Death-associated protein kinase DAPK and signaltransduction: blebbing in programmed cell death Miia Bovellan1,2,*, Marco Fritzsche1,3,*, Craig Stevens4and Guillaume Charras1,2 1 London C
Trang 1Death-associated protein kinase (DAPK) and signal
transduction: blebbing in programmed cell death
Miia Bovellan1,2,*, Marco Fritzsche1,3,*, Craig Stevens4and Guillaume Charras1,2
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.
Trang 2particularly 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 elevnucle-ated 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
Trang 3two 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.
Trang 4that 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 organilocali-zation 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-bundlunbind-ing 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
Trang 5occur 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 blebbinhibit-ing 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
Trang 6was 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
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