Download A role for actin in regulating apoptosis/programmed cell death

Biochem. J. (2008) 413, 389–404 (Printed in Great Britain)
389
doi:10.1042/BJ20080320
REVIEW ARTICLE
A role for actin in regulating apoptosis/programmed cell death: evidence
spanning yeast, plants and animals
Vernonica E. FRANKLIN-TONG* and Campbell W. GOURLAY†1
*School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K., and †Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, U.K.
Achieving an understanding of how apoptosis/PCD (programmed
cell death) is integrated within cellular responses to environmental
and intracellular signals is a daunting task. From the sensation of a
stimulus to the point of no return, a programme of cell death must
engage specific pro-death components, whose effects can in turn
be enhanced or repressed by downstream regulatory factors. In
recent years, considerable progress has been made in our understanding of how components involved in these processes function.
We now know that some of the factors involved in PCD networks
have ancient origins that pre-date multicellularity and, indeed,
eukaryotes themselves. A subject attracting much attention is the
role that the actin cytoskeleton, itself a cellular component with
ancient origins, plays in cell death regulation. Actin, a key cellu-
lar component, has an established role as a cellular sensor,
with reorganization and alterations in actin dynamics being a
well known consequence of signalling. A range of studies have
revealed that actin also plays a key role in apoptosis/PCD regulation. Evidence implicating actin as a regulator of eukaryotic cell
death has emerged from studies from the Animal, Plant and Fungal
Kingdoms. Here we review recent data that provide evidence for
an active, functional role for actin in determining whether PCD
is triggered and executed, and discuss these findings within the
context of regulation of actin dynamics.
INTRODUCTION
tissue surrounding the seed endosperm during germination [12]
and senescence in leaves and flowers [13]. PCD in plants is
also triggered in response to biotic and abiotic stimuli, such as
pathogens or environmental stresses, where it is responsible for
the removal of damaged or infected cells. Examples include UV-C
light [14], temperature stress [15], pathogen attack [16,17] and
SI (self-incompatibility) ‘self’ interactions [18,19]. In yeast,
apoptosis is triggered in response to oxidative stress [20] and
ageing [21], a variety of environmental and intracellular triggers
[22, 23], and plays an important role in the differentiation of
colonies [24].
Compelling evidence that PCD and apoptosis have common
origins comes from the observation that some of the molecular
components of death mechanisms are highly conserved [25,26].
Some genes involved in regulating apoptosis have homologues
in plants, such as BI-1 (Bax-1 inhibitor gene) [27-29] and dad1
(Defender Against Death gene) [30,31]. Other genes, such as
homologues of the Bcl-family genes, such as Bcl-2 or BAX,
are not present in plants or yeast. However, several proteins
that regulate apoptosis in animals, such as the BCl2 family of
pro- and anti-apoptotic proteins, display the expected function
(survival/death) if expressed in plants or yeast (Saccharomyces
cerevisiae), even though their homologues are not present (see,
for example, [14,28,32–36]).
We now know that many of the factors involved in the regulation
of apoptosis and PCD have ancient origins and can be found
in the genomes of unicellular eukaryotes and prokaryotes, with
Apoptosis/PCD (programmed cell death)
The fact that cells are selectively executed during normal development has been recognized and revisited a number of times
over the last century [1]. However, it was not until the recent
discovery of regulators of the most studied form of PCD, apoptosis
(reviewed in [2]), that this became a ‘red hot’ field of study. PCD/
apoptosis is an essential process in the development and maintenance of multicellular organisms. Although the terminology is
increasingly complex and contentious, for simplicity’s sake we
will use the term ‘apoptosis’ for animal and yeast studies and
PCD in relation to an analogous process in plants, as these are
generally accepted and used terms within the respective fields.
Cell death regulation is required during a wide range of processes in animal cells, such as embryogenesis [3], tissue maintenance of somatic cell populations [4] and immune system regulation [5], to name but a few examples. The importance of PCD/
apoptosis is highlighted by the fact that de-regulation of apoptosis is associated with a host of diseases such as cancer (reviewed in [6]), autoimmune diseases (reviewed in [7]) and neurodegenerative disorders [8]. In plants, PCD plays a key role during
embryogenesis, development and senescence, sculpting organ
shape and enabling differentiation of specific cell types. Examples
include the formation of xylem vessels, which are highly
specialized organs involved in the transport of water and solutes
[9], leaf patterning [10], embryogenesis [11], PCD of the aleurone
Key words: actin, apoptosis, mitochondrion, programmed cell
death (PCD), reactive oxygen species (ROS).
Abbreviations used: ABP, actin-binding prtoein; ActMAp, actin-mediated apoptosis; ADF, actin-depolymerization factor; AIF1, apoptosis-inducing factor
1; Arp2/3, actin-related protein 2/3; AtCP, Arabidopsis capping protein; CAP, cyclase-associated protein; CD44s, CD44 standard form; CytB, CytD and
CytE, cytochalasins B, D and E; DISC, death-inducing signalling complex; Dlk, DAP (death-associated protein)-like kinase; Endo G, endonuclease
G; E-Tmod, E-tropomodulin; FADD, Fas-associated death domain; HR, hypersensitive reponse; Hxk1, hexokinase isoenzyme 1; IL-2, interleukin-2; Jasp,
jasplakinolide; LatA and LatB, latrunculins A and B; MLC, myosin light chain; NHR, non-host resistance; OYE, ‘old yellow enzyme’; Par-4, prostate apoptosis
response-4; PCD, programmed cell death; PKA, protein kinase A; PtdA, phophatidic acid; ROCK1, Rho-associated coiled-coil containing protein kinase
1; ROS, reactive oxygen species; SI, self-incompatibility; SRK, S6 kinase-related kinase; S-RNase, S-locus RNase; Sr2v2, suppressor of RasVal19; TB10,
thymosin B10; VDAC, voltage-dependent anion channel; Vpr, HIV viral protein R.
1
To whom correspondence should be addressed ([email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
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V. E. Franklin-Tong and C. W. Gourlay
evidence that even the simplest cells have the capacity to be
programmed to die [2,26]. For example, at its simplest level,
a programme of cell death could arise via the engagement of a
single protein, for example a nuclease, which, when diverted from
its ‘normal’ cellular function under certain circumstances, adopts
an unregulated and destructive pattern of activity.
Mitochondria and the intrinsic pathway of apoptosis
The mitochondria represent a major force in the apoptotic machinery of eukaryotic cells. Mitochondria are thought to have evolved
from a bacterial progenitor as endosymbionts of a primitive
eukaryote [37]. A logical progression of this theory follows
that modern-day eukaryotes of divergent lineages, but derived
from a common ancestor, may exhibit common mitochondrial
activity. In support of this notion, the mitochondrial, or intrinsic,
pathway of cell death has been observed as an active component
of apoptotic/PCD pathways in yeast [38], humans [39] and plants
[40,41].
During apoptosis/PCD driven by the intrinsic pathway, the
reception of an apoptotic stimulus leads to an increase in the permeability of the mitochondrial outer membrane, allowing proapoptotic molecules such as cytochrome c, AIF1 (apoptosisinducing factor 1) and Endo G (endonuclease G) to be released
from the intermembrane space into the cytoplasm. Cytochrome
c release from the mitochondria regulates the formation of the
apoptosome in mammalian cells. However, although cytochrome
c release may play a role in apoptosis in other organisms, such
as in the yeast S. cerevisiae [38], apoptosome-like structures are
yet to be described outside of mammalian systems. Nevertheless,
the conservation of other regulators of mitochondrial function in
apoptosis has been clearly demonstrated. For example, AIF is
present in all eukaryotes, and both AIF1 and Endo G release from
the mitochondria and translocation to the nucleus during H2 O2
induced apoptosis occurs during apoptosis in animals [42] and
yeast [38,43,44].
The production of ROS (reactive oxygen species) from the
electron transport chain of the mitochondria is also a key element of many apoptosis/PCD pathways. Loss of mitochondrial
function, involving a decrease of mitochondrial transmembrane
potential and ROS production, is a central component of
apoptosis/PCD in animals [45], plants [46,47] and yeast [20,38].
ROS are produced as an inevitable by-product of an aerobic
metabolism and have themselves been incorporated as important
signalling molecules [48]. As ROS accumulation can be damaging, cells regulate their production and removal using powerful
antioxidant machinery [45,49]. ROS production and release has
also been employed within the mechanisms of cell death, as overproduction, which swamps the cells’ antioxidant systems during
PCD, leads to cellular damage from which they cannot recover [49].
Thus conservation of the mitochondrial/intrinsic pathway of
apoptosis, may go some way to explain why regulated pathways
of cell death can be found in highly divergent eukaryotes.
However, it is noteworthy that a key downstream component of the
mammalian intrinsic pathway, the apoptosome does not appear to
be conserved, highlighting the fact that there are also likely to be
crucial differences in how these signalling cascades operate.
Caspases
A good example of appropriation into PCD mechanisms lies
in the key regulators of apoptosis in animals, the caspases.
These proteins, when activated, cleave substrates following an
aspartate residue and accelerate the progression of cell death
[50]. Interestingly, a number of caspases do not have a role in the
execution of apoptosis, whereas others function in both apoptotic
c The Authors Journal compilation c 2008 Biochemical Society
and non-apoptotic signalling [50]. This suggests that, in some
cases at least, caspases may have been ‘hijacked’ from their
original cellular function to participate within apoptotic pathways.
However, at a genetic level, some key differences between apoptosis/PCD in animal cells, yeast and plant cells exist. Although
caspases do not appear to be present within the plant or fungal
genomes investigated to date, both fungal and plant apoptosis/
PCD have been shown to associated with the induction of caspaselike activities (for examples, see [19,51–53]), and it has been
demonstrated that caspase inhibitors can block PCD in plants and
yeast [53].
The nature of caspase-like proteases in plant cells is currently
the subject of considerable debate. Good candidates have arisen
in the discovery of a caspase-related family of proteases, termed
the metacaspases, in plant and fungal genomes [54]. Metacaspases structurally and functionally resemble animal caspases [55]
and have been identified in yeast, fungi, protozoa and plants
[54–57] and their involvement in PCD has been demonstrated.
However, they do not cleave authentic caspase-specific substrates
[58,59], and there are clearly several further caspase-like activities with activities similar to those found in animal cells (e.g.
caspases 1, 3 and 6) identified in plants and yeast for which
genes have not been identified. YVADase (caspase-1-like; enzyme
cleaving the substrate Tyr-Val-Ala-Asp), VEIDase (caspase-6like) and DEVDase (caspase-3-like) activities have been relatively
well characterized and shown to regulate PCD in plant cells
[18,19,60–63]. Recently a LEVDase (caspase-4-like) has been
identified [19]. VPEs (vacuolar processing enzymes), which
exhibit YVADase activity, represent the first cloned putative caspase-like genes in plants with an established role in PCD [61–
64]. Caspase-3-like (DEVDase) activities, which are closest to the
key caspase involved in the execution of animal apoptosis, have
been identified in several plant systems using fluorescent caspase3-specific substrates [14,19,66], and DEVD tetrapeptide inhibitors have been shown to alleviate PCD [18,19,51], implicating this
type of activity in mediating PCD in several higher-plant systems.
However, the gene encoding this caspase-3-like/DEVDase activity remains elusive and, for obvious reasons, its discovery remains
a key goal.
The actin cytoskeleton
The actin cytoskeleton plays an essential role in a plethora of
cellular functions, including endocytosis, motility, organelle and
vesicle trafficking, cytokinesis and signal–response coupling. The
appropriation of actin into these processes reflects the plasticity
that exists within the systems of assembly and disassembly of
filaments, and the range of actin binding and regulatory molecules.
In a healthy cell, actin filaments are assembled in a polarized
manner with ATP-bound actin monomers preferentially added
to fast-growing (+) or barbed ends. Actin possesses a weak
intrinsic ATPase activity, and the hydrolysis of ATP to ADP + Pi
is accompanied by a conformational change that destabilizes
the actin filament and promotes monomer dissociation from the
slow growing (−) or pointed end. Released ADP-bound actin
monomers then undergo nucleotide exchange, which is facilitated
by ABPs (actin-binding proteins), enabling re-charged ATPbound actin to re-enter the cyclical process commonly called
‘treadmilling’ (for recent reviews, see [67]). A huge number of
ABPs have evolved to facilitate and manipulate actin filament
formation and turnover, a subject that has been reviewed in detail
and will not be discussed here (for recent reviews on the topic,
see [67–71]). Some of the central orchestrators of eukaryotic
actin assembly are also well conserved. For example, the actinnucleating Arp2/3 (actin-related protein 2/3) complex has been
Actin regulation of apoptosis/programmed cell death
shown to regulate the formation of branched filament networks in
fungal [70] and animal cells [67]; however, although it is present
in plants [72,73], whether it nucleates or makes branched networks
has not yet been established. In yeast, Arp2/3 function regulates
cortical actin patch assembly [70], whereas in mammalian cells it
is required for the branching filamentous networks underlying the
leading edge of motile cells [74]. In higher plants the ARP2/3
complex has emerged as a pivotal player in determining cell
shape [73], although there are notable exceptions to this rule
(see [68]). Other examples of ABPs with conserved functions in
filament bundling, severing, capping, cross-linking and monomer
sequestration are also evident and beyond the scope of the present
review (see [68–70,76] for reviews). ABPs known to play a
role in apoptosis in animal cells include gelsolin, cofilin/ADF
(actin-depolymerization factor), coronin and β-thymosins, and
are discussed below.
The regulation of actin assembly and disassembly is under the
control of complex signalling systems that link external signals
to remodelling events, which result in altered cellular activities
that adapt cell shape and/or behaviour to suit new environmental
conditions. In the last decade, evidence that actin plays an
important role in regulating apoptosis/PCD has emerged. Here
we review data from yeast, plants and animal cells, which places
actin firmly as a key player at the interface between environmental
sensing mechanisms, controlling the cell death decision apparatus
in each of these groups, despite their divergence.
ACTIN AND APOPTOSIS IN MAMMALIAN CELLS
In mammalian cells, shape and organization is facilitated by the
cytoskeleton, which typically comprises filamentous networks
of actin, microtubule and intermediate filaments. The assembly of
actin into higher-order structures in response to environmental
cues is co-ordinated members of the Rho family of GTPases,
the best studies being Rho, Rac and Cdc42 (cell division cycle
42), which regulate stress fibres, lamellipodia and filopodia
formation respectively when activated (reviewed in [77]). These
actin-dependent structures are essential for cellular processes
such as the regulation of motility, cell shape and plasma-membrane integrity. Another more recently discovered role for actin
in mammalian cells lies in the regulation of some of the morphological features associated with apoptosis [78], as well as in the
regulation and triggering of apoptosis itself (see below).
Actin dynamics and apoptosis
The use of drugs to manipulate actin dynamics has been a useful
tool to investigate links between actin and apoptosis signalling cascades. Although the drugs used in these studies are
widely accepted to influence the dynamic capability of the actin
cytoskeleton, it is worth highlighting the possibility that indirect
or uncharacterized actin-independent effects may also occur. As
drug treatment that indisciminantly disrupts actin structures will
disrupt a host of cellular processes, the interpretation of such
data in isolation must be viewed cautiously. However, results
produced from studies involving a number of actin regulatory
proteins, as dicussed below, support the results generated by the
use of actin-disrupting drugs. Taken together, the results of these
studies convincingly place actin within pathways that regulate a
cells commitment to apoptosis in mammalian cells.
The addition of the F-actin-stabilizing drug Jasp (jasplakinolide) to Jurkat T-cells rapidly induced apoptosis, accompanied
by an increase in caspase-3 activation [79,80]. In CTLL-20 cells
[an IL-2 (interleukin-2)-dependent T-cell line that shows early
commitment to apoptosis after IL-2 removal], Jasp enhanced
apoptosis when added during IL-2 deprivation [80]. This could
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be inhibited by over-expression of the anti-apoptotic protein
Bcl-xL, which functions at mitochondria [80]. In MCF10A
cells, inhibition of actin depolymerization stimulated apoptosis
and provided evidence for a novel role for Bcl-2 in cell
death induced by direct disruption of the actin cytoskeleton
[81]. In articular chondrocytes, disruption of F-actin by CytD
(cytochalasin D) inhibited apoptosis [82]. Actin stabilization by
Jasp has also been observed to induce apoptosis in HL-60 cells
[83,84] Thus, although the exact mechanisms are unknown, these
data suggest that actin stabilization can induce mitochondriadependent apoptosis (see Figure 1).
The link between apoptosis triggering and actin dynamics is not
restricted to scenarios in which F-actin structures are stabilized,
as there is also evidence for a similar effect in some animal cells
when actin is actively depolymerized (see Figure 1). In T-cells, for
example, CytD treatment resulted in elevated caspase-3 activity,
suggesting that actin depolymerization can regulate apoptosis
[85]. More recently, it has been shown that CytD induced rapid
cytochrome c release from mitochondria and consequent caspase
activation in murine cell lines [86]. Apoptosis in ischaemic
kidney cells, which involves actin depolymerization and can be
stimulated by the actin depolymerizing drug LatB (latrunculin B)
can be alleviated by Jasp [87]. Phalloidin, another actinstabilizing drug, with the same binding site as Jasp, also prevents
both cisplatin-mediated apoptosis and actin depolymerization
in porcine kidney proximal-tubule cells [88]. In Jurkat T-cells
undergoing Fas-mediated apoptosis, CytD enhanced the execution
phase of apoptosis [89]. As the authors had also shown that
Jasp can stimulate apoptosis in this system, they suggested
that this might hint that alteration of actin dynamics might be
responsible for modulating the apoptotic signal [89]. Thus both
actin stabilization and depolymerization can stimulate apoptosis
in animal cells.
In addition to the actin stabilization/depolymerization influencing entry into apoptosis debate, there are other apparent ‘anomalies’. In some cases, simply stimulating alterations to F-actin
status is sufficient to induce apoptosis, whereas in other cell
types, altering F-actin dynamics can only influence apoptosis
if it has already been stimulated. These data argue that actin
may influence apoptosis by different mechanisms in differing cell
types. However, it is possible that it is the alteration of actin
dynamics (i.e. the rate of actin polymerization/depolymerization)
that modulates the signalling to apoptosis, with alterations to
actin providing the sensory mechanism, involving either changes
in polymer levels, changes in the flux of actin through the filament pool, or both [66]. One striking (and probably important)
difference between different cell types is the ratio of G-actin to Factin, where it has been measured. In budding yeast S. cerevisiae,
the majority of the total actin pool is considered to be in the Factin form [90]. By contrast, pollen from plants has high ratio of
G- to F-actin, with as little as 5–10 % of the total actin protein
in the filamentous form [91,92]. Similarly high levels of G-actin
are maintained under normal growth conditions in mammalian
cells where this has been assessed [93]. Thus the endogenous
balance between monomer and polymer in different eukaryotic
cells is likely to vary considerably, depending on function and
status, and this will reflect the nature of actin dynamics and their
regulation in different cell types and species. Interestingly, actin
is a known target of caspase activities during apoptosis, leading
to the production of actin N-terminal 32 kDa (Fractin) and
15 kDa (tActin) fragments. After cleavage, tActin can undergo Nmyristoylation, which targets it to mitochondria [94]. Moreover,
expression of tActin, but not Fractin, is sufficient to trigger morphological changes resembling those observed in apoptotic cells
[95].
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Figure 1
V. E. Franklin-Tong and C. W. Gourlay
Model outlining links between actin dynamics and apoptotic signalling in animal cells
Actin has been demonstrated to play a role at multiple stages of apoptosis in mammalian cells. Clustering of the CD95/Fas and CD44 death receptors has been shown to require actin; this action is
important to elicit a full apoptosis response upon stimulation of these death receptors. Treatment of mammalian cells with actin-disrupting drugs such as Jasp and CytD has been shown to affect
the sensitivity of a number of different cell types to apoptotic stimuli. The regulation of actin by accessory proteins also plays an important role in the apoptosis response in mammalian cells. Actin
monomer concentration, as regulated by the thymosins and the promotion of dynamic actin filaments by gelsolin, cofilin and coronin 1 have all been convincingly shown to regulate the commitment
to apoptosis in response to stimuli. Both cofilin and gelsolin are known to regulate mitochondrial influence upon the commitment to apoptosis, probably by influencing membrane permambility via
the VDAC. Mice lacking coronin 1, a haematopoietic specific isoform of coronin, display elevated levels of cell death displaying markers of apoptosis in cells of this lineage. After the initiation of
apoptosis, membrane blebbing and the formation of apoptotic bodies is facilitated by the cytoskeleton, and actin is a key component in the manipulation of the plasma membrane required to elicit
these phenotypes.
Actin regulatory proteins and apoptosis
A tightly controlled dynamic equilibrium between monomeric Gactin and F-actin exists within cells. An important part of this
regulation lies within the activity of a number of actin monomer
sequestration and regulatory proteins. Recent data suggests that
the activity of actin regulatory proteins such as gelsolin, cofilin/
ADF, coronin and β-thymosins, play a crucial role in the regulation of apoptosis in animal cells. The fact that an ever-increasing
list of actin regulators are implicated in apoptosis regulation
(see below) reflects the importance of cytoskeletal control in
maintaining cellular homoeostasis in response to environmental
change.
Gelsolin
Gelsolin is a member of the gelsolin superfamily, a conserved
family defined by the presence of the gelsolin-like domain.
Gelsolin itself is an ABP capable of severing and capping actin
filaments in a manner which is responsive to changes in Ca2+ ,
intracellular pH, tyrosine phosphorylation and phosphoinositides
in vivo [96,97]. Of the seven members of this protein family,
which includes adseverin, CapG, villin, advillin, supervillin
and flightless, only gelsolin has been shown to play a role
in the regulation of apoptosis to date. The overexpression of
c The Authors Journal compilation c 2008 Biochemical Society
gelsolin has been demonstrated to inhibit the induction of
apoptosis in Jurkat cells [98], most likely by blocking the
loss of mitochondrial membrane potential. In another study
the overexpression of gelsolin in HeLa cells, which do not
normally produce gelsolin, led to an increase in susceptibility
to apoptosis [99]. It has been proposed that gelsolin protects cells
from apoptosis by regulating VDACs (voltage-dependent anion
channels), mitochondrial membrane pores that regulate the release
of pro-apoptotic factors and that are important for maintaining
mitochondrial membrane potential [100], via an actin-regulatory
mechanism (see Figure 1; [101]). One possibility is that a
gelsolin–PtdInsP2 complex has the capacity to inhibit caspase3 activity and so influence the execution phase of apoptosis
[102]. The anti-apoptotic activity of gelsolin may also operate
at the level of the mitochondria via a proposed role in the
regulation of the VDAC. The overexpression of gelsolin in
Jurkat cells was also found to impart resistance to Vpr (HIV
viral protein R)-induced apoptosis [103]. This study and a
previous study by the same author [104] showed that a specific
C-terminally located domain of gelsolin, the G5 segment,
was required for the anti-apoptotic effect. It was proposed that
the G5 domain bound to the mitochondrial VDAC and prevented
Vpr-induced loss of mitochondrial membrane potential [103,104].
A recent report convincingly demonstrated that gelsolin has an
Actin regulation of apoptosis/programmed cell death
anti-apoptotic pro-survival role in pancreatic β-cells [105].
Yermen and colleagues were able to demonstrate both a decrease
in apoptosis in gelsolin-overexpressing primary mouse β-cells
and an increase in in apoptosis when gelsolin levels were depleted
by RNA intereference [105]. It has also been suggested that a
newly established N-RAS (neuroblastoma RAS viral oncogene
homologue)–gelsolin complex may play a role in the promotion of
cell survival [106]. Interestingly gelsolin-knockout mice are more
susceptible to brain injury following ischaemia [107], an effect
that has been attributed to the gelsolin/actin-mediated regulation
of Ca2+ channels. As the regulation of Ca2+ is known to play an
important part in the regulation of apoptosis, it is tempting to
speculate that a loss of apoptosis/PCD regulation may play a part
in the reduced ability of gelsolin knockout mice to recover from
injury.
Gelsolin is targeted and cleaved by caspases during apoptosis to
yield an N-terminal product [99]. This accelerates apoptosisassociated morphological changes by severing actin filaments in
an unregulated manner [99]. A recent in vitro study provided evidence that the pro-apoptotic N-terminal gelsolin fragment can
compete or inhibit DNase 1 (a well known G-ABP and one
of the principle nucleases involved in nuclear fragmentation
during apoptosis) binding to actin [108]. The authors proposed
an interesting positive-feedback mechanism, suggesting that the
cleavage of actin and gelsolin by caspase activity during apoptosis
will promote terminal DNA destruction, as DNase 1 is released
from actin and activated [108]. Gelsolin is cleaved during apoptosis to yield both the pro-apoptotic N-terminal fragment and
a C-terminal fragment (tGelsolin) that is N-myristolated and
exhibits anti-apoptotic activities [101,109]. Although tGelsolin is
N-myristolated, this post-translational modification was not found
to target the C-terminal fragment to the mitochondria, as is the case
for the N-myristolated tActin fragment also generated by caspase
cleavage during apoptosis [94,109]. The authors established that
overexpression of tGelsolin significantly reduced apoptosis when
COS-1 cells were treated with etoposide in a manner that was
dependent on its N-myristolation [109]. Thus it is becoming
increasingly apparent that gelsolin is a key regulator of cell
survival in mammalian cells, operating at multiple points within
the regulation of apoptosis pathways.
Cofilin/ADF
Cofilin is a member of the cofilin/ADF family. It promotes
the depolymerization and severing of actin filaments, and is
involved in the recycling of the G-actin monomers [110,111].
The active (dephosphorylated) form of cofilin has been shown
to be targeted to mitochondria after initiation of apoptosis [112].
A mitochondrial-targeted cofilin was able induce cytochrome c
leakage and trigger apoptosis, an activity that requires the actinbinding domain of cofilin [112]. The association of cofilin and its
effects on mitochondrial function have been further highlighted
by recent work from the laboratory of James Bamburg on a
stress-induced structure called the ‘ADF/cofilin rod’ [113,114].
ADF/cofilin rods are actin-containing structures that rapidly form
in response to stresses, leading to ATP depletion as a result of
increased phosphorylation of cofilin [113,114]. Cofilin rods have
been shown to associate with mitochondria under conditions of
stress and elicit a transient protective role in primary hippocampal
neurons [114,115]. However, the persistence of ADF/cofilin rods
was also shown to have a detrimental effect on mitochondrial
function and to cause a loss of membrane potential [114,115].
This research is highly significant in the light of the role actin and
cofilin have in apoptosis regulation, as actin-rich aggregates, such
as Hirano bodies and ADF/cofilin rods, are associated with, and
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implicated in, many diseases (reviewed in [116]). Interestingly, a
recent study in which β-actin was knocked down in HeLa cells
using short interfering RNA, revealed a 2–5-fold increase in the
level of phosphorylated cofilin [117]. However, cell death in HeLa
cell cultures was not affected by β-actin knockdown in this study
[117]. It may be the case that, as mentioned above, alterations to
the actin cytoskeleton itself, or to key regulatory proteins such as
cofilin, may not be sufficient to induce apoptosis in certain cell
lines, or that additional initiating stimuli are required.
Coronin
Coronin is an important regulator of the evolutionarily conserved
Arp2/3 complex, an important component of actin filament
networks [67,68,70,72]. Arp2/3 filament nucleation activity is
regulated by several proteins, one of which is coronin [118,119].
Of the seven mammalian coronin family members, coronin 1
is preferentially expressed in cells of haematopoietic origin,
where it is co-expressed with other members (2, 3 and 7)
[119]. A recent study in which coronin-1-knockout mice were
generated suggested that coronin 1 exerted an inhibitory effect on
cellular steady-state F-actin formation via an Arp2/3-dependent
mechanism [120] (see Figure 1). The authors found that coronin 1 was also required for chemokine-mediated migration. Interestingly, actin dynamics, through a mitochondrial pathway, were
linked to lymphocyte homoeostasis in this study. T-cells from the
knockout mice had increased levels of annexin V (an indicator of
apoptosis)-positive CD4+ and CD8+ thymocytes. An increased
rate of in vivo apoptosis associated with caspase-3 and -9 cleavage,
which could be reversed by caspase inhibitors, was also observed
in these cells. Elevated cytochrome c levels in the cytoplasm
and reduced mitochondrial membrane potential were also noted
in coronin-knockout T-cells (see Figure 1). Moreover, actin
stabilization was also linked to apoptosis, as it could be triggered
in T-cells treated with Jasp [120] (see Figure 1).
β-Thymosins
A further example of the importance of regulation of the actin
pool in the regulation of apoptosis has emerged from studies of
the regulation of actin monomer sequestration by β-thymosin
[121,122]. The β-thymosins are a conserved family of 5 kDa
polypeptides that specifically bind monomeric G-actin, preventing
its incorporation into filaments [123]. A key observation was that
the overexpression of TB10 (thymosin B10) in ovarian tumour
cells increased the rate of cell death [122]. TB10 and actin share
the same binding site on E-Tmod (E-tropomodulin), a pointedend actin-capping protein that modulates the length of the actin
filament. Overexpression of E-Tmod was shown to block TB10dependent apoptotic activity, supporting the hypothesis that a
dynamic equilibrium among pools of actin, TB10 and E-Tmod
regulates an apoptotic homoeostasis [122].
Signalling systems, actin and apoptosis
Actin and Fas-mediated apoptosis
The binding of a Fas ligand to the CD95 (APO-1/Fas) death
receptor leads to clustering, subsequent DISC (death-inducing
signalling complex) assembly and initiation of apoptosis (see
Figure 1, where we attempt to draw together the links between
actin and apoptosis in mammalian cells). The DISC comprises
an adaptor molecule, FADD (Fas-associated death domain), and
the initiator caspase 8 [124]. Two types of cells have been
described in terms of CD95/Fas-induced apoptotic signalling.
In cells classified as type 1, Fas activation leads to caspase 8
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recruitment to the DISC in quantities that result in the activation
of caspase 3, resulting in an apoptotic response that does not
require mitochondrial function [125]. In type II cells, however,
DISC formation is less efficient, and small quantities of active
caspase 8 are produced. In this case, the mitochondria are required
to elicit a complete cell-death response [126–128].
A number of studies have highlighted the importance of
actin filaments in regulating the susceptibility to stimuli that
induce CD95/Fas-induced apoptosis [129–134]. For example, the
disruption of actin filaments was shown to inhibit Fas-induced
apoptosis in type I cells [129], as a result of DISC formation
inhibition and reduction of caspase-8 activation [129]. Actin was
also found to regulate Fas-induced apoptosis in activated human
T-lymphocytes [131,132], an effect mediated by ezrin, a member
of the ERM (ezrin–radixin–moesin) family [131,133]. Further
evidence for the role of actin in Fas-mediated apoptosis has
come from a recent study investigating the CD44–ezrin complex
[135,136]. The transmembrane receptor CD44 also conveys
extracellular signals to within the cell and binds to ezrin. Of
the several isoforms of CD44 that exist, only CD44s (the CD44
standard form) modulates Fas-mediated apoptosis, forming a
complex with ezrin and actin (see Figure 1). The authors suggested
that the interaction between the cytoplasmic domain of CD44s and
ezrin and the consequent organization of the actin cytoskeleton
may function as a scaffold, which stabilizes DISC formation,
thereby enhancing apoptotic sensitivity. As well as functioning
within the process of DISC formation, actin is an important part
of the endocytotic machinery that internalizes Fas receptor after
induction [137].
Actin may not play such a major role in type II Fas-mediated
apoptosis, as actin depolymerization did not affect CD95/Fasinduced apoptosis in a type II Jurkat cell line [89]. However,
cytokine-withdrawal-induced apoptosis was enhanced in the
same cell line when actin polymerization was inhibited [89]. In
addition, disruption of actin by CytD or LatA reduced Fas-induced
apoptosis of HIV-specific CD8+ T-cells [138]. CD95/Fas-induced apoptosis of HIV-specific CD8+ T-cells is regulated by
mitochondria-related factors [139,140], which is recognized as a
feature of type II cells. This adds weight to the idea that actin
plays an important role in Fas-mediated apoptosis in both type I
and type II cells.
Actin, plasma-membrane stability and apoptosis
Upon initiation of apoptosis, the cortical cytoskeleton is targeted
by caspase activity (summarized in [78]), leading to a reduction
in plasma-membrane integrity and blebbing (irregular bulges in
the plasma membrane formed during apoptosis). Actin-dependent
blebbing is promoted by myosin II-based contractile activity
[141,142], which occurs as a result of the cleavage of ROCK1
(Rho-associated coiled-coil containing protein kinase 1), subsequent phosphorylation of its light chain MLC (myosin light chain),
and activation of myosin II contractility [141,142]. The activity
of caspase-cleaved ROCK I and a constitutively active MLC
is necessary and sufficient for membrane blebbing [141–144].
The regulation of MLC phosphorylation during apoptosis has
also been shown to involve the association of Par-4 (prostate
apoptosis response-4), a protein known to regulate cellular
response to apoptotic stimuli, with actin [145,146]. Deregulated
expression of Par-4 has been observed to occur in a variety of
tumour types [147,148], and it has been reported that downregulation of Par-4 is essential for Ras-induced tumour progression [148]. Disruption of the actin cytoskeleton with CytD led
to a significant decrease in Par-4-mediated apoptosis in rat
fibroblasts [145,146]. Par-4 also recruits Dlk [DAP (death-asso
c The Authors Journal compilation c 2008 Biochemical Society
ciated protein)-like kinase], which belongs to the novel subfamily
of DAP kinases, from the nucleus to the cytoplasm, and particularly to actin filaments [146,149]. Par-4-mediated recruitment
of Dlk leads to enhanced phosphorylation of MLC and dramatic
cytoskeletal rearrangements [146]. Par-4 has also been shown to
interact with the Amida protein (named after Amida, a Buddhist
god), identified as an interaction partner of Arc (activity-regulated
cytoskeleton-associated protein) [150]. Co-expression of Par-4
and Amida led to the movement of Amida from the nucleus to
cytoplasmic actin filaments, an increased MLC phosphorylation
and enhanced induction of apoptosis. These studies highlight the
importance of Par-4 recruitment of regulatory nuclear proteins to
the actin filament system and suggest a mechanism through which
apoptosis is triggered.
Together, these findings strongly implicate regulation of actin
dynamics in playing a critical decision-making role in deciding
whether cells go into apoptosis in animal cells. It seems likely
that other ABPs responsible for regulating actin dynamics may
also be involved in triggering apoptosis. Moreover, as a variety
of cells exhibit this phenomenon, it is possible that this may be a
widespread or universal feature.
ACTIN AND APOPTOSIS IN YEAST
Yeast cells have been shown to undergo regulated cell death that
displays a number of the hallmarks of apoptosis, including DNA
fragmentation, annexin V exposure, ROS build-up and the loss of
mitochondrial membrane potential [53]. Apoptosis in the budding
yeast S. cerevisiae can be triggered by a variety of exogenous
stimuli or cellular dysfunctions, indicating that pathways that lead
to death exist in these simple eukaryotes [38,151]. Importantly, it
has been shown that, as has been found in many mammalian cells,
the stabilization of cortical actin structures induces apoptosis in
yeast [152]. Mutations in actin regulatory proteins that lead to the
accumulation of aggregates of stabilized F-actin have been shown
to trigger a process termed ‘actin mediated apoptosis’ (ActMAp)
[152–154]. In actin-aggregating cell lines, ActMAp leads to a
loss of mitochondrial membrane potential and the production and
release of ROS into the cell which results in an apoptotic cell
death (see Figure 2, where we attempt to draw together the links
between actin and apoptosis in yeast). Interestingly, mutations that
lead to an increase in the dynamic nature of the actin cytoskeleton
were shown to result in reduced levels of ROS [152]. In addition,
deletion of a gene encoding the actin bundling protein Scp1p,
the yeast homologue of mammalian SM22/transgelin, which also
destabilizes cortical actin structures, reduced ROS levels and led
to a significant increase in replicative lifespan [152].These data
suggest that, in yeast, actin dynamics are linked to processes that
regulate mitochondrial function and ROS regulation.
From the evidence available to date, ActMAp in yeast occurs as
a result of an interaction that exists between the actin cytoskeleton
and a key signalling pathway called the ‘Ras–cAMP–PKA (protein kinase A) cascade (see Figure 2). This signalling pathway
is required for the co-ordination of cell growth and proliferation
in response to the nutritional environment [155,156]. In yeast the
regulation of Ras activity, like mammalian Ras, links extracellular
signals to proliferation and growth [157]. When yeast cells
experience nutritional depletion, the Ras–cAMP–PKA pathway
activity is down-regulated, allowing effective cell-cycle exit and
initiation of the cellular stress response. The Ras–cAMP–PKA
pathway is linked to mitochondrial activity, as cells expressing the
constitutively active rasala12val19 allele exhibit elevated ROS levels
[158,159]. Mutants that accumulate aggregates of F-actin were
shown to trigger hyperactivity of the Ras–cAMP–PKA signalling
pathway, which is responsible for the mitochondrial dysfunction
Actin regulation of apoptosis/programmed cell death
Figure 2
395
Model outlining links between actin and apoptotic signalling in yeast
Stabilization of the actin cytoskeleton, by treatment with drugs such as Jasp or using strains carrying specific mutations, has been shown to lead to the apoptosis in the budding yeast S. cerevisiae . In
actin-stabilized cells an inappropriate hyperactivation of the Ras–cAMP–PKA signalling cascade is observed when cells attempt to arrest their growth in the face of environmental stress. This results
in severe mitochondrial dysfunction characterized by the loss of mitochondrial membrane potential and the production of ROS at a level capable of killing the cell. Actin is linked to cAMP signalling
via the cyclase-associated protein Srv2p/CAP, which acts as a regulator of both actin dynamics and Ras–cAMP–PKA signalling. A consequence of ROS production is that the actin cytoskeleton
can be further stabilized as a result of disulfide linkage between cysteine residues present within actin. Under normal circumstances the oxidoreductase, Oye2p, prevents actin stabilization under
conditions of oxidative stress, and in this capacity possesses anti-apoptotic properties.
and ROS accumulation observed [153,154]. The formation of
aberrant aggregations of F-actin was dependent on the activity
of the protein Srv2p (suppressor of RasVal19)/CAP (cyclaseassociated protein) [152]. Srv2p/CAP is a highly conserved actin
regulatory protein that binds preferentially to ADP–G-actin via
its C-terminal domain [160,161] and can associate with actin
filaments through an interaction between a proline region and
the SH3 domain of Abp1p (ABP 1) [162]. Srv2p/CAP is an
attractive candidate to link Ras signalling to actin reorganization,
as the N-terminus of Srv2p/CAP can bind to adenylate cyclase
(Cyr1p) and facilitate cAMP/PKA activation [163,164]. Actin
aggregation that triggered Ras–cAMP–PKA pathway activity was
shown to require only the C-terminal actin-binding region of
Srv2p/CAP; however, the presence of the N-terminal cyclasebinding domain enhanced PKA stimulation, leading to higher
levels of ROS production [152]. The accumulation of ROS and
subsequent death in actin-aggregating cells was demonstrated to
require the activity of the PKA subunit Tpk3p, an enzyme known
to play an important role in mitochondrial function (see Figure 2).
Yeast lacking Tpk3p exhibit altered mitochondrial enzymatic
content, including decreased levels and activity of cytochrome
c, a constituent of the electron-transport chain [165]. There is
also evidence that the activity of Tpk3p within mitochondria
can control transcription of mitochondrially encoded genes in a
cAMP-dependent manner, by phosphorylation of mitochondrialocated target proteins [166,167]. One possibility is that PKA
activity is compartmentalized in yeast. This has been widely
reported in higher eukaryotes, where PKA is sequestered to
particular locations and cellular compartments by AKAPs (Akinase anchoring proteins) [168].
Ras signalling may be a conserved mechanism by which yeasts
are able to regulate cell death. Evidence to support this comes
from data showing that the pathogenic yeast Candida albicans has
also been shown to regulate apoptosis via Ras/cAMP signalling
[169,170]. The significance of these findings also reinforces the
potential for the study of yeast apoptosis within the field of
medical microbiology, with respects to the development of new
approaches to anti-fungal therapeutics.
Actin and mitochondrial regulation in yeast
The mitochondria are well established as important regulators of
apoptosis/PCD [38,171]. The actin cytoskeleton has been demonstrated to be an important factor in the movement, deployment
and function of mitochondria in a variety of systems [172].
Examples of this include the use of the actin cytoskeleton to ensure
the concentration of mitochondria in areas of cells with high
energy demands [173] and their correct distribution during cell
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V. E. Franklin-Tong and C. W. Gourlay
division [174]. The mitochondrial network consists of a dynamic
tubular network whose distribution throughout unicellular and
multicellular organisms is tightly controlled. The maintenance of
an appropriate balance between mitochondrial fission and fusion
events is known to be an important factor in the the regulation
of PCD in yeast. The promotion of fragmentation, by the fission
protein Dnm1 (dynamin 1), is a crucial element in the execution of
apoptosis when yeasts are subjected to acetic acid or H2 O2
treatment [175]. In S. cerevisiae, the mitochondria have been
shown to physically interact with parallel bundles of F-actin
cables. This occurs via a protein complex known as the mitochore,
which consists of three integral mitochondrial-outer-membrane
proteins Mmm1p, Mdm10p and Mdm12p [176,177]. The force
generated by the power of actin polymerization within cortical
actin patches promotes mitochondrial movement through the cell
[178,179]. The type V myosin Myo2 is also required to facilitate
stable mitochondrial inheritance into newly emerging daughter
cells [178,179].
Evidence to support the strong interaction between actin and
the mitochondria comes from the use of either actin-depolymerizing drugs such as LatA [179] or actin mutants [180]. Both studies
demonstrated that actin disruption leads to aberrant mitochondrial
morphology and distribution. A connection between actin dynamics, mitochondrial function and the regulation of cell death
came from studies using yeast cells expressing the actin allele
act1-159 [152]. Cells expressing act1-159 form filaments that
depolymerize slowly as a result of a failure to undergo a conformational change after Pi release [181]. Under conditions of environmental stress, act1-159-expressing cells undergo mitochondriadependent apoptosis, characterized by the accumulation of high
levels of intracellular ROS [152]. The relationship between actin
dynamics, ROS production and the mitochondria was further
highlighted by the fact that cells carrying the act1-157 allele,
which leads to increases in the dynamic capability of the cytoskeleton, exhibited reduced levels of ROS during growth
[152].
The permeability of mitochondrial membranes is an important
feature of apoptosis in yeast and other eukaryotes and is regulated
in part by the VDAC [182]. Increasing evidence suggests that
the actin cytoskeleton, and its accessory proteins, can influence
VDAC function and so influence apoptotic pathway regulation.
In the filamentous fungus Neurospora crassa, monomeric actin
has been shown to bind to, and modulate, VDAC gating [183]. Gactin binding was found to significantly reduce the VDAC pore’s
conductance, reducing metabolic flux across the mitochondrial
membrane [183]. In addition, an interaction between G-actin and
the VDAC was demonstrated using surface plasmon resonance,
which showed physiologically relevant dose-dependent reversible
binding between the S. cerevisiae VDAC and rabbit G-actin proteins [184]. Although the physiological relevance of actin binding
to VDAC has not been demonstrated, it may be that this interaction
contributes to VDAC channel regulation and mitochondrial
membrane permability, with actin acting as a stabilizing molecule
to maintain the closed conformation. The fact that actin regulatory
proteins, such as cofilin and gelsolin, are thought to influence
apoptosis via an actin-dependent interaction with the VDAC
in mammalian systems (discussed above) suggests that such a
mechanism may be conserved within eukaryotes.
Actin and oxidative stress in yeast: mechanisms of protection
It has been known for some time that the actin cytoskeleton is
sensitive to the oxidative status of cells [185]. A disulfide bond
may be formed, under conditions of elevated ROS, between
cysteine residues at positions 284 and 373 of actin [185–187].
c The Authors Journal compilation c 2008 Biochemical Society
The formation of such bonds is an important factor in the reduced
flexibility observed in red blood cells from patients suffering from
sickle-cell anaemia as a result of a decrease in the dynamic
capability of the cytoskeleton [188,189]. The high degree of
conservation observed for actin means that a similar disulfide
bond can be formed in S. cerevisiae in response to oxidative stress.
This promotes yeast as an attractive model with which to study
the mechanisms involved in, and the effects of, actin stabilization
as a consequence of oxidative stress (see Figure 2). A recent study
has found that the oxidoreductase Oye2p, which is also known as
OYE (‘old yellow enzyme’), serves to regulate oxidation between
Cys285 and Cys374 on yeast actin [190]. The authors proposed that
OYE can oppose the damaging effects of ROS by preventing
disulfide-bond formation between these two cysteine residues
[190]. Mutants expressing an ACT1 gene in which Cys285 and
Cys374 were replaced with the uncharged residue alanine were
shown to be more resistant to oxidative stress than wild-type
strains. In addition, these actin alleles could suppress the oxidative
sensitivity displayed by cells lacking the OYE2 gene. As the literature points to a general phenomenon whereby stabilization of
the actin cytoskeleton increases the likelihood of apoptotic cell
death, the authors investigated whether the OYE2 actin protection
mechanisms also protected cells from apoptosis [191]. As was
expected, cells lacking OYE2 died, showing markers of apoptosis
such as ROS accumulation and DNA fragmentation [191] which
could be suppressed by the C284A and C374A mutations. These
data add weight to the growing evidence that the dynamic nature
of the actin cytoskeleton is tightly linked to the regulation of
apoptosis in yeast cells.
ACTIN AND PCD IN PLANTS
There is considerable evidence that plants use the actin cytoskeleton as a biosensor to monitor the environment, translating
this into alterations in polymerization status, which result in
changes in morphogenesis (see [192]). Plants probably also use
actin as a sensor to monitor stress, translating this into alterations
in polymerization status, which, if large enough and held for a long
enough period of time, triggers PCD. Figure 3 provides a model
summarizing the evidence relating to the involvement of ROS,
changes to actin dynamics and PCD. As this topic has not been
reviewed before, we discuss specific examples in some detail
below. Notably, as mentioned above, although there is extensive
evidence that ROS plays a key role in PCD in plants, in contrast
with the undisputed link in yeast and animal cells, there appears to
be no existing evidence in the literature indicating a possible link
between ROS and actin (see the right-hand side of Figure 3). This
is likely to be due to a lack of investigation rather than negative
evidence. This will be an interesting area to follow in the next
few years, as it would be surprising, given the other similarities
between these systems, if there were no involvement.
Actin, PCD and plant–pathogen interactions in plants
A classic example of PCD is the response of plants to pathogens.
The plant HR (hypersensitive response) represents a rapid PCD
that is initiated by the plant in response to pathogen attack. The
HR occurs in a limited area around the pathogen entry site and
often goes hand-in-hand with the activation of disease resistance.
This has been intensely studied, and some of the most important
findings relating to PCD execution have been established in this
system. The reader is referred to [16,17,47,193] for reviews of
some aspects of this huge topic.
Plant responses to pathogen attack are known to involve major
reorganization of the actin cytoskeleton, with actin filaments
bundling and aligning close to the site of infection [194–197]. A
Actin regulation of apoptosis/programmed cell death
Figure 3
397
Model outlining alterations to actin dynamics and ROS involvement in PCD in plants
A range of stimuli are known to trigger PCD in plants. This is known to involve mitochondria and ROS production. This is shown to the right of the Figure. Several key second messengers involved in
signalling to PCD include Ca2+ , ROS and NO. Other components (not shown) include lipids, such as PtdA, are also known to act as signals involved in mediating HR and PCD; PtdA has been shown
to decrease H2 O2 -promoted PCD [227]. Ceramides, important second messengers involved in signalling to apoptosis in animal cells, have recently been found also to play a role in signalling to PCD
in plant cells [231,232]. Several of these components co-operate with ROS and/or NO to mediate PCD. See [47,193,233,234] for recent reviews. Early common PCD events in plants include changes
to the mitochondria, such as loss of mitochondrial membrane potential (ψ m ), cytochrome c leakage and increases in ROS. Although plants use ROS to regulate plant PCD, very little is currently
understood about how ROS interacts with other signaling molecules during PCD. Crucially, whether this pathway interacts with actin (as in yeast and mammalian cells; see Figures 1 and 2) in plant
cells is not known. Thus this pathway is depicted as separate from actin dynamics known to lead to PCD. However, recent data show a link between hexokinase (associated with mitochondria) and
PCD [222]. Since association between F-actin and hexokinase has also been reported independently, there may be a link between these pathways. Involvement of F-actin depolymerization in PCD has
been shown in several plant systems (shown in the left-hand side of the Figure). Embryogenesis in Picea involved actin depolymerization and PCD; CytD and LatB have been shown to induce PCD in
this system [205]. In a cryptogein-induced HR, bistheonellide (Bis), which inhibits actin polymerization, further stimulated cryptogein-induced PCD [202]. In the Papaver pollen, SI stimulates actin
depolymerization and, later, actin stabilization. Both actin depolymerization, using LatB, and stabilization, using Jasp, were sufficient to trigger caspase-like activities and PCD in pollen. Moreover,
inhibiting SI-stimulated actin alterations using Jasp gave a significant alleviation of SI-induced PCD in incompatible pollen [66]. Mimicking SI-stimulated actin depolymerization by using LatB in
combination with Jasp also alleviated PCD, providing firm evidence for actin polymerization status being crucial to initiating PCD in pollen. In NHR, although there is no direct link between actin and
PCD, there is evidence that actin stabilization is involved in mediating preventing pathogen infection; CytB and CytE, which depolymerize actin, result in reduced resistance/increased infection in
several systems. Though direct evidence linking phosphatidic acid to actin and PCD are lacking to date, PA triggers actin stabilization [228] and is known to signal to PCD, so may play a role
in actin-mediated PCD.
role for the actin cytoskeleton in HR-mediated PCD and NHR
(non-host resistance), which provides immunity against many
pathogens, has been implicated by several studies. However,
generally, actin filaments have been reported to bundle and
persist until the onset of HR-mediated PCD. This hints that actin
stabilization may play a role in mediating PCD. Although there is
no direct link between actin alterations and PCD in this system,
evidence for a crucial role of the actin cytoskeleton in cellular
defence has been provided by several laboratories. Inhibitors
of actin polymerization, such as cytochalasin, which
causes depolymerization of actin microfilaments, have been
demonstrated to prevent defence responses, allow enhanced
penetration efficiency of non-host pathogenic fungi that normally
cannot invade the plant. The existing data suggest that alterations
in actin polymerization status may be a general mechanism
used by plants to mediate PCD (see Figure 3). For example,
potato (Solanum tuberosum) cells treated with CytB resulted
in suppression of HR cell death triggered by the potato-blight
fungus Phytophthora infestans [198], and the incidence of PCD
in cowpea (Vigna unguiculata) by cowpea rust fungus (Uromyces
phaseoli) infection was significantly reduced by CytE [195].
These data first suggested an involvement of alterations to the
actin cytoskeleton in HR-mediated PCD and imply a possible
causal relationship between the changes in actin cytoskeletal
dynamics/organization and defence against fungal pathogens
utilizing the PCD mechanism. However, later studies suggest that
actin dynamics is not involved in R (race-specific resistance)gene-mediated resistance in barley (Hordeum vulgare) [199].
There is considerably more evidence that the actin cytoskeleton
is involved in NHR. Treatment of barley with cytochalasins significantly increased penetration of the non-pathogen [200]. Several
other studies have recently added further information and,
together, they provide compelling evidence for a role for actin
dynamics in NHR. For example, a decrease in NHR was observed
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V. E. Franklin-Tong and C. W. Gourlay
following treatment of Arabidopsis (thale cress) plants with
CytE, and in eds1 mutants, which are defective in host resistance. Use of CytE treatment to disrupt the actin cytoskeleton
resulted in severely compromised NHR in Arabidopsis infected
with wheat powdery mildew (Blumeria graminis), allowing infection [201]. This has helped establish a requirement for actin
polymerization for NHR. Also, in Arabidopsis, cytE treatment
significantly reduced callose papilla formation (a key feature of
infection) and allowed pathogen penetration, suggesting that actin
depolymerization is important for infection. Similarly, in barley,
treatment with either CytE or ectopic expression of ADF in
barley epidermal cells gave increased levels of infection [199].
Since actin-depolymerizing drugs generally seem to inhibit
PCD and allow greater infectivity by the pathogen, this strongly
suggests that actin stabilization (as in yeast) is an important trigger
for PCD in some plant systems. However, a recent study of HRmediated PCD in cryptogein elicitor-induced tobacco (Nicotiana)
BY-2 cells showed that the actin polymerization inhibitor bistheonellide significantly accelerated and increased the incidence of
PCD [202], suggesting that actin depolymerization can also play
a role in HR-PCD (see Figure 3). Together these studies provide
strong evidence that the actin cytoskeleton plays a key role in
plant–pathogen interactions and resistance. However, focus on
what is exactly involved has not been thoroughly pursued to date,
although it has been suggested that dynamic disassembly and
reassembly of the actin cytoskeleton may play a role as well as
its influence in organizing key cellular events in the HR [196].
The recent finding that cryptogein-induced HR-mediated PCD
is enhanced by effectively preventing actin stabilization substantiates this notion that either stabilization or depolymerization (or
perhaps gross alterations in actin dynamics) can play important
roles in regulating this phenomenon.
Actin, PCD and embryogenesis in plants
PCD involving metacaspases [11] plays an important role during
embryogenesis in plants. PCD has at least two important functions
during this early developmental phase, including elimination
of the suspensor (the structure connecting the endosperm to
the embryo), which is only required during early development
[203] and also in selective embryo abortion [204]. A role for
the actin cytoskeleton in PCD in embryogenic tissues in Picea
abies (Norway spruce) has been implicated. Depolymerization of
F-actin, using LatB or CytD, resulted in abnormal embryos,
accompanied by a high incidence of cell death and DNA fragmentation [205] (see Figure 3). This provided the first hint
that actin depolymerization might play a role in PCD, though
the possibility that PCD might be induced indirectly, owing to
development being disrupted as a result of cytoskeletal collapse,
could not be ruled out. The authors suggested that actin may be
a negative regulator of PCD in the developing embryo and that
preservation of thick longitudinal F-actin cables in the suspensor
cells may regulate the timing of PCD-related events.
Actin, PCD and the SI response
Pollen–pistil interactions often involve SI, a key mechanism preventing self-fertilization, resulting in inhibition of incompatible
pollen-tube growth and consequent inbreeding. Several distinct
types of SI have evolved (see [206,207]). SI in Papaver rhoeas
L. (the field poppy) involves PCD of incompatible pollen [18].
SI triggers a Ca2+ -dependent signalling cascade (see [208] for a
review). PCD events include leakage of cytochrome c, dramatic
morphological alterations to organelles [209], activation of a
c The Authors Journal compilation c 2008 Biochemical Society
mitogen-activated protein kinase [210], activation of DEVDase,
VEIDase and LEVDase caspase-like activities [18,19] and, later,
DNA fragmentation [18]. In this SI system, extensive and
sustained F-actin depolymerization, followed later by a striking
stabilization of actin into punctate foci, is stimulated by SI
interactions in incompatible pollen [92,211].
A causal link between actin depolymerization and PCD in pollen was established, using pretreatment with the actin-stabilizing
drug Jasp prior to SI induction. Counteracting depolymerization
in this way resulted in an alleviation of SI-induced PCD in incompatible pollen [66] and implicated SI-induced actin depolymerization in triggering PCD in Papaver pollen (see Figure 3).
This represents the first report of a specific causal link between
actin-polymerization status and initiation of PCD in a plant
cell. This was confirmed by using the actin-depolymerizing drug
LatB to mimic the levels of depolymerization stimulated by SI,
establishing the notion that actin depolymerization is sufficient
to trigger PCD (see Figure 3). A similar experiment using
Jasp to counteract the effect of LatB-induced depolymerization
demonstrated that Jasp could ‘rescue’ pollen from entry into PCD
[66]. This demonstrated the involvement of actin polymerization
status in initiating PCD in pollen. Surprisingly, it does not seem
to be actin depolymerization per se that stimulates PCD in this
system, as Jasp treatment also triggers PCD. Thus sustained
changes to actin filament levels or dynamics appear to play a
functional role in initiating PCD in Papaver pollen. Moreover,
a relatively transient, but substantial, F-actin depolymerization
(∼ 50 % reduction for ∼ 10 min) can trigger PCD, which involves
a caspase-3-like activity [66]. Whether the later formation of
stabilized actin foci also play a role in regulating PCD is not yet
known. However, the fact that actin stabilization can mediate PCD
in this system suggests that it may. Moreover, it is well established
that, in yeast, actin stabilization triggers PCD. Thus in Papaver
it is possible that both of these F-actin alterations stimulated by
SI play a key role in different phases of the PCD cascade, with
sustained deploymerization being followed by reorganization and
stabilization of F-actin.
Together, these data imply a key role for the actin cytoskeleton as a sensor of cellular stress in pollen tubes. Although
studies have not explored this aspect fully, candidate ABPs that
could mediate this event potentially include PrABP80, a candidate
SI-mediated ABP that exhibits potent Ca2+ -dependent severing
[212]. In vitro kinetic actin polymerization assays showed that
PrABP80 has the properties of a gelsolin; MS of PrABP peptides
confirmed this identification. It is thought that PrABP80 acts
synergistically with profilin to mediate the Ca2+ -dependent Factin depolymerization stimulated by SI [212].
Interestingly, SI also triggers very rapid apparent depolymerization of cortical microtubules [213]. Moreover, actin depolymerization triggers apparent microtubule depolymerization [213],
and recently obtained results show that, although disruption of
microtubule dynamics alone does not trigger PCD, SI-induced
PCD is alleviated by taxol. This suggests that signal integration
between microfilaments and microtubules is required for
triggering of PCD.
Pyrus (pear) uses a different SI mechanism, involving S-RNases
(S-locus RNases) and F-box proteins as the pistil and pollen
determinants respectively (see [206]). Interestingly, dramatic
reorganization of the actin cytoskeleton in incompatible Pyrus
pollen growing in vitro has recently been reported to be stimulated
by S-RNases [214]. Because of the very different S-components
and pollen-inhibition system, it is thought to use a completely
different mechanism from Papaver to regulate incompatible
pollen-tube growth [206,207]. Despite this, the punctate actin
foci formed appear rather similar to those observed in the late
Actin regulation of apoptosis/programmed cell death
phase of Papaver SI [92,211]. However, the nature and role of
these punctate actin foci is unknown. Thus, further investigation
of the nature of these aggregates is needed.
In Brassica, SI is controlled using yet another regulatory
system, utilizing different genes, involving interaction between
a receptor-like kinase, SRK (S6 kinase-related kinase) in the
pistil and a small ligand [SP11/SCR (S-locus protein 11 or
S-locus cysteine-rich)] carried by the pollen (see [206]). The
interaction is the reverse of that found in Papaver; the pollen
ligand interacts with SRK in the pistil, triggering signalling in the
pistil (though this presumably then signals back to the pollen
in order to inhibit it). Despite the apparent difference between
the Brassica and Papaver SI systems, a surprising parallel has
recently been observed. It has been shown that, in an incompatible
interaction, the actin filaments in the papilla cells are reorganized
and apparently depolymerized, in a manner very reminiscent of
early events involving actin in incompatible Papaver pollen [215].
It would be very interesting to establish if PCD is stimulated in
papilla cells by an incompatible SI interaction in Brassica. If
this were the case, this would presumably prevent the growth
of incompatible pollen tubes through this tissue, in much the
same way as a pathogen is prevented entry by an HR. Such a
finding would really provide a firm basis for the suggestion of
parallels between SI pollen–pistil interactions and plant–pathogen
interactions [216]. However, in this case it is unlikely that this
signals to PCD, as it has been reported that incompatible pollen
can be ‘rescued’ when moved on to a compatible stigma [217].
Actin, PCD and environmental adaptation
A study on cold acclimation in olive tree (Olea europea) protoplasts provides hints suggesting a role for changes in the actin
cytoskeleton in acclimation-related PCD. Cold shock in nonacclimated wild-type protoplasts resulted in a decrease in actin
signal, suggesting depolymerization, whereas after acclimation
no cold-shock-induced change in F-actin signal was observed.
The authors suggested a role for osmotin in influencing actin
cytoskeleton organization, acclimation and PCD [218]. However,
a direct link between actin depolymerization and PCD remains to
be established here. Studies of an Arabidopsis ACT2 dominantnegative mutation, which disturbs F-actin polymerization, also
hint at a potential role for actin in PCD, as the mutant not
only affected initiation and elongation of root hairs, but cell
death in specific cells of the root epidermis was also observed
[219].
Links between actin, PCD and other components
Relatively little is known about links between the actin cytoskeleton with other components involved in PCD in plants, and
this is an avenue that needs to be explored in the future. There are
some components that may be involved in linking actin dynamics
to PCD. A potential connection between hexokinase, PCD, mitochondria and actin dynamics has been implied by recent findings. Hexokinase is involved in sugar signalling and is involved in
many aspects of plant growth and senescence [220]. Virusinduced gene silencing of a plant hexokinase, Hxk1 (hexokinase
isoenzyme 1) in Nicotiana resulted in PCD involving caspase-9and caspase-3-like proteolytic activities. Moreover, overexpressing HXK1 and HXK2 resulted in increased resistance to PCD.
Interestingly, the Hxk1 was associated with the mitochondria
[221,222], and addition of recombinant Hxk1 to mitochondriaenriched fractions prevented cytochrome c release and loss of
mitochondrial membrane potential [222]. These data firmly
suggests that hexokinase plays a key role in modulating PCD in
399
plant cells. The mitochondrial localization fits nicely with recent
findings suggesting a pivotal role for mitochondria-associated
hexokinase in the regulation of apoptosis in animal cells by
binding with BAD (Bcl-2/Bcl-XL -antagonist, causing cell death)
[223,224]. Moreover, actin has been shown to interact with
mitochondrial VDAC in maize (Zea mays) [225]. Although a
direct link between PCD, hexokinase and actin has not been made
in plants yet, in other systems, it has been shown that hexokinase
interacts with cortical actin filaments and that actin depolymerization prevents hexokinase translocation [226]. Recent data
support the idea that this may be the case in plants too, as in
Arabidopsis Hxk1 also interacts with F-actin [221]. Moreover,
sugar signalling is compromised by disruption of F-actin, and
addition of glucose to Arabidopsis seedlings rapidly disrupted
F-actin [221]. Interestingly, although hexokinase and actin are
functionally diverse proteins, they have a structurally similar
ATPase domain with conserved residues. Thus these recent data
clearly suggest a possible role for plant hexokinases in actindepolymerization-mediated PCD (see Figure 3), although the
connections remain indirect to date and mechanisms unknown.
This certainly makes this an area to watch with respect to the
unfolding story of actin–PCD links in plant cells.
Another component that may link actin dynamics to PCD
responses is PtdA (phosphatidic acid), which is known to operate
in signalling involved in mediating HR and PCD in plants; PtdA
has been shown to decrease H2 O2 -promoted PCD [227]. It has
recently been shown that PtdA regulates an important regulator
of actin filament polymerization, AtCP (Arabidopsis capping
protein), in suspension cells and pollen grains [228]. These studies
showed that exogenous PtdA application resulted in significant
increases in F-actin levels and that PtdA significantly affected
the properties of AtCP by inhibiting the actin-binding activity
of AtCP [228]. These data strongly imply a role for inhibition of
AtCP activity by increased levels of PtdA in stimulating actin
polymerization (see Figure 3). Thus stabilization of actin during
PCD may operate through this type of mechanism, thus hinting at
a further possible involvement of alteration of actin dynamics in
signalling to PCD in plant systems.
A general role for actin alterations mediating PCD in plants?
From the relatively sparse, but emerging, data there seems no
doubt that there exists a role for regulation of actin dynamics in
regulating PCD in plant cells. Moreover, data suggest that the
alterations in actin polymerization status may be a widespread
or universal mechanism used by plants to mediate PCD, though
details may vary. For example, it appears that, generally in the
NHR, actin stabilization is important for regulating PCD, whereas
in other systems, such as embryogenesis [205] and the SI
response [66], it appears to be actin depolymerization that is
the main physiological stimulus triggering PCD in incompatible
pollen, though actin stabilization can also stimulate PCD (see
Figure 3). Thus, although the data may appear skimpy and perhaps
contradictory at this early stage of investigation, the consistent
theme is of F-actin alterations mediating a decision-making role
in regulating PCD in a wide range of plant cells in variety of
different different circumstances.
PERSPECTIVES: ACTIN AS A COMMON MECHANISM REGULATING
APOPTOSIS/PCD IN EUKARYOTES?
The regulation of the cytoskeleton forms an essential link between
signal and appropriate response in all eukaryotic cells. The overwhelming evidence suggests that cells from lower to more
highly evolved systems have adopted the dynamic state of the
c The Authors Journal compilation c 2008 Biochemical Society
400
V. E. Franklin-Tong and C. W. Gourlay
cytoskeleton as an indicator of the cells’ overall health. It has
been shown that, at least in some cases, actin polymerization
status plays a role in mediating PCD, clear differences exist
between plants, yeast and animal cells with respect to both
components involved in actin and PCD regulation. For example,
in yeast, actin stabilization triggers apoptosis [89,152]. In animal
cells, depending on the cell type, either actin stabilization or
depolymerization (see above) can stimulate apoptosis. In plant
cells also, it seems that either depolymerization or stabilization
of F-actin can stimulate PCD. Because of these differences, it has
been proposed that the dynamics of actin polymerization may be
responsible for modulating apoptotic signalling cascades, rather
than absolute requirement for one mechanism or the other. This
needs further investigation.
The question as to whether core interactions and regulatory
mechanisms between cytoskeletal components and pathways that
influence apoptosis remains unanswered. However, a strong body
of evidence points to a close relationship between the actin and
microtubule cytoskeletons and the regulation of mitochondrial
function as a possible point of conserved apoptotic regulation.
From the available data, the strongest conserved point of regulation may come from the regulation of mitochondrial membrane
permeability, a prime candidate being the regulation of the
VDAC. Mitochondrially derived apoptosis regulatory factors,
such as ROS production, outer membrane permeability and mitochondrial morphology, have all been linked to cytoskeletal
function in disparate systems, lending weight to the possibility
that conserved regulatory mechanisms exist. Another interesting
emerging theme is that aberrant actin formations appear to have
detrimental effects upon downstream signalling mechanisms that
retard a cell’s ability to respond to environmental change.
Although the mechanisms involved will differ markedly in
terms of signalling components between organisms, physiologically relevant links between environmental sensing, the actin
cytoskeleton and apoptosis/PCD may be present in all eukaryotic
systems. Although many actin-binding/regulatory proteins are
conserved throughout the eukaryotes, there are notable exceptions
known to play a role in apoptosis in mammalian cells that have
not been identified in yeast and are not present in the Arabidopsis
genome database. Notably, β-thymosin and gelsolin genes/sequences (see the section above relating to these proteins in animal
cells) do not appear to be present in plants [75] or yeast, and coronins have not been found in plants. However, others, e.g. cofilin/
ADF are present in both yeast and plants. Moreover, although a
gelsolin sequence is not found in plants, a gelsolin-like activity has
been measured [212]. Thus, it will be of considerable interest to
explore whether these proteins or alternative proteins with similar
activities also play a similar role in regulating apoptosis/PCD in
these organisms, providing evidence for universal mechanisms in
controlling apoptosis/PCD.
We are grateful to Dr Christopher J. Staiger (Department of Biological Sciences and
The Bindley Bioscience Center, Purdue University, West Lafayette, IN, U.S.A.), Dr Tobias
von-der-Haar and Dr Martin Carden (both of the Department of Biosciences, University
of Kent, Canterbury, Kent, U.K.) for their helpful comments, advice and thoughts on
the manuscript before its submission. Work in the laboratory of V. E. F.-T. is funded by the
BBSRC (Biotechnology and Biological Sciences Research Council). C. W. G. is supported
by an MRC (Medical Research Council) Career Development Award (no. 78573).
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