A "primer"-based mechanism underlies branched actin filament network formation and motility.
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ABSTRACT: Motile cells extend a leading edge by assembling a branched network of actin filaments that produces physical force as the polymers grow beneath the plasma membrane. A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion. Signaling pathways converging on WASp/Scar proteins regulate the activity of Arp2/3 complex, which mediates the initiation of new filaments as branches on preexisting filaments. After a brief spurt of growth, capping protein terminates the elongation of the filaments. After filaments have aged by hydrolysis of their bound ATP and dissociation of the gamma phosphate, ADF/cofilin proteins promote debranching and depolymerization. Profilin catalyzes the exchange of ADP for ATP, refilling the pool of ATP-actin monomers bound to profilin, ready for elongation.Cell 03/2003; 112(4):453-65. · 32.40 Impact Factor
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ABSTRACT: Actin-based cell motility is a complex process involving a dynamic, self-organizing cellular system. Experimental problems initially limited our understanding of this type of motility, but the use of a model system derived from a bacterial pathogen has led to a breakthrough. Now, all the molecular components necessary for dynamic actin self-organization and motility have been identified, setting the stage for future mechanistic studies.Nature Reviews Molecular Cell Biology 12/2000; 1(2):110-9. · 39.12 Impact Factor
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ABSTRACT: Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.Nature 11/1999; 401(6753):613-6. · 36.28 Impact Factor
Current Biology 20, 423–428, March 9, 2010 ª2010 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2009.12.056
A ‘‘Primer’’-Based Mechanism Underlies
Branched Actin Filament Network
Formation and Motility
Ve ´rane Achard,1,3Jean-Louis Martiel,2,3Alphe ´e Michelot,1,4
Christophe Gue ´rin,1Anne-Ce ´cile Reymann,1
Laurent Blanchoin,1,* and Rajaa Boujemaa-Paterski1,*
1Laboratoire de Physiologie Cellulaire Ve ´ge ´tale,
Institut de Recherches en Technologies et Sciences pour le
Vivant, CNRS/CEA/INRA/UJF, 38054 Grenoble, France
2Laboratoire Techniques de l’Inge ´nierie Me ´dicale et de la
Complexite ´, CNRS/UJF, Pavillon Taillefer,
Faculte ´ de Me ´decine, 38706 La Tronche, France
Cells use actin assembly to generate forces for membrane
protrusions during movement  or, in the case of patho-
, or in mixtures of actin and other purified proteins .
Significant progress has been made in understanding the
mechanism of actin-based motility at a macroscopic level
by using biomimetic systems in vitro [4–6]. Here, we com-
bined such a system with evanescent wave microscopy
to visualize Arp2/3-mediated actin network formation at
single-actin-filament resolution. We found that actin fila-
catalytic and propagative formation of the actin network.
In the presence of capping protein, multiple ‘‘primers’’ gen-
erate independent networks that merge around the object
to form an outer ‘‘shell’’ made of entangled and capped fila-
ments. Simultaneously, newly created filaments on the
surface of the particle initiate mechanical stress, which
develops until symmetry breaking. Our results and exten-
sive modeling support that the stress, which releases into
propulsive forces , is controlled not by any specific orien-
tation of actin filaments toward the nucleation sites but only
by new monomers added near the load surface.
Results and Discussion
We used total internal reflection fluorescence microscopy
(TIRFM) to study the dynamic formation of Arp2/3 complex
branched actin networks in real time at single-actin-filament
rods coated with pWA, the C-terminal region of WASP/Scar
protein  that initiates actin polymerization in the presence
of Arp2/3 complex and G-actin. The initial steps of actin
assembly and the network extension around the particles
were characterized with Alexa 488-labeled G-actin . Addi-
tionally, the use of photosensitive Alexa 532-labeled actin
allowed us to track individual actin filaments during the poly-
merization within such a dense and branched actin network.
Therefore, we were able to monitor in real time the sustained
and autocatalytic branch formation, which continuously prop-
agates along the particle surface.
Actin Filament ‘‘Primers’’ Are Necessary to Initiate Arp2/3
Complex-Mediated Actin Polymerization around
the NPF-Coated Particle
As a first step, and because nonmuscle cells maintain unpoly-
merized actin at concentrations as high as 100–300 mM, we
used a profilin-G-actin buffered medium containing a minimal
set of purified actin-binding proteins to reconstitute sustained
actin-based motility of nucleation-promoting factor (NPF)-
coated beads and glass rods. The recorded velocities were
8–33 nm/s (Figures 1A and 1B; see also Movies S1A and S1B
available online). In these conditions where the actin monomer
pool is bound to profilin, actin filaments nucleated by Arp2/3
complex elongate strictly at free barbed ends. Although this
macroscopic reconstitution under physiological conditions
was an important step toward identifying the nature of the
minimal set of purified proteins essential to generate actin-
based motility, most of the molecular mechanisms involved in
Using evanescent wave microscopy, we observed specifi-
cally the elementary molecular reactions that control actin
dynamics leading to symmetry breaking and motility at a
microscopic level and addressed the central role of heterodi-
meric capping protein (CP; the muscle isoform is also called
CapZ) in these processes. We first investigated how assembly
ated. We discovered that NPF-coated particles only induced
autocatalytic actin assembly after an initial contact with a drift-
ing actin filament in the medium (Figure 1C, red and green
arrowheads; Movie S1C) or with an actin filament emanating
from an adjacent branched network (Figure S1A; Movie S1D),
which is consistent with biochemical measurements .
Accordingly, the time to first contact between the NPF-coated
particles and a drifting actin filament decreases as a function
of the density of ‘‘primers’’ in solution (Figure 1D). The obser-
vation of this activation by a drifting ‘‘primer’’ responsible for
the initiation of protruding actin networks was buried because
of the high concentration of actin filaments present in the
media of reconstituted systems used previously [2–5]. More-
over, the long functionalized rods allowed us to observe that
several actin filament ‘‘primers’’ were required to achieve the
rapid and overall spread of an actin-branched network around
the particle (Figure 1C). We found that the actin-branched
network often drifted around the NPF-coated glass fiber, con-
tacted the rod, initiated branches, and finally detached, so the
interactions of filaments with the nucleation sites on the fiber
were transient (Figure 1E; Movie S2A); this was consistent
with biochemical measurements of rapid dissociation of VCA
from Arp2/3 complex .
Barbed Ends of Branched Actin Filaments Initiated
at the Nucleation Sites Grow away from the NPF-Coated
To further characterize the geometry of the branched
network of actin filaments assembled at the surface of
*Correspondence: email@example.com (L.B.), firstname.lastname@example.org
3These authors contributed equally to this work
4Present address: Department of Molecular & Cell Biology, University of
California, Berkeley, Berkeley, CA 94720-3202, USA
functionalized glass rods by Arp2/3 complex, we followed
the growth of the network by using G-actin covalently
labeled with Alexa 532. Rapid photobleaching of polymerized
Alexa 532-actin allows one to follow barbed ends elongating
either outwards or, interestingly, within the branched net-
work, because they are much brighter than older regions
0 20 4060 min
0 3254 min
35 s50 s60 s 100 s
200 s 250 s290 s350 s
Time to first
Primers in 2000 µm2
020 40 60
µ ( e
a t s i
Figure 1. Dynamic Architecture of an Arp2/3 Complex-
Generated Actin Filament Network
(A and B) Reconstituted motility of 2 mM GST-pWA-coated
beads or 9 mM GST-pWA-coated rods, imaged by phase-
contrast microscopy, with 4 mM G-actin, 12 mM profilin,
75 nM Arp2/3 complex, 25 nM capping protein (CP),
and 1 mM ADF/cofilin. G-actin was 7% Alexa 488 labeled.
(C–F) Actin assembly around nucleation-promoting factor
(NPF)-coated particles followed by total internal reflection
fluorescence microscopy (TIRFM).
(C) Actin filament triggers polymerization around 1 mM GST-
pWA-coated rods with 50 nM Arp2/3 complex and 1.2 mM
Alexa 488-G-actin. Red and green arrowheads indicate
initial contact of an actin filament ‘‘primer’’ with the particle,
and arrows show the direction of actin filament elongation.
Kymographs, measured between the dotted lines, illustrate
the fluorescence increase due to polymerization; corre-
sponding fluorescence intensity distribution was calculated
at different times. Color coding in graph at right is associ-
ated with times indicated by colored numbers under panels
to the left.
(D) Actin polymerization around NPF-coated rods was
analyzed (in conditions similar to C), and the time to
first contact by a ‘‘primer’’ was determined for different
densities of filaments measured in the evanescent wave
field (2000 mm2). The spontaneous nucleation process
of monomeric actin in the reconstituted medium was
tuned with increasing amount of profilin. The actin fil-
ament ‘‘primers’’ that initiate actin assembly along the glass
rod range between 1 and 2 mm in length. Kymographs
illustrate the fluorescence increase due to polymerization.
(a)–(d) represent activation events. The graph at right
represents the comparison between experimental results
(blue dots) and the theoretical prediction (solid line)
based on a diffusion-controlled capture of ‘‘primers’’ by
(E) 0.5 mM Alexa 532-G-actin polymerized in the presence of
1.5 mM profilin, 0.5 mM ADF/cofilin, and 33 nM Arp2/3
complex into actin filaments (red arrowheads) that initiate
branches (yellow arrowheads) on 0.5 mM GST-pWA-coated
(F) Actin network assembly on 5 mM GST-WA-coated rods
mixed with 0.8 mM Alexa 532-G-actin and 40 nM Arp2/3
complex; kymograph highlights branching activity emerging
from the rod.
(G) Zoomed region of actin network indicated by red box in
(F) (color code as in E). The cartoon at right represents the
two actin networks generated on the rod.
Scale bars represent 5 mm. (See also Figure S1 and Movies
S1 and S2.)
of the filaments . Whereas previous stud-
ies described actin incorporation sites around
branched actin network or by speckle micros-
copy [12, 13], here we monitored actin filament
network formation in real time at single-actin-
filament resolution and obtained a chronological
description of the elementary events that lead to
actin-based motility. We found that the actin-
branched network elongated with all barbed ends growing
away from the NPF-coated glass rod (Figure 1F; Movie
S2B; see also Figure S1B for modeling), subsequent to the
stimulation of Arp2/3 complex branching activity on actin
filaments growing along the NPF-coated rod (Figure 1G;
Current Biology Vol 20 No 5
Capping Protein Shortens the Growth of Actin Filament
‘‘Primers’’ and Creates Independent Networks around
the NPF-Coated Particle
We investigated the role of CP in the architecture of a growing
Arp2/3 complex-mediated actin network prior to symmetry
breaking. CP was identified as one of three actin-binding
proteins (ABPs) that precisely choreograph actin polymeriza-
tion and organization to generate ‘‘comet tail’’ motility in vitro
. Heterodimeric CP binds with high affinity (0.1 nM) to fila-
ment barbed ends and prevents subunit loss or addition. First,
we followed Arp2/3 complex-mediated branch formation in
solution (Figure 2A) to quantify the kinetic formation of a
dendritic filament network (Figure 2B; Figure S1C). Both
the total number of branches and their cumulative length
increased exponentially over time. Increasing CP concentra-
tion did not affect this autocatalytic behavior but reduced the
Cumulative number of branches
Cumulative length (µm)
0 1 2 3 4
Figure 2. Capping Protein Limits the Average Network
Extension Radius around Particles by Reducing the Average
(A) Dynamic assembly of actin-branched structures in
solution observed by TIRFM with 1 mM Alexa 568-G-actin,
10 nM Arp2/3 complex, 113 nM GST-WA, and CP at the indi-
(B) The total number of branches counted correlates linearly
with the cumulative length (dots) as predicted by simulation
concentration. Error bars are the error of each slope calcu-
lated from the linear regressions in (B).
(D) Decrease of the radius of actin networks around 4 mM
GST-WA-coated beads with 1.5 mM Alexa 532-G-actin,
30 nM Arp2/3 complex, and CP at the indicated concentra-
tions; each reaction was followed by TIRFM. Rightmost
radius around beads versus time (blue dots = Rmax, green
dots = Rmin) and in silico simulation (solid line) (see Fig-
ure S1C and Supplemental Experimental Procedures).
(E) Cartoon illustrating the Rmaxand Rmindetermination.
(F) Experimental (blue dots) and simulated (red triangles)
mean actin network radius decreases with CP concentra-
tion (n = 20 for each condition). Error bars represent the
standard deviation of the radii measured for each CP
Scale bars in (A) and (D) represent 5 mm. (See also
average filament length (Figure S1C). As a con-
sequence, the average distance between two
branching points on a mother branch was nega-
tively correlated with increasing CP concentration
as a result of less available actin filament length to
host the nucleation of new branches (Figure 2C).
Second, when NPFs were located on beads or
glass rods, increasing the concentration of CP
reduced the radius of the actin network assem-
bled around the particle (Figures 2D and 2E), in
agreement with our kinetic model implemented
with the capping activity (Figure 2F; Supplemental
Capping actin filament barbed ends con-
strained actin filament elongation near the NPF
in agreement with previous studies [6, 12], but
without necessarily affecting the orientation of
branches in the network assembled around
particles. To increase the spatial resolution, we followed
Arp2/3 complex-mediated actin-branched network assembly
around glass rods, instead of around beads, with Alexa 532-
actin monomers. The branched actin filaments grew away
from the rod until being capped at their barbed end (Fig-
ure 3A). Using photobleaching of Alexa 532-actin, we followed
appeared after a capping event (Figure 3A; Movie S3A). When
the CP concentration was increased, the actin-branched net-
work did not propagate efficiently along the rod (Figure 3C).
In order to obtain a homogenous spread of branched filament
network along the functionalized rod, actin assembly must be
initiated by multiple actin filament ‘‘primers’’ at several NPF
coating sites (Figure 3C).
We simulated the kinetics of nucleation, branching, and
capping of new filaments on glass rods (Figure 3D; Movie
Mechanism of Branched Actin Network Formation
S3B) or beads (Figures 2D–2F) by using the different kinetic
steps illustrated in Figure 3E (see also the detailed description
of the model in Supplemental Experimental Procedures and
Figures S2A and S2B). Based on the experimental settings in
which neither the density of NPFs nor the concentrations of
Arp2/3 complex or actin monomers were limiting, nucleation
0714 20 min
10 nM CP
25 nM CP
25 27 30 32 36 min
022 48 min
5 nM CPs
Actin filament “primers”
Nucleation-promoting factor (NPF)
Autocatalytic and propagative assembly of merging actin networks
Figure 3. Spreading of Actin-Branched Network
along Glass Rods Emanates from Actin Filament
‘‘Primers’’ and Is Limited by Capping Protein
(A–C) Actin assembly is followed by TIRFM. Scale
bars represent 5 mm.
(A) 0.8 mM Alexa 532-G-actin polymerizes around
5 mM GST-WA-coated glass rods with 80 nM
Arp2/3 complex and 5 nM CP. Barbed ends
grew away from the rod (green arrowheads)
before been capped (red arrowheads). The fluo-
rescence of the Alexa 532 then disappears as
a result of photobleaching, as shown in the
zoom of the red boxed area and in its associated
(B and C) 2 mM Alexa 488-G-actin polymerizes
around 1 mM GST-pWA-coated glass rods with
25 nM Arp2/3 complex and CP as indicated.
filament ‘‘primer’’ with the NPF-coated particle.
The color code used in the kymographs in (B)
and (C) is associated with the indicated times.
(D) Simulated network growth. Activated Arp2/3
complex (magenta dots) initiates branches (gray
lines) on the actin filament ‘‘primers.’’ Free barbed
ends (green dots) escape the rod surface or are
stalled against it (blue dots). However, because
of thermal fluctuations, stalled barbed ends are
oriented tangentially to the particle surface.
Thereby, actin filament barbed ends resume their
growth (yellow dots). In the presence of CP, the
spatial actin network extension is reduced
(capped barbed ends; red dots). The rightmost
column magnifies the yellow boxed regions of
each row. Scale bars represent 10 mm.
(E) Model of branched network formation around
the motile particle. The molecular mechanism of
Arp2/3 complex-mediated network formation
consists of an initial ‘‘primer’’-based activation
(‘‘primer’’ trigger step) followed by the autocata-
lytic and propagative spread of the network.
Each ‘‘primer’’ creates an independent network.
These networks merge to cover the particle.
Each new branch can transiently be stalled
against the load (see Figure S2Ab and ) or
elongate away from it before being capped.
Networks are viewed from the top. The encircled
area attopis azoomed side viewof some branch-
ing points. (See also Figure S2 and Movie S3.)
of new filament branches was a self-sus-
tained process that depended on CP
concentration (Figure 3D; Movie S3B).
Barbed-end capping restricted the max-
imal growth of the network, whereas it
had little or no effect on the self-sus-
tained nucleation process at low CP
concentration (Figure 3D; Movie S3B).
Additionally, capping modified the distri-
bution of filament branch lengths, but
barbed-end orientation remained isotro-
pic. Because CP reduced the average
filament length, both the network radius and the rate of
network extension were smaller with than without CP. More-
over, CP increased the density of actin filaments inside the
was sufficient at low CP concentration to trigger an explosive
generation of branches (Figure 3D, top and middle panels;
Current Biology Vol 20 No 5
Movie S3B). Conversely, experimental data and numeri-
cal simulations showed that above a CP concentration of
w10 nM (in our experimental setup), the shortened actin fila-
ment ‘‘primers’’ and reduced duration of branch elongation
were unable to sustain autocatalytic branching (Figures 3C
and 3D, bottom panel; Movie S3B). For CP concentrations
above 10 nM, the model predicts that up to 20 independent
actin filament ‘‘primers’’ were necessary to generate a fully
developed filament meshwork comparable to experimental
data (Figure 1; Figure 4).
300 seconds 230 16010
048 1216 min
3 152750 min39
38 12 1928 min
Figure 4. Network Rupture Occurs through a ‘‘Multiple Shell-
Breaking’’ Process of Randomly Oriented Actin Filaments
(Aa–Ac) An isotropic actin network assembles around 2 mM GST-
pWA-coated beads mixed with 2 mM Alexa 488-G-actin and 50 nM
Arp2/3 complex (Aa). Addition of 25 nM CP triggers a ‘‘multiple
shell-breaking’’ process (Ac); arrowheads indicate the successive
shells. We changed the ratio of Arp2/3 complex to CP and still
observed the multiple shell-breaking process for 4 mM GST-pWA-
coated beads mixed with 4 mM Alexa 488-G-actin, 12 mM profilin,
100 nM Arp2/3 complex, and 25 nM CP. However, insufficient
capping leads to a ‘‘fishbone’’ pattern in the actin ‘‘comet’’ (Ab).
Arrowheads highlight elongating filaments or bundles growing
away from the bead. Scale bars represent 10 mm.
mental Experimental Procedures) shows that after an isotropic and
homogeneous growth of filaments nucleated on the bead (10–170 s),
the internal stress fractures the network (170 s) before the filaments
are displaced (between 170 s and 230 s). Newly nucleated actin fila-
ments spread over the bead ‘‘empty zone’’ (after 230 s), leading to
the reconstruction of a fully developed network. The free barbed
ends in the network that move away from the bead (170–230 s) are
rapidly capped and form the ‘‘comet tail.’’ Scale bar represents
5 mm. (See also Figures S2 and S3 and Movies S4 and S5.)
Evidence for a ‘‘Multiple Shell-Breaking’’ Process
during Actin-Based Motility
To bridge the molecular events of actin filament mesh-
work formation and actin-based motility of NPF-coated
beads, we used evanescent wave microscopy to follow
ends growing away from the bead (Figure 4Aa). Surpris-
ingly, in thepresenceof sufficientCP, we found that sus-
tained motility results from a ‘‘multiple shell-breaking’’
process (Figure 4Ac; Movie S4B) during ‘‘continuous
movement’’ observed by phase contrast or epifluores-
cence microscopy (Figures 1A and 1B).
Based on this observation, we propose the following
model centered on the control of actin filament length
by CP (Figure S2C): (1) CP creates a ‘‘dead zone’’ at
the outer actin ‘‘shell’’ by inhibiting all actin filament
elongation beyond the shell radius; (2) Arp2/3 nucleation
occurs strictly at the bead surface because NPF is
immobilized on the particle; this will create an active
polymerization zone embedded within the constrained
dead zone; (3) continuous actin nucleation in the vicinity
of the bead will generate an internal stress that breaks
the former dead zone; and (4) CP will block elongation
of actin filaments in the ‘‘active zone,’’ creating a new
dead zone, while Arp2/3 complex nucleates new actin
filaments at the surface of the bead, building a new
active zone. The dead zone breaking repeats, leading
to the multiple shell-breaking process. The existence
of cyclic versus continuous network breakage was assigned
to different mechanisms controlled by either particle size 
or the role of fluctuations behind symmetry breaking [6, 14].
Based on our observations, we propose that the actin network
oscillates between expansion and rupture phases, but de-
pending on the network thickness at rupture, this process
may or may not be observable via conventional microscopy
methods but is always observable via TIRFM.
Moreover, when the ratio between Arp2/3 complex and CP
is low, multiple shell breaking still occurs, but some actin
Mechanism of Branched Actin Network Formation
filaments elongate beyond the dead shell radius (Figure 4Ab;
Figure S2C; Movie S4A), giving rise to the ‘‘fishbone’’ pattern
observed previously .
Symmetry Breaking: The Elastic Continuum Made of
Elementary Merging Actin Meshworks Breaks upon
Mechanical and Spatial Constraints
We modeled dynamic organization of actin filaments during
network formation and shell breakage (Supplemental Experi-
mental Procedures). Simulations presented in Figure 3 show
about 30 independent ‘‘primers’’ are required to generate full
coverage of the bead by growing actin filaments. Extension
and merging of these elementary networks (Figure 3E; Fig-
ure S2E), each generated by a single ‘‘primer’’ filament, pro-
Movie S5). Extension of this network is accompanied by a
slow but constant displacement of the actin filaments away
from the bead (Figure 3E; Figure 4B; Movie S5). However,
because entanglements between networks tend to oppose
the forces generated at the bead surface, the progression of
the network slows down and it reaches equilibrium. At 170 s,
the filaments away from the bead and leaving an empty space
(Figure S2E). This rapid movement (between 180 s and 230 s)
relieves the stress applied to the actin filaments, which in turn
results in a marked increase in the generation of new filaments
(Figure S2Fa). However, the orientation of actin filaments
remains isotropic (Figure S2Fc). The occurrence of symmetry
breaking of the actin filament network around a NPF-coated
and in the absence of CP (Figure S3) confirms our model’s
fundamental hypothesis of barbed-end steric constraint.
based on a simple but fundamental steric constraint wherein
elongating barbed ends tend to escape when they are aimed
directly at the load and simultaneously entangled into a broad
actin meshwork. Thus, filaments either growing along the load
or growing outwards create a steric hindrance and a stress,
which releases into propulsive forces, consistent with the
mechanics of symmetry breaking of actin gel [6, 7]. Therefore,
a challenge for future investigations will be to constrain our
molecular model with previous observations on the symmetry
breaking time  to predict at the molecular level the mechan-
ical properties of the active gel around the motile particle.
We have demonstrated that actin filaments that we call
‘‘primers’’ initiate the formation of the actin network. Physio-
logical concentration of capping proteins shortens the growth
of these ‘‘primers’’andcreates independent networksmadeof
isotropically oriented actin filaments that merge around the
motile particle. Moreover, the movement of NPF-coated parti-
cles then results from a multiple shell-breaking process con-
trolled by the tight tiling of these independent networks,
tions suggest that force production necessary to propel cyto-
plasmic particles or to protrude the plasma membrane relies
not necessarily on any preferential orientation of actin fila-
ments inside the network, but on simple and universal physics
laws. The ‘‘primer’’-based mechanism likely emerges as a
general feature of branched network assembly involved in
‘‘comet tail’’ or lamellipodium formation during pathogen or
cell motility. Based on this mechanism, actin filaments parallel
to the nucleating surface constitute an efficient and optimal
way to initiate branched network formation upon signaling.
Supplemental Information includes Supplemental Experimental Proce-
We are grateful to T.D. Pollard, J. Van der Gucht, J.-F. Joanny, C.J. Staiger,
and D. Vignjevic for helpful discussions and insightful suggestions. This
work was supported by Agence Nationale de la Recherche grant ANR-06-
PCV1-0022 to L.B. and J.-L.M.
Received: October 1, 2009
Revised: December 22, 2009
Accepted: December 23, 2009
Published online: February 25, 2010
1. Pollard, T.D., and Borisy, G.G. (2003). Cellular motility driven by
assembly and disassembly of actin filaments. Cell 112, 453–465.
2. Cameron,L.A., Giardini, P.A.,Soo, F.S., and Theriot, J.A. (2000). Secrets
of actin-based motility revealed by a bacterial pathogen. Nat. Rev. Mol.
Cell Biol. 1, 110–119.
3. Loisel, T.P., Boujemaa, R., Pantaloni, D., and Carlier, M.F. (1999).
Reconstitution of actin-based motility of Listeria and Shigella using
pure proteins. Nature 401, 613–616.
4. Akin, O., and Mullins, R.D. (2008). Capping protein increases the rate of
actin-based motility by promoting filament nucleation by the Arp2/3
complex. Cell 133, 841–851.
5. Bernheim-Groswasser, A., Wiesner, S., Golsteyn, R.M., Carlier, M.F.,
and Sykes, C. (2002). The dynamics of actin-based motility depend on
surface parameters. Nature 417, 308–311.
6. Dayel, M.J., Akin,O.,Landeryou,M., Risca,V.,Mogilner, A.,andMullins,
R.D. (2009). In silico reconstitution of actin-based symmetry breaking
and motility. PLoS Biol. 7, e1000201.
7. van der Gucht, J., Paluch, E., Plastino, J., and Sykes, C. (2005). Stress
release drives symmetry breaking for actin-based movement. Proc.
Natl. Acad. Sci. USA 102, 7847–7852.
8. Machesky, L.M., Mullins, R.D., Higgs, H.N., Kaiser, D.A., Blanchoin, L.,
May, R.C., Hall, M.E., and Pollard, T.D. (1999). Scar, a WASp-related
protein, activates nucleation of actin filaments by the Arp2/3 complex.
Proc. Natl. Acad. Sci. USA 96, 3739–3744.
9. Michelot, A., Berro, J., Gue ´rin, C., Boujemaa-Paterski, R., Staiger, C.J.,
Martiel, J.L., and Blanchoin, L. (2007). Actin-filament stochastic
dynamics mediated by ADF/cofilin. Curr. Biol. 17, 825–833.
10. Marchand, J.B., Kaiser, D.A., Pollard, T.D., and Higgs, H.N. (2001). Inter-
action of WASP/Scar proteins with actin and vertebrate Arp2/3
complex. Nat. Cell Biol. 3, 76–82.
11. Michelot, A., Derivery, E., Paterski-Boujemaa, R., Gue ´rin, C., Huang, S.,
Parcy, F., Staiger, C.J., and Blanchoin, L. (2006). A novel mechanism for
Biol. 16, 1924–1930.
12. Cameron, L.A., Svitkina, T.M., Vignjevic, D., Theriot, J.A., and Borisy,
G.G. (2001). Dendritic organization of actin comet tails. Curr. Biol. 11,
13. Vignjevic, D., Yarar, D., Welch, M.D., Peloquin, J., Svitkina, T., and
Borisy, G.G. (2003). Formation of filopodia-like bundles in vitro from
a dendritic network. J. Cell Biol. 160, 951–962.
14. Cameron, L.A., Robbins, J.R., Footer, M.J., and Theriot, J.A. (2004).
Biophysical parameters influence actin-based movement, trajectory,
and initiation in a cell-free system. Mol. Biol. Cell 15, 2312–2323.
15. Pantaloni, D., Boujemaa, R., Didry, D., Gounon, P., and Carlier, M.F.
(2000). The Arp2/3 complex branches filament barbed ends: functional
antagonism with capping proteins. Nat. Cell Biol. 2, 385–391.
16. Berg, O.G., and von Hippel, P.H. (1985). Diffusion-controlled macromo-
lecular interactions. Annu. Rev. Biophys. Biophys. Chem. 14, 131–160.
17. Mogilner, A., and Oster, G. (1996). Cell motility driven by actin polymer-
ization. Biophys. J. 71, 3030–3045.
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