The most important discovery in the field is that the Arp2/3
complex nucleates assembly of actin filaments with free barbed
ends. Arp2/3 also binds the sides of actin filaments to create a
branched network. Arp2/3’s nucleation activity is stimulated by
WASP family proteins, some of which mediate signaling from
small G-proteins. Listeria movement caused by actin
polymerization can be reconstituted in vitro using purified
proteins: Arp2/3 complex, capping protein, actin
depolymerizing factor/cofilin, and actin. actin depolymerizing
factor/cofilin increases the rate at which actin subunits leave
pointed ends, and capping protein caps barbed ends.
Department of Cell Biology, Washington University, Box 8228, 660
S. Euclid Ave, St Louis, MO 631110, USA
Current Opinion in Cell Biology 2000, 12:97–103
0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
actin depolymerizing factor
Actin filaments grow or shrink by virtue of the addition or
loss, respectively, of actin subunits from either end of the
filament. The two ends, barbed and pointed, are both able
to add and lose subunits. Polymerization is favored at the
barbed end over the pointed end in terms of steady-state
binding affinity. Kinetic rate constants for both polymer-
ization and depolymerization are greater at the barbed end.
Proteins that bind filament ends control actin polymeriza-
tion. First, they cap filament ends, such that subunits
cannot be added or removed. Second, some can nucleate
the formation of new filaments, which is otherwise unfa-
vorable. A number of end-binding proteins have been
defined biochemically. How they function in vitro and in
vivo is a current focus of the field.
Proteins that bind the sides of actin filaments can also
influence how fast subunits leave the filament. Proteins,
such as tropomyosin, which bind to multiple subunits
along the side of a filament and proteins that crosslink fil-
aments decrease the rate of subunit loss from ends. An
exciting discovery is that ADF/cofilin proteins increase the
rate of subunit loss from pointed ends.
In vivo, these proteins work simultaneously to produce
motility. Understanding how their individual actions on
actin are coordinated is an essential goal, thus the field has
been investigating how combinations of actin-binding
proteins affect actin polymerization. One exciting exam-
ple is the discovery that three actin-binding proteins, plus
actin, are sufficient for Listeria to induce actin polymeriza-
tion and motility [1••].
This field has been the subject of a number of reviews in
recent years. We reviewed this topic in more depth sever-
al years ago . More recent reviews have addressed
filament length in muscle , actin polymerization dynam-
ics and signaling , how WASP links small G proteins to
Arp2/3 complex , how Arp2/3 complex functions [6–8],
how ADF/cofilin proteins function [9,10] and how gelsolin
and its relatives function . In this minireview, we focus
on the most recent advances in understanding how actin-
binding proteins control the addition and loss of subunits
at filament ends.
Barbed ends: capping protein (Cap Z)
When cells induce actin polymerization, especially to
cause movement at the cell periphery, free barbed ends are
created and add subunits. Over time, capping protein
binds barbed ends to stop filament growth. The cessation
of filament growth by capping protein may be necessary
for polymerization to occur at specific times and places in
vivo (i.e. free barbed ends probably exist only when they
are newly created or when capping protein is inhibited in
their vicinity). Since nearly all barbed ends are capped by
capping protein, polymerization will be confined or ‘fun-
neled’ to the free barbed ends. This hypothesis for
localized actin assembly, proposed by Carlier and Pantaloni
, has received experimental support with the discovery
that capping protein is necessary for actin polymerization
and motility of Listeria in a system comprising pure pro-
teins [1••]. In those experiments, as the concentration of
added capping protein was decreased to zero, Listeria
motility fell to zero, which is consistent with the idea of
‘funneling’. In support of this hypothesis in vivo, capping
protein is a component of the actin filament tails whose
polymerization drives the movement of Listeria . The
model also can explain the observation in Dictyostelium that
loss of capping protein led to increased F-actin but
decreased motility .
Actin filaments with capping protein at their barbed ends
can be uncapped by polyphosphoinositides in vitro ,
and this may occur during platelet activation .
However, uncapping appeared not to occur during
chemoattractant-induced actin assembly in Dictyostelium;
capping protein only terminated polymerization .
Some studies suggest that capping protein may be inhibit-
ed or may be unable to cap barbed ends under certain
conditions in vivo. Barbed ends of red cell membrane
preparations seemed to be resistant to capping protein
Control of actin assembly and disassembly at filament ends
John A Cooper* and Dorothy A Schafer†
; barbed ends in red cells may be capped by adducin
instead . Also, barbed ends induced in neutrophil
extracts by Cdc42 appeared to be protected from capping
protein [20•]. Perhaps some factor inhibits capping protein
or prevents it from binding in these cases.
In striated muscle, capping protein binds to barbed ends of
actin filaments at the Z line, the inspiration for the name
‘capZ.’ This topic was recently reviewed . In new work
consistent with this view, expression of a mutant capping
protein unable to bind actin led to severe sarcomere dis-
ruption in hearts of transgenic mice . In addition,
expression of a nonsarcomeric isoform of capping protein
(beta2) had the same severe effect on sarcomere assembly,
indicating that the nonsarcomeric isoform cannot substi-
tute for the sarcomeric isoform, beta1 [20•].
Gelsolin and its superfamily
Gelsolin, which was thoughtfully reviewed in Current
Opinion in Cell Biology last year , appears to play an
important role in certain situations where actin assembly
is induced. In most cells at rest, gelsolin is not bound to
actin. However, stimulation, especially with increased
Ca2+or H+, can cause gelsolin to sever and cap actin fila-
ments. If gelsolin-capped filaments can be uncapped to
create free barbed ends, polymerization may be induced.
Activation of heterotrimeric and small G proteins dissoci-
ated gelsolin from barbed ends . Fibroblasts from
gelsolin knockout mice were defective for formation of
lamellipodia in response to Rac signaling , which
depends on actin polymerization.
Depletion or inhibition of gelsolin in cultured cells led to
loss of stress fibers and contractility ; in contrast,
fibroblasts from gelsolin-knockout mice had increased F-
actin in stress fibers . These observations confirm a
role for gelsolin in remodeling the actin cytoskeleton but
reveal the potential complexity of gelsolin’s effects on
actin assembly in vivo.
During apoptosis, gelsolin is proteolyzed by caspase-3 to a
form that no longer needs Ca2+for activity. This active
fragment of gelsolin contributes to progression of apopto-
sis, presumably through severing actin filaments .
Villin, which is related to gelsolin, has a small headpiece
that bundles actin filaments in vitro, in addition to the sev-
ering and capping activities of the gelsolin-related portion
of the protein. This bundling activity of villin is clearly
able to function in vivo as seen in transfection experiments
in cultured cells  and overexpression in Drosophila
oocytes , but whether the bundling activity of endoge-
nous villin is an important element of its function in vivo
remains unclear. Notably, in a villin knockout mouse, the
assembly of actin bundles in microvilli of intestinal epithe-
lial cells, which normally contain villin, was normal
[27,28•]. However, microvilli also have other bundling pro-
teins, and the intestinal lining expresses a villin relative,
advillin, whose function may overlap with that of villin and
thereby mitigate the severity of the phenotype in the villin
knockout . Another new relative, albeit more distant,
which also bundles actin filaments at the plasma mem-
brane of villin is supervillin .
On the other hand, the severing activity of villin may have
a more essential role in vivo. Brush borders from intestinal
epithelial cells of a villin knockout mouse did not disas-
semble their actin bundles in response to high Ca2+
concentrations [28•]. Moreover, in vivo, in an experimental
model for intestinal epithelial injury that includes loss of
filamentous actin from the brush border, a villin-knockout
mouse showed decreased loss of actin, increased severity
of epithelia injury and greater probability of death [28•].
Pointed ends: Arp2/3 complex
Several exciting discoveries about Arp2/3 complex have
now given it a central role in our view of how actin poly-
merization occurs in cells. Purified Arp2/3 complex
binds pointed ends and nucleates the formation of actin
filaments with free barbed ends [31••]. Creation of free
barbed ends has been up until now a poorly understood
feature of actin polymerization in cells. Arp2/3 simulta-
neously binds to the sides of actin filaments, creating a
branching network of filaments [31••]. The filament
network created in vitro is very similar to the actin fila-
ment network observed in vivo in the cortex of
migrating cells. In the cortex, Arp2/3 was localized to
branch points where pointed ends meet the sides of fil-
aments [32••]; this observation provides a major piece of
evidence that the biochemical model is correct in vivo.
In addition, in studies localizing actin polymerization in
living cells, Arp2/3 was localized at sites of actin poly-
merization and motility [33•]. Arp2/3 was also found in
the zone of actin polymerization and motility at the
leading edge of lamellopodia [32••,34,35].
Another exciting discovery is that the nucleating activity of
Arp2/3 is greatly stimulated by signaling proteins that are
implicated in inducing actin polymerization. WASP and its
relative Scar1 bind directly to Arp2/3 , which greatly
increases Arp2/3’s nucleation activity [37•]. WASP-coated
beads placed in a cell extract polymerized actin and
moved, which required Arp2/3 [38•]. In cell extracts, actin
polymerization can be stimulated by Cdc42, which also
required Arp2/3 complex [39,40]. N-WASP appeared to
mediate the interaction between Cdc42 and Arp2/3 [41•].
In Listeria-induced actin polymerization and movement,
the Listeria protein ActA interacts with Arp2/3 complex and
increases its actin nucleation activity [42•]. This interaction
is predicted to be necessary for the bacterial motility caused
by actin polymerization in host cell cytoplasm. The pres-
ence of Arp2/3 was necessary for Listeria motility . Also,
Arp2/3 was one of a minimal set of three actin-binding pro-
teins sufficient to reconstitute the actin polymerization and
movement associated with Listeria [1••].
In yeast, the WASP relative Bee1/Las17 also stimulates
Arp2/3 biochemically ; however, a truncated form of
Bee1 that lacks a region necessary for Arp2/3 binding func-
tions normally in vivo, showing that the Bee1–Arp2/3
interaction is not necessary in vivo . Arp2/3 mutants
show loss of actin patch movement , but this move-
ment appears not to depend on actin assembly [46,47].
Therefore, the roles of Arp2/3 in yeast and animal cells
may be different.
Tropomodulin caps the pointed ends of the actin-based
thin filaments in striated muscle, playing an essential role
in the assembly of the sarcomere. Tropomodulin also binds
tropomyosin, which greatly increases its affinity as a point-
ed end cap . This topic was recently reviewed . In
newer work, the distribution of tropomodulin early in
myofibrillogenesis suggests that pointed ends of actin fila-
ments are capped early. Thus, whole actin filaments, with
both ends capped, may slide into place as the sarcomere
matures, as opposed to filaments polymerizing and depoly-
merizing at their ends .
The function of tropomodulin in nonmuscle cells is not
understood well. In Drosophila, the tropomodulin homo-
logue sanpodo is necessary for Notch-based signaling of
cell fate in the peripheral nervous system [50,51] and mus-
cle . The affected cells have an altered actin
distribution. Most likely, actin is necessary for asymmetric
localization of cell fate determinants, and the loss of san-
podo/tropomodulin impairs the assembly and function of
the actin cytoskeleton. In vertebrates, tropomodulin has
been described as part of the actin cytoskeleton in ery-
throcyte membranes and eye lens cells .
Many proteins that bind to the sides of actin filaments are
polyvalent and thus stabilize filaments by inhibiting the
loss of subunits from ends. Such proteins include
tropomyosin, filament bundlers and filament crosslinkers.
The actin dynamics that underlie cell motility and mor-
phogenesis require the disassembly of actin filaments as
well as their assembly. Proteins of the ADF/cofilin family,
which includes ADF, cofilin, actophorin, depactin and
destrin, mediate actin filament disassembly [9,10]. Actin
filaments shorten in the presence of ADF/cofilin proteins,
which could occur in two ways: by severing, thereby creat-
ing more filament ends that disassemble; and by increasing
the rate of subunit loss from filament ends.
Strong evidence for the existence of severing came from
films of fluorescent actin filaments treated with ADF/cofil-
in, in which single long filaments clearly broke into smaller
pieces . Additional evidence supporting severing
includes analysis of the kinetics of actin assembly in the
presence of cofilin [55•,56,57]. These data were best fit by
a severing mechanism, based on kinetic modeling.
Severing is more likely to occur when filaments are long.
ADF/cofilin had little severing activity on short filaments
prepared by capping with gelsolin [55•,58,59].
On the other hand, Carlier et al.  observed a 25-fold
increase in the turnover of F-actin in the presence of
ADF/cofilin that could not be accounted for solely by sev-
ering. They proposed an alternative mechanism whereby
ADF/cofilin accelerates the rate-limiting step in filament
disassembly by increasing the off-rate of actin subunits
from pointed ends. This view was supported by analyses of
the effect of ADF/cofilin on actin disassembly when
barbed ends were capped [55•,58]. This ability of
ADF/cofilin to increase F-actin turnover can account for
the level of actin assembly/disassembly required for the
rates of cell motility observed in vivo . As predicted
using this model, Arp2/3 complex binding to pointed ends
decreased but did not eliminate the effect of ADF/cofilin
on F-actin turnover .
One complexity in the field is that effects of ADF/cofilin
proteins on actin vary considerably depending on the
source of the ADF/cofilin protein, as well as on the source
of the actin and conditions of ionic strength and pH.
Despite these difficulties, the field is approaching a con-
sensus view that both filament severing and increased
pointed-end subunit loss occur [9,60]. A quantitative
understanding of the relative contributions of the two
mechanisms, especially in vivo, is a current challenge for
Structural analysis of ADF/cofilin binding to actin fila-
ments has provided important information about how it
affects filament dynamics . ADF/cofilin bound cooper-
atively along the side of an actin filament, at a 1:1 ratio.
ADF/cofilin binding changed the orientation of actin sub-
units within the filament, resulting in a change in the twist
of the filament. This change in filament architecture may
alter subunit contacts and weaken the lateral and longitu-
dinal interactions between subunits, making the filament
more likely to break and subunits more likely to dissociate
from ends . In addition, the altered filament structure
induced by ADF/cofilin may induce the dissociation of
pointed end-binding proteins, such as Arp 2/3 complex,
leading to filament disassembly.
Small GTPases induce actin dynamics in cells by stimulating
factors that promote actin assembly and by inhibiting factors
that promote its disassembly. Several new observations indi-
cate that ADF/cofilins are regulated downstream of the Rho
family of GTPases. It has long been appreciated that phos-
phorylation of ADF/cofilin at a specific serine residue
abolishes its actin-binding activity . LIM kinase was
identified as the kinase that phosphorylated cofilin at this
serine residue in vitro and in vivo, where its ability to phos-
phorylate ADF/cofilin was linked to the activation of the
small GTPase, Rac [63,64]. The ability of LIM kinase to
Control of actin assembly and disassembly at filament ends Cooper and Schafer 99
phosphorylate ADF/cofilin also is regulated by phosphoryla-
tion via Pak1 [65•], an effector of Rac, and via Rho-associated
kinase (ROCK) [66•], an effector of Rho. These multiple
pathways for ADF/cofilin regulation may contribute to the
spatial and temporal specificity of regulation of actin assem-
bly associated with various cells and signals.
Overexpression of LIM kinase, which should inhibit cofil-
in, blocked actin assembly and motility at the leading edge
of the cell and at sites within the lamella [33•]. This result
is consistent with the hypothesis that ADF/cofilin pro-
motes actin depolymerization globally, which is necessary
for localized polymerization. This view is also supported
by the observation that ADF/cofilin is found only in the
proximal portion of the cortex and not at the distal edge
where polymerization occurs [32••]. On the other hand, the
actin filaments in the ADF/cofilin-containing region of the
cortex were stable during a cell extraction procedure that
caused depolymerization in general [32••]. Therefore,
extracted factors may be necessary for ADF/cofilin to pro-
A second protein regulator of ADF/cofilin increases its
actin disassembly activity. In vitro, actin-interacting pro-
tein 1 (Aip1) from yeast and Xenopus increased the ability
of ADF/cofilin to sever filaments and increased the rate of
filament disassembly [67,68]. Injection of Xenopus Aip1
caused the disassembly of filamentous actin in the con-
tractile ring of cytokinesis . In Dictyostelium, mutants
lacking Aip1 are defective in endocytosis, motility, cytoki-
nesis and growth, which involve actin assembly .
A synthetic system of actin-based motility
The combination of purified Arp2/3, capping protein and
ADF/cofilin was able to polymerize actin on the surface of
Listeria and caused the bacteria to move [1••]. This discov-
ery represents the first description of pure proteins
sufficient for motility based on actin polymerization. All
three actin-binding proteins were necessary for Listeria
motility; when the concentration of any one protein was
zero, the speed of Listeria movement was zero [1••]. As the
concentration of each protein increased, the speed of Listeria
movement increased to a maximum and then decreased.
Listeria is a good model for molecular analysis of actin
polymerization. The movement of Listeria in cytoplasm is
powered by actin polymerization, using host cell proteins.
The actin polymerization and motility induced by Listeria
are similar in many respects to the polymerization and
motility that occur during extension of the leading edge of
a cell. Furthermore, Arp2/3, capping protein and
ADF/cofilin have all been implicated as important for actin
polymerization and motility in cells.
Why is each protein necessary for motility? A proposed
model for localized actin polymerization is shown in
Figure 1. A localized stimulus activates Arp2/3 to nucleate
actin polymerization with free barbed ends. For Listeria,
the stimulus is the bacterial surface protein ActA. The free
barbed ends polymerize for a time and are then capped by
capping protein. The need for capping protein seems odd;
it should inhibit polymerization. One explanation is that
capping protein is bound mainly to the older barbed ends,
which are located away from the activation zone, based
simply on the fact that newly created barbed ends are free
and some time is required for capping protein to associate
with them. Therefore, in this situation, the presence of
A current model for stimulation of actin polymerization, illustrating flux
through a system not at equilibrium. The polymerization of actin (black
chevrons) is activated within a zone of signals (yellow oval) that
activate Arp2/3 complex (green oval) and may also inhibit capping
protein (red circle). Arp2/3 complex binds to sides of actin filaments,
which also increases its nucleation activity. Filaments nucleated by
Arp2/3 have their pointed ends capped and their barbed ends free.
Filaments grow by addition of subunits to the free barbed ends. After
some time, the barbed ends become capped by capping protein,
which stops their growth. The lifetime of the free barbed ends depends
simply on the time of the association reaction with capping protein.
However, if capping protein is inhibited in the active zone, however,
barbed ends will only become capped when they leave the active zone,
perhaps moved by the force of polymerization. ADF/cofilin (blue circle)
binds cooperatively and preferentially to older actin filaments that
contain ADP. This increases the dissociation rate of actin subunits from
pointed ends. To lose subunits, pointed ends need to be uncapped,
losing Arp2/3 complex. Arp2/3 dissociates at its normal rate or at an
increased rate owing to the altered filament structure caused by
ADF/cofilin. The subunits lost from the pointed end diffuse and are
available to add to new free barbed ends. Thus, actin polymerization is
‘funneled’ to the active zone because free barbed ends are created
there and because older barbed ends are capped by capping protein.
Current Opinion in Cell Biology
capping protein restricts, or ‘funnels’, polymerization to
the free barbed ends near the bacterium. ADF/cofilin is
proposed to increase the rate of loss of subunits from point-
ed ends, increasing flux into the pool of free subunits,
which then add to free barbed ends. This step requires
removal of Arp2/3 from the pointed ends. Arp2/3 may dis-
sociate spontaneously or in response to the change in
filament structure upon ADF/cofilin binding.
The synthetic system may not provide a valid representa-
tion of how actin polymerization provides for motility in
vivo. The system does reveal one way that this particular
set of proteins can work together to provide motility, how-
ever, the cell is more complex, so these proteins may
function differently and other proteins may play an impor-
tant role. For example, in cell extracts, altering the activity
of cofilin did not affect the speed of Listeria motility, only
the length of the tail . Perhaps in the cell extract, pro-
teins other than ADF/cofilin, such as profilin and
thymosin, provide for increased flux of subunits to free
barbed ends . In addition, Rickettsia in host cells dis-
plays actin-based motility with tails whose structure is
different from that of Listeria [72,73]. The actin filaments
in their tails are longer and less branched, resembling
those of filopodia rather than lamellae; Rickettsia tails also
do not contain Arp2/3 .
Actin assembly involves the addition and loss of subunits
at barbed and pointed ends of filaments. Actin-binding
proteins have specific effects on these reactions. We are
beginning to understand how these proteins, alone and in
combination, can provide spatial and temporal control of
actin polymerization in vitro and in vivo.
For critical discussions and unpublished work, the authors are grateful to
Velia Fowler, Pekka Lappaleinen, Laura Machesky, Marie-France Carlier,
Amy McGough, Laurent Blanchoin, Alan Weeds, Michael Way and Dyche
Mullins. The writing of this article was supported by National Institutes of
Health grant 38542.
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Control of actin assembly and disassembly at filament ends Cooper and Schafer 103