Báo cáo khoa học: Death-associated protein kinase (DAPK) and signal transduction: blebbing in programmed cell death docx

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:
*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