkind DJ, Cao LG & Wang YL (1991) Microinjec- tion of the catalytic fragment of myosin light chain kinase into dividing cells: effects on mitosis and cytoki- nesis. J Cell Biol 114, 967–975. 15 Burton K & Taylor DL (1997) Traction forces of cyto- kinesis measured with optically modified elastic sub- strata. Nature 385, 450–454. 16 Boucrot E & Kirchhausen T (2007) Endosomal recy- cling controls plasma membrane area during mitosis. Proc Natl Acad Sci USA 104, 7939–7944. 17 O’Connell CB, Warner AK & Wang YL (2001) Distinct roles of the equatorial and polar cortices in the cleavage of adherent cells. Curr Biol 11, 702–707. 18 Barros LF, Kanaseki T, Sabirov R, Morishima S, Castro J, Bittner CX, Maeno E, Ando-Akatsuka Y & Okada Y (2003) Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency. Cell Death Differ 10, 687–697. 19 Barros LF, Stutzin A, Calixto A, Catalan M, Castro J, Hetz C & Hermosilla T (2001) Nonselective cation channels as effectors of free radical-induced rat liver cell necrosis. Hepatology 33, 114–122. 20 Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J & Morishima S (2001) Receptor-mediated control of regu- latory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532, 3–16. 21 Cheung A, Dantzig JA, Hollingworth S, Baylor SM, Goldman YE, Mitchison TJ & Straight AF (2002) A small-molecule inhibitor of skeletal muscle myosin II. Nat Cell Biol 4 , 83–88. 22 Mitchison TJ, Charras GT & Mahadevan L (2008) Implications of a poroelastic cytoplasm for the dynam- ics of animal cell shape. Semin Cell Dev Biol 19, 215–223. 23 Charras GT, Coughlin M, Mitchison TJ & Mahadevan L (2008) Life and times of a cellular bleb. Biophys J 94, 1836–1853. 24 Sheetz MP, Sable JE & Dobereiner HG (2006) Continu- ous membrane–cytoskeleton adhesion requires continu- ous accommodation to lipid and cytoskeleton dynamics. Annu Rev Biophys Biomol Struct 35, 417–434. 25 Cunningham CC, Gorlin JB, Kwiatkowski DJ, Hartwig JH, Janmey PA, Byers HR & Stossel TP (1992) Actin- binding protein requirement for cortical stability and efficient locomotion. Science 255, 325–327. 26 Nakamura F, Osborn TM, Hartemink CA, Hartwig JH & Stossel TP (2007) Structural basis of filamin A func- tions. J Cell Biol 179, 1011–1025. 27 Boal DH (2002) Mechanics of the Cell. Cambridge University Press, Cambridge. 28 Hamill OP & Martinac B (2001) Molecular basis of mechanotransduction in living cells. Physiol Rev 81, 685–740. 29 Dai J & Sheetz MP (1999) Membrane tether formation from blebbing cells. Biophys J 77, 3363–3370. 30 Hochmuth RM & Marcus WD (2002) Membrane tethers formed from blood cells with available area and determination of their adhesion energy. Biophys J 82, 2964–2969. 31 Murthy K & Wadsworth P (2005) Myosin-II-dependent localization and dynamics of F-actin during cytokinesis. Curr Biol 15, 724–731. 32 Hannemann S, Madrid R, Stastna J, Kitzing T, Gaste- ier J, Schonichen A, Bouchet J, Jimenez A, Geyer M, Grosse R et al. (2008) The diaphanous-related formin FHOD1 associates with ROCK1 and promotes Src- dependent plasma membrane blebbing. J Biol Chem 283, 27891–27903. 33 Bretscher A, Edwards K & Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3, 586–599. 34 Bialik S & Kimchi A (2006) The death-associated pro- tein kinases: structure, function, and beyond. Annu Rev Biochem 75, 189–210. 35 Gozuacik D & Kimchi A (2006) DAPk protein family and cancer. Autophagy 2, 74–79. 36 Inbal B, Cohen O, Polak-Charcon S, Kopolovic J, Vadai E, Eisenbach L & Kimchi A (1997) DAP kinase links the control of apoptosis to metastasis. Nature 390, 180–184. 37 Raveh T, Droguett G, Horwitz MS, DePinho RA & Kimchi A (2001) DAP kinase activates a p19ARF ⁄ p53- mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol 3, 1–7. 38 Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C et al. (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075. 39 Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S & White E (2007) Autophagy miti- gates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 21, 1621–1635. Blebbing in programmed cell death M. Bovellan et al. 64 FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS 40 Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K, Chen G, Jin S & White E (2007) Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev 21, 1367–1381. 41 Cohen O, Feinstein E & Kimchi A (1997) DAP-kinase is a Ca2+ ⁄ calmodulin-dependent, cytoskeletal-associ- ated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J 16 , 998– 1008. 42 Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, Feinstein E & Kimchi A (1999) DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol 146, 141–148. 43 Shani G, Marash L, Gozuacik D, Bialik S, Teitelbaum L, Shohat G & Kimchi A (2004) Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions. Mol Cell Biol 24, 8611–8626. 44 Mills JC, Stone NL, Erhardt J & Pittman RN (1998) Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation. J Cell Biol 140, 627–636. 45 Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A & Olson MF (2001) Membrane blebbing during apopto- sis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3, 339–345. 46 Bialik S, Bresnick AR & Kimchi A (2004) DAP-kinase- mediated morphological changes are localization dependent and involve myosin-II phosphorylation. Cell Death Differ 11, 631–644. 47 Mills JC, Stone NL & Pittman RN (1999) Extranuclear apoptosis. The role of the cytoplasm in the execution phase. J Cell Biol 146, 703–708. 48 Rudolf E & Cervinka M (2005) Membrane blebbing in cancer cells treated with various apoptotic inducers. Acta Medica (Hradec Kralove) 48, 29–34. 49 Kondo T, Takeuchi K, Doi Y, Yonemura S, Nagata S & Tsukita S (1997) ERM (ezrin ⁄ radixin ⁄ moesin)- based molecular mechanism of microvillar breakdown at an early stage of apoptosis. J Cell Biol 139, 749– 758. 50 Sebbagh M, Renvoize C, Hamelin J, Riche N, Bertoglio J & Breard J (2001) Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol 3, 346–352. 51 Sebbagh M, Hamelin J, Bertoglio J, Solary E & Breard J (2005) Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase- independent manner. J Exp Med 201, 465–471. 52 Kuo JC, Lin JR, Staddon JM, Hosoya H & Chen RH (2003) Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase. J Cell Sci 116, 4777–4790. 53 Wang WJ, Kuo JC, Yao CC & Chen RH (2002) DAP- kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals. J Cell Biol 159, 169–179. 54 Inbal B, Bialik S, Sabanay I, Shani G & Kimchi A (2002) DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles dur- ing programmed cell death. J Cell Biol 157, 455–468. 55 Harrison B, Kraus M, Burch L, Stevens C, Craig A, Gordon-Weeks P & Hupp TR (2008) DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing. J Biol Chem 283, 9999–10014. 56 Rubinsztein DC, Gestwicki JE, Murphy LO & Klionsky DJ (2007) Potential therapeutic applications of autophagy. Nat Rev Drug Discov 6, 304–312. 57 Cooper JA (2002) Actin dynamics: tropomyosin provides stability. Curr Biol 12, R523–R525. 58 Houle F, Rousseau S, Morrice N, Luc M, Mongrain S, Turner CE, Tanaka S, Moreau P & Huot J (2003) Extracellular signal-regulated kinase mediates phosphor- ylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing. Mol Biol Cell 14, 1418–1432. 59 Houle F, Poirier A, Dumaresq J & Huot J (2007) DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress. J Cell Sci 120, 3666–3677. 60 Kuo JC, Wang WJ, Yao CC, Wu PR & Chen RH (2006) The tumor suppressor DAPK inhibits cell motil- ity by blocking the integrin-mediated polarity pathway. J Cell Biol 172, 619–631. 61 Segundo C, Medina F, Rodriguez C, Martinez-Palencia R, Leyva-Cobian F & Brieva JA (1999) Surface mole- cule loss and bleb formation by human germinal center B cells undergoing apoptosis: role of apoptotic blebs in monocyte chemotaxis. Blood 94, 1012–1020. 62 Rosenblatt J, Raff MC & Cramer LP (2001) An epithe- lial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mecha- nism. Curr Biol 11 , 1847–1857. M. Bovellan et al. Blebbing in programmed cell death FEBS Journal 277 (2010) 58–65 ª 2009 The Authors Journal compilation ª 2009 FEBS 65 . 2009) doi:10.1111/j.1742-4658.2009.07412.x Death- associated protein kinase (DAPK) regulates many distinct signalling events, including apoptosis, autophagy and membrane blebbing. The role of DAPK in the blebbing process is only beginning. filamin-deficient blebbing cells has not been exam- ined in blebs of cells undergoing cell death. Although the proteins involved in the execution of blebbing appear similar in apoptotic and autophagic cell death, . MINIREVIEW Death- associated protein kinase (DAPK) and signal transduction: blebbing in programmed cell death Miia Bovellan 1,2, *, Marco Fritzsche 1,3, *, Craig Stevens 4 and Guillaume