112
Review
TRENDS in Cell Biology Vol.12 No.3 March 2002
The lamellipodium: where motility
begins
J. Victor Small, Theresia Stradal, Emmanuel Vignal and Klemens Rottner
Lamellipodia, filopodia and membrane ruffles are essential for cell motility, the
organization of membrane domains, phagocytosis and the development of
substrate adhesions. Their formation relies on the regulated recruitment of
molecular scaffolds to their tips (to harness and localize actin polymerization),
coupled to the coordinated organization of actin filaments into lamella
networks and bundled arrays. Their turnover requires further molecular
complexes for the disassembly and recycling of lamellipodium components.
Here, we give a spatial inventory of the many molecular players in this dynamic
domain of the actin cytoskeleton in order to highlight the open questions and
the challenges ahead.
A supplementary movie
is available at:
http://archive.bmn.com/
supp/tcb/small.avi
J. Victor Small*
Emmanuel Vignal
Dept of Cell Biology,
Institute of Molecular
Biology, Austrian
Academy of Sciences,
Billrothstrasse 11,
Salzburg, 5020, Austria.
*e-mail: jvsmall@
imb.oeaw.ac.at
Theresia Stradal
Klemens Rottner
GBF, National Research
Center for Biotechnology,
Dept for Cell Biology,
Mascheroder Weg 1,
D-38124 Braunschweig,
Germany.
Thirty years ago, when concepts of non-muscle cell
structure were rudimentary, Abercrombie identified
the thin layer of cytoplasm (~0.2 µm thick) that
protrudes at the front of spreading and migrating
cells as the primary ‘organelle’ of motility. When such
protrusions were parallel to the substrate, he referred
to them as the ‘leading lamella’, the ‘leading edge’ or
the ‘lamellipodium’ (Fig. 1); when they curled
upwards, he referred to them as ‘ruffles’ [1].
Subsequent studies over the next two decades [2]
revealed the presence of concentrated arrays of polar
actin filaments in lamellipodia and demonstrated
that protrusion was based on actin polymerization.
Experiments in which fluorescent actin was injected
into fibroblasts showed that lamellipodia were, in
fact, the primary sites of actin incorporation [3],
marking them as the major ‘filament factory’ of the
cell [4]. Alongside their protrusive activity,
lamellipodia serve other important roles. They are
involved in the development of adhesions to the
substrate and, as ruffles, serve in macropinocytosis
and phagocytosis. They must therefore recruit all the
components required for these functions. Also,
adhesion itself entails reorganization of
lamellipodium filaments, leading to the development
of different classes of adhesion complexes.
As far as motility is concerned, interest currently
focuses on how actin polymerization is localized and
controlled. Because lamellipodia are not easily
isolated for biochemical analysis, ideas on this front
first developed from in vitro studies of actin
polymerization and from the characterization of the
proteins recruited by pathogens to enable their
movement in cytoplasm [5,6]. From these studies, the
Arp2/3 complex has emerged as an important player
in the initiation of actin polymerization for actinbased pathogen motility [5,6], and other findings
support a role for Arp2/3 in lamellipodium protrusion
[7,8]. However, the Arp2/3 complex is just one player
http://tcb.trends.com
among many implicated in initiating, organizing and
disassembling the lamellipodium network. More
recent progress in characterizing other players has
come in part from the use of green-fluorescent protein
(GFP) to tag putative components, combined with
live-cell microscopy to localize them in vivo. This
approach, which has rapidly gained in importance, is
particularly relevant to the question of lamellipodium
organization because chemical fixation can easily
lead to the loss of resident components and, under
inappropriate conditions, to the gross distortion of
lamellipodium structure; unfortunately, this is
common in published pictures. Here, we attempt to
produce a current molecular inventory of
(a)
GFP–actin
*
0
(b)
*
68
(c)
GFP–VASP
(d)
Actin
TRENDS in Cell Biology
Fig. 1. (a,b) The lamellipodium and associated microspikes seen
in two video frames of a B16 mouse melanoma cell expressing
green-fluorescent protein (GFP) fused to actin (see supplementary
video at: http://archive.bmn.com/supp/tcb/small.avi). In addition to the
forward translation of the lamellipodium, there is a lateral motion of
microspikes, indicated by the asterisks: two microspikes (a) fuse into
one (b). The numbers indicate time in seconds. (c,d) The localization of
VASP at the tips of protruding lamellipodia. (c) The last video frame of a
living cell expressing GFP–VASP before fixation with glutaraldehyde.
(d) The fixed cell after labeling of actin with phalloidin. Bar, 5 µm.
0962-8924/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(01)02237-1
Review
TRENDS in Cell Biology Vol.12 No.3 March 2002
D
D
A
E
E
Microspike
C
Filopodium
Lamellipodium
B
F
TRENDS in Cell Biology
Fig. 2. Schematic representation of subdomains in lamellipodia and
filopodia: (A) tip of lamellipodium; (B) actin meshwork; (C) region of
major disassembly; (D) tip of filopodium; (E) bundle; (F) undegraded
filament that contributes to the cytoplasmic network. According to
localization studies, zones B and C overlap considerably, but the activity
of disassembly increases towards the base of the lamellipodium.
lamellipodia, with the aim of highlighting their
subdomains and composition (Figs 2–4) and to discuss
the functional implications and open questions.
However, first, a note on nomenclature. Depending
on cell type and condition, the lamellipodium can vary
in breadth from ~1 µm to 5 µm and can exhibit highly
variable numbers of radiating bundles 0.1–0.2 µm in
diameter and many micrometers long. When
contained within the breadth of the lamellipodium,
the bundles have often been referred to as ‘ribs’ and,
when they extend beyond the edge of the lamellipodium,
as either ‘microspikes’ or ‘filopodia’. Here, we use
‘microspikes’ (rather than ribs) [9] to describe bundles
that do not project beyond the cell edge and ‘filopodia’
when they do. According to this nomenclature,
microspikes are part of the lamellipodium and can be
potential precursors of filopodia. The term ‘cortical
actin’, often misused to describe lamellipodium
networks, will be reserved for actin-associated
complexes at the cell membrane, involving proteins
such as spectrin, dystrophin and ezrin.
Lamellipodium tip engages protein complexes to drive
actin polymerization
Pathogens that usurp the machinery of the cell to
move in cytoplasm do so by recruiting to their
surface the complexes involved in driving actin
polymerization [6] (Table 1). A growing body of
evidence indicates that the tips of lamellipodia and
filopodia serve an analogous function of localizing and
harnessing actin polymerization for cell motility.
This was highlighted by studies of the dynamics of
GFP-tagged vasodilator-stimulated phosphoprotein
(VASP; a member of the Ena/VASP family of proteins)
in melanoma cells. VASP, which binds to the surface
protein ActA of Listeria [6], was found to accumulate
at the tips of lamellipodia and filopodia (Fig. 1),
corresponding to the sites where the fast-growing
ends of the actin filaments abut the cell membrane.
The amounts of VASP recruited to lamellipodium tips
increased with the protrusion rate [10], pointing to a
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113
positive role of Ena/VASP proteins for actin assembly.
Such a role for VASP was demonstrated for the
actin-based motility of Listeria [11] and for
phagocytosis [12]. The apparent incompatibility of
these findings with the increased motility of cells
lacking Ena/VASP proteins [13] might be explained
by changes in their ruffling and adhesion dynamics,
or by a combination of these, leading to more efficient
net translocation. Nevertheless, VASP represents the
first of a growing list of proteins marking
lamellipodium tips as sites of assembly of protein
complexes engaged in driving and regulating actin
polymerization (Fig. 3, Table 1).
Actin-driven pathogen motility involves the
activation of the Arp2/3 complex at the pathogen
surface, but different pathogens recruit alternative
combinations of molecular adaptors to achieve this
[6]. Thus, ActA of Listeria can activate Arp2/3 directly,
whereas Shigella and Vaccinia virus recruit the
Wiskott–Aldrich-syndrome protein family member
N-WASP to activate Arp2/3. For vertebrate cells,
another family member (Scar/WAVE) has been
implicated in activating Arp2/3 in lamellipodium
formation [8,14]; this is supported by the localization
of Scar/WAVE1 at lamellipodium tips [15,16].
Scar/WAVE proteins interact directly with the
Abelson tyrosine kinase, c-Abl [17], which has
previously been implicated in actin dynamics,
suggesting that Scar/WAVE proteins might recruit
this kinase to lamellipodia. In addition to Scar/WAVEproteins, c-Abl also interacts with a protein family
termed Abi proteins (Abl-interacting proteins), which
localize exclusively to the tips of lamellipodia and
filopodia [18]. Because fibroblasts deficient in Abl
and its relative Arg (Abl-related gene) can still form
lamellipodia [19], c-Abl is not essential but might
serve in the modulation of lamellipodium protrusion.
This would be in line with findings implicating this
kinase in cell motility [19,20]. Another protein that
accumulates at the surface of Listeria and at the tips
of lamellipodia and filopodia is profilin [21], which is
known to enhance the treadmilling of actin filaments
in vitro by shuttling monomeric actin to the barbed
ends of actin filaments [5]. Its localization here is thus
consistent with bulk addition of actin monomer close
to the membrane [22].
Signaling at the tip through Rho GTPases
The assembly of actin-based membrane projections is
regulated by small GTPases of the Rho family [23,24].
Two members of this family, Rac1 and Cdc42,
signal the formation of lamellipodia and filopodia,
respectively [25]. The activation of Rac and Cdc42 can
be mediated by stimulation of both growth factor [23]
and integrin [26] receptors and requires GDP–GTP
exchange factors (GEFs), many of which have been
described [27,28]. Rho GTPases are synthesized as
cytosolic proteins but can be targeted to membranes
by a series of posttranslational modifications [29].
General membrane localization cannot explain the
114
Review
TRENDS in Cell Biology Vol.12 No.3 March 2002
focal induction of lamellipodia or filopodia at the cell
periphery, and so it is tempting to speculate that Rac
and Cdc42 might be locally activated to induce these
protrusions. Indeed, a fluorescence resonance energy
transfer (FRET) approach to visualizing GTP-bound
Fig. 3. The locations
of molecules and
complexes in the zones
corresponding to those
depicted in Fig. 2: (a) tip of
lamellipodium, (b) actin
meshwork, (c) region of
major disassembly,
(d) tip of filopodium,
and (e) bundle.
(a)
?
Rac1 in live cells recently revealed an accumulation of
the activated state of this GTPase in membrane
ruffles upon growth factor stimulation [30].
Upon ligand binding, growth-factor receptors can
activate phosphoinositide 3-kinases, a product of
(d)
?
P
?
?
?
Cargo
(b)
(e)
Cargo
Cargo
Key:
(c)
Cargo
F-Actin
Myosin X
Myosin I
Profilin
Profilactin
Arp2/3
Myosin VI
ADF Cofilin
Scar/WAVE
c-Abl
Capping
protein (gelsolin)
Cortactin
GEF
Filamin
Abp1p
Cdc42/Rac
α-Actinin
Aip
Receptor
Fascin
Ena/VASP
Adhesion
receptors
Fimbrin
Abi
Irsp53
Protein
(unknown)
Unknown protein with
phosphotyrosine
P
TRENDS in Cell Biology
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Review
TRENDS in Cell Biology Vol.12 No.3 March 2002
which – phosphatidylinositol (3,4,5)-trisphosphate –
in turn activates GEFs such as Vav [31] and Sos [28].
Interestingly, lipid products of phosphoinositide
3-kinases [such as phosphatidylinositol
(3,4,5)-trisphosphate] accumulate in a polarized way
at the protruding membranes of chemotactic
leukocytes and are thought to contribute to the
spatial activation of Rho GTPases [32]. It is therefore
an exciting question whether GEFs are present at the
sites of actin assembly or whether the Rho GTPases
are recruited to these sites after being activated by
GEFs elsewhere in the cell. An indication that the
first of these is true comes from the recent
demonstration that Vav-1, a GEF for both Rac and
Cdc42, is recruited to the tips of filopodia [33].
Recently, potential pathways for the transduction
of signals from active Rac and Cdc42 to actin
polymerization into lamellipodia and filopodia have
been uncovered. Of the many effector proteins that
interact specifically with GTP–Cdc42, only the
haematopoietic Wiskott–Aldrich-syndrome protein
(WASP) and its ubiquitous family member
N-WASP provide a direct link to actin assembly
through activation of the nucleating activity of the
Arp2/3 complex. In vitro, phosphatidylinositol
(4,5)-bisphosphate and GTP–Cdc42 can activate
N-WASP in a cooperative manner, and it has
therefore been proposed that N-WASP could, upon
recruitment to and activation at the membrane, effect
the protrusion of filopodia and/or lamellipodia [5].
However, recent studies of cells derived from
N-WASP-knockout models demonstrated that
N-WASP is not essential for Cdc42-based
filopodium formation [34,35] and therefore call for a
revision of current models of N-WASP function in
actin assembly [5,20].
As opposed to the direct interaction of N-WASP and
Cdc42, Scar/WAVE (which transduces Rac-mediated
lamellipodium formation via the Arp2/3 complex)
cannot bind to Rac directly [14]. A search for the link
between Rac and Scar/WAVE led to the identification
of the insulin receptor substrate Irsp53 (also known
as IRS-58) [36]. This adaptor protein links activated
Rac and Scar/WAVE to induce lamellipodia [36] but is
recruited to the tips of both lamellipodia and filopodia
(H. Nakagawa and J.V. Small, unpublished). In line
with the additional localization of Irsp53 at filopodium
tips, overproduction of this protein has been found to
induce the formation of Cdc42-based filopodia [37].
More recently, a direct interaction of Irsp53 with
Cdc42 was attributed to a partial Cdc42/Rac
interactive binding (CRIB) motif, and filopodium
formation was proposed to involve binding of Irsp53
to the Ena/VASP-family protein Mena [38]. Together,
these results led to the proposal of a novel,
Arp2/3-independent, pathway for filopodium
induction, which would be consistent with the
findings from N-WASP-defective fibroblasts [34].
Another signaling pathway implicated in
lamellipodium formation involves the p21-activated
http://tcb.trends.com
115
kinase (PAK) protein family. These serine/threonine
kinases were identified as direct downstream
effectors of Rac and Cdc42. PAKs are engaged in
multiple signaling pathways, some of which might be
coupled directly to lamellipodium protrusion. For
instance, PAK interaction with Cdc42/Rac increases
the levels of phosphorylated myosin light chain
(MLC) thought to be required for the anchorage of
lamellipodia. In addition, PAKs were shown more
recently to activate Lim kinases to phosphorylate and
thereby block the severing/depolymerizing activity of
cofilin, which is proposed to effect lamellipodium
turnover [39,40].
Forming and stabilizing the actin network
Actin polymerization at the lamellipodium tip must
be tightly coupled to the establishment of molecular
linkages that constrain the generated actin filaments
within a membrane sheet, through filament–filament
and filament–membrane interactions. Emphasis has
recently been placed on the possible role of the Arp2/3
complex in initiating and structuring actin networks.
In vitro experiments have shown that Arp2/3 can
promote the branching of actin filaments, but
conflicting models have been proposed for how this
occurs [41,42]. Nevertheless, evidence for the in vivo
relevance of filament branching by Arp2/3 has been
extracted from appealing images of lamellipodium
meshworks prepared for electron microscopy by an
improved critical-point drying method [43].
Accordingly, a dendritic branching model of
actin-based protrusion has been widely accepted to
explain cell motility [43,44]. Although it is attractive,
this model still requires rigorous testing, especially by
the use of alternative methods of electron microscopy
to re-evaluate the existence and frequency of filament
branching in lamellipodia.
Two of the proteins shown to bind to and activate
the Arp2/3 complex in vitro, cortactin and Abp1, also
co-distribute with Arp2/3 across the lamellipodium.
Because cortactin can activate Arp2/3 when bound to
F-actin and inhibits debranching of in vitro
Arp2/3–actin complexes, it has been suggested to
serve as a stabilizer of the putative actin filament
branches in the lamellipodium [45]. Cortactin is also
found along the length of actin comet tails of
pathogens, where it might play a similar role in
network stabilization [46]. As a potential receptor
linker, cortactin might couple actin flow to receptors
on the surface of the lamellipodium [47]. Abp1 has
similar properties to cortactin, and complementary
data from yeast and mammalian cells suggest
that it might link actin polymerization with
endocytosis [48,49]. The localization of the related
protein drebrin in the lamellipodium and in close
proximity to the plasma membrane [50] is consistent
with such a role.
Other candidates for actin network stabilization
are the classical actin crosslinking proteins filamin
and α-actinin. A structural role for filamin in
116
Arp2/3 complex
Arp2
Arp3
3
1
2
5 4
CH1CH2
(a)
TRENDS in Cell Biology Vol.12 No.3 March 2002
CH1CH2
Fig. 4. Domain
organization of proteins
in lamellipodia and
filopodia. (a) Actinbinding and -remodeling
proteins. (b) Modular
structure of signaling
proteins. (c) Modular
structure of myosin
motors.
Review
Filamin
ABP - 280
FLNa
Fascin
ABD
Fimbrin
EF
EF
CH1
CH2
Gelsolin
G1
G2
G3
G4
G5
G6
α-Actinin
CH1
EF
CH2
EF
Cortactin
Ac.
Spc.
Spc.
Spc.
EF
Spc.
Spc.
Spc.
Spc.
CH1
Helix
PPPP
SH3
ADF-H/C
EF
CH2
XAip1
Coronin
WD
(b)
Spc.
WD
WD
WD
WD
WD
C.coil
WD
WD
Abp1p
WD
WD
WD
WD
WD
WD
Ena/VASP
ADF-H/C
Ac. Ac.
Helix
PPPP
EVH1
SH3
PPPP
EVH2
IRSp53
Scar/WAVE
SHD
Basic
Abi proteins
Abi N. R.
c-Abl
SH3
GR
PPPP
WH2
HHR
SH2
ADF-H/C
PPPP
Tyr kinase
PPPP
RacBD
Ac.
Vav
CH
SH3
DNA
G
CdcBD
Ac.
DH
SH3
PH
SH3
SH2
SH3
F
Eps8
PTB
PPPP
PPPP
SH3
PPPP
Effector domain
(c)
Myosin X
IQ
IQ
C.Coil
C.Coil
PEST
PEST
PH
PH
PH
PH
PH
PH
MyTH4
MyTH4
FERM
FERM
Myosin V
Myosin VI
IQ
C.Coil
IQ
C.Coil
+/–
+/–
IQ
IQ
C.Coil
C.Coil
C.coil
C.coil
C.coil
C.coil
C.coil
C.coil
Myosin VII
IQ
IQ
Key:
C.Coil
C.Coil
MyTH4
MyTH4
FERM
FERM
SH3
SH3
PTB
CdcBD
RacBD
EVH1
EVH2
SHD
WH2
SH2
SH3
PPPP
PH
Protein tyrosine binding domain
Cdc42 binding domain
Rac binding domain
Ena/VASP homology domain 1
Ena/VASP homology domain 2
Scar/WAVE homology domain
WASP homology domain 2
Src homology 2 domain
Src homology 3 domain
Proline-rich region
Pleckstrin-homology domain
DH
WD
GR
Basic
Ac.
F
G
Dbl-homology domain
Tryptophan aspartate repeat
Glutamine-rich domain
Basic sequence
Acidic sequence
F-actin binding site
G-actin binding site
ADF-H/C
MyTH4
MyTH4
FERM
FERM
Effector domain
DNA
HHR
Myosin I
IQ
Basic
Actin and SOS binding
DNA-binding domain
Homeobox homology region
PEST
Target for calpain and other proteases
IQ
Calmodulin-binding motif
EF
FERM
Spc.
G1
Calcium-binding EF hand domain
Ezrin/radixin/moesin homology domain
Spectrin-homology domain
Gelsolin repeats
Ig-like domain
Calponin-homology domain
CH
Abi N. R.
MyTH4
Abi N-terminal region
Myosin tail homology 4 domain
C.coil
Coiled coil (dimerization) region
Helix
Helix region
Tyr kinase
Tyr kinase domain
Myosin motor domain
ADF/cofilin homology region
TRENDS in Cell Biology
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Review
TRENDS in Cell Biology Vol.12 No.3 March 2002
117
Table 1. Comparative localization of proteins involved in actin-based motility
Protein
Lam.
Fil.
Pathogen Putative functions
Scar/WAVE
Profilin IIa
Tip
Tip
Tip
Tip
n.d.a
Pole
Ena/VASP
Tip
Tip
Pole
Irsp53
Abi
c-Abl
Vav-1
Arp 2/3 complex
Tip
Tip
Tip
–
Tip and
Mesh.
Mesh.
Mesh.
Mesh.
Tip
Tip
n.d.
Tip
–
n.d.
n.d.
n.d.
n.d.
Tail
n.d.
n.d.
n.d.
Tail
Tail
Tail
Coronin
Cofilin
Cortactin
Capping proteins (CapZ) Mesh.
Fascin
Mesh.
n.d.
Tail
Microspike n.d.
Fimbrin (Plastin)
Mesh.
Microspike Tail
Talin
Filamin
Front zone Tip region Tail
Mesh.
Microspike Tail
α-Actinin
Mesh.
Microspike Tail
Selected binding partners
Activates Arp2/3.
Also in focal adhesions. Shuttles actin
monomers onto filaments.
Also on stress fibers and in focal adhesions
Modulate actin polymerization.
Links Rho GTPases to effector proteins.
Adaptor protein engaged in multiple complexes.
Tyrosine kinase that modulates actin protrusions.
GEF for Rho GTPases.
Nucleates and branches actin filaments.
Arp2/3 complex, G-actin
WASp/Scar, Ena/VASP, G-actin,
Arp2/3 complex, dynamin
Zyxin, vinculin, profilin, F-actin,
c-Abl
Rac1, Cdc42, Scar/WAVE, Mena
Abl, Arg, Eps8, Sos, Nap
Abi, Ena/VASP, Scar/WAVE
Rac1, Cdc42, Rho, Nck, Ack, Fyn
WASp/Scar cortactin, Abp1,
profilin, actin
Promotes actin polymerization.
F-actin
Severs and depolymerizes actin filaments.
F-actin, G-actin, Aip, Lim kinase
c-Src substrate.
F-actin, Arp2/3, dynamin, c-Src,
Stabilizes Arp2/3-induced actin filament network. shank-2
Block growth from actin filament barbed ends.
F-actin
Regulated by phosphorylation.
F-actin
Bundles actin filaments.
F-actin, Ca2+
Also found in microvilli. Ca2+ sensitive.
Bundles actin filaments.
Links receptors to the actin cytoskeleton.
Layilin (in lamellipodium)
Stabilizes actin filament meshworks.
Small GTPases, integrins, RalA,
Trio
Bundles actin filaments, links receptors to
Integrins, F-actin, Ca2+, PIP2
actin cytoskeleton.
Refs
[15,16]
[21]
[10,13,
16]
[36] –b
[18]
–c
[33]
[8,43]
[62,60]
[62]
[46,47]
[62]
[65,66]
[69]
[98,99]
[98]
[56]
aAbbreviations: Lam., lamellipodium; Fil., filopodium; Mesh., lamellipodial actin meshwork; n.d.: not determined; (–), not localized; PIP2, phosphatidylinositol
(4,5)-bisphosphate.
bH. Nakagawa and J.V. Small, unpublished.
cP. Hahne and J.V. Small, unpublished.
lamellipodia is supported by findings with cells of a
human line deficient in the filamin isoform FLNa,
which spread poorly and bleb actively at their edges
but revert to normal morphology on transfection with
FLNa cDNA [51]. Recent studies reconfirm the
localization of filamin in the actin filament network of
lamellipodia and raise the question of the relative
contributions of filamin and Arp2/3 in network
formation and stabilization [52]. In addition to
binding to F-actin, filamin can associate with
transmembrane proteins through its C-terminal
region [53,54]. Thus, filamin could serve as a linker
between the membrane and the cytoskeleton to
recruit signaling proteins to the vicinity of sites of
actin polymerization and remodeling. The reported
association of filamin with the Rho GEF Trio [55] and
small GTPases [53] supports an involvement of
filamin as a docking site for signaling molecules,
although the significance is unclear. α-Actinin
crosslinks actin filaments into bundles and networks
in vitro and localizes throughout the lamellipodium
[56]. Different isoforms are partially segregated
between different actin compartments, with actinin 1
and actinin 4 in ruffles [57]. α-Actinin-null cells of
Dictyostelium show no motility defects except in a
null background of the filamin homolog ABP120 [58],
suggesting structural complementation between
these crosslinkers in the lamellipodium. In synthetic
comet tails of actin [11], a lack of α-actinin results in a
less compacted tail, supporting a crosslinking
function for this protein.
http://tcb.trends.com
Coronin, an actin-binding and crosslinking
protein, is similarly homogeneously distributed in
lamellipodia, and its deletion in Dictyostelium leads
to decreased motility and impaired cytokinesis [59].
Xenopus coronin remains bound to fibroblast
cytoskeletons after Triton extraction, and coronin
overproduction amplifies lamellipodium formation
[60]. The nature of the interplay between coronin and
other actin-binding proteins is unknown, but the
β-propeller-forming WD domains in coronin could
mediate interactions with such partners [61].
Coronin is present throughout the Listeria actin tail
[62], consistent with a membrane-independent
structural function.
Microspikes and filopodia
According to antibody labeling [43], Arp2/3 is
excluded from filopodia and microspikes. This
situation might reflect the elongation of pre-existing
filaments in filopodia during protrusion [63] with no
new filament generation, as in lamellipodia.
Microspikes and filopodia are probably generated by
bundling of lamellipodium filaments; fascin and
fimbrin (plastin), which both bundle actin filaments
in vitro, have been implicated in this process [64].
Fascin is a ubiquitous protein involved in stabilizing
actin bundles in prominent cellular processes,
including stereocilia and hair bristles [64], where
additional crosslinkers cooperate in bundling.
Bundling of actin by fascin is inhibited by serine
phosphorylation in vitro [65] and also in vivo [66],
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TRENDS in Cell Biology Vol.12 No.3 March 2002
but the details remain to be clarified [67]. An
additional regulatory pathway of bundle formation is
suggested by the inhibition of the actin binding of
fascin by drebrin [68]. Bundling by fimbrin might
be regulated by calcium or phosphorylation, but
we must admit that almost nothing is known
about the way filopodia are assembled and
disassembled in vivo.
However, because fascin and fimbrin are found not
only in microspike bundles but also in the intervening
lamellipodium network [66,69], we assume that
they exist in these two locations in dormant and
active states. A further interesting aspect is the
often-observed rapid lateral mobility of microspikes
and filopodia (see supplementary video at:
http://archive.bmn.com/supp/tcb/small.avi). How does
this occur? A simple explanation is provided by the
geometry of the lamellipodium network, which
suggests that there is a lateral flow of filaments
during protrusion [70]. Taking this idea one step
further, the rate of lateral movement of bundles in
neuronal growth cones increases with their angle to
the lamellipodium front [71]. From correlated
measurements of retrograde flow rate, the lateral
movement could readily be explained by the extension
of bundles by polymerization at the tip, whereby the
polymerization rate increased with angle [71].
An important feature distinguishing microspikes
and filopodia from lamellipodia is the difference in the
complement of proteins at their tips. For example,
microspike and filopodium tips harbor an unknown
protein that is heavily phosphorylated [72], lack
Scar/WAVE-1 [15] and selectively recruit Vav [33].
Further characterization of the proteins specifically
resident at filopodium tips should contribute
to a clarification of the targets downstream of
Cdc42 that specify filament bundling and
filopodium protrusion.
Lamellipodium disassembly
In a steadily migrating lamellipodium, the
actin meshwork remains essentially constant in
breadth (Fig. 1 and supplementary video at:
http://archive.bmn.com/supp/tcb/small.avi),
indicating a balance between assembly at the front
and disassembly at the rear. Protrusion and
retraction rates can be regulated at the level of actin
assembly, apparently through the recruitment or
dissociation of regulatory scaffolds [10,15,18].
Disassembly is thought to be achieved by proteins of
the ADF/cofilin family [73] and possibly severing
proteins like gelsolin, probably in cooperation with
factors that break filament crosslinks [5]. These ideas
stem more or less entirely from in vitro data but
receive circumstantial support from localization studies.
Depolymerization of actin by cofilin is inhibited
through phosphorylation by LIM kinase [73] and
enhanced by the actin-interacting protein Aip1 [74].
Cofilin localizes throughout the lamellipodium in
Dictyostelium [75] and in fibroblasts [43]. This
http://tcb.trends.com
suggests that depolymerization is not restricted to
the rear of the lamellipodium [43], consistent with a
graded distribution of filament lengths from front
to rear [76].
So that unproductive actin polymerization away
from the cell edge is avoided, it has been suggested
that any fast-growing free filaments ends that might
be exposed in the lamellipodium network (by severing,
for example) are excluded from the polymerization
pool through ‘capping’ by the actin filament capping
protein [5]. This suggestion is consistent with the
presence of capping protein in lamellipodia and
membrane ruffles [77]. For the in vitro propulsion of
Listeria or Shigella [11], capping protein has been
successfully used to limit the polymerization of actin
to the pathogen surface. Also, in this assay, capping
protein can be functionally replaced by gelsolin,
implying that gelsolin family members might
play a complementary capping role in lamellipodia
[78]. However, is such a role required? It must be
admitted that the problems of free plus-ends of
actin filaments away from the cell edge is most
simply solved if they do not exist at all. Indeed,
their existence in lamellipodia has yet to be
convincingly demonstrated.
Shunting to the front with myosin motors
Several non-filament-forming members of the myosin
family have been localized in lamellipodia and
filopodia, following the first observation of myosin I
in Dictyostelium [79]. Myosin is not required for
Listeria motility in vitro [11], suggesting a specific
need for myosin-linked processes in the membrane
leaflet environment of the lamellipodium. In addition
to myosin I, myosins V and VI [80], VII [81], and X
[82] localize to lamellipodia and membrane ruffles.
Myosin I proteins in both budding yeast and
Dictyostelium bind through their Src-homology 3
(SH3) domains, directly or indirectly, with Arp2/3 and
other components of the actin polymerization
machinery [83–86], lending support to the idea that
these myosins might act by carrying cargo to the
plus-ends of actin filaments, thus acting as
cofactors in protrusion. Both myosin V and
myosin VI localize to the lamellipodia of human
carcinoma cells after stimulation with epidermal
growth factor [80]. Myosin V has been generally
implicated in vesicle transport [87], but a role
in protrusion has also been suggested by an
antibody-linked approach to disable it through
chromophore-assisted laser inactivation (CALI) [88].
Significantly, myosin VI moves in a direction on
actin that is opposite to that of all other known
myosins [89], raising interesting questions about its
function. Because there is rapid retrograde flow in
lamellipodia linked to actin treadmilling, presumably
potentiated by plus-end-directed myosin [90], there
seems to be little need for a retrograde myosin motor
for transport in lamellipodia. An alternative role for
myosin VI could be organizing actin filaments [91],
Review
Acknowledgements
Our work was supported
by funds from the
Austrian Science
Research Council (to
J.V.S.). K.R. is the holder
of an EMBO postdoctoral
fellowship, and K.R. and
T.S. thank J. Wehland for
allowing time to
contribute to this article.
We thank H. Nakagawa
and P. Hahne for allowing
us to cite unpublished
work.
TRENDS in Cell Biology Vol.12 No.3 March 2002
perhaps as a cofactor in ‘zipping’ filaments together
from the tip in the generation of filopodia.
Myosin X localizes to lamellipodia and to the
tips of filopodia in epithelial MDCK cells [82], and
myosin VII, its close relative in Dictyostelium, is
found in lamellipodia, filopodia and phagocytic cups
[81]. Deletion of the gene encoding myosin VII in
Dictyostelium leads to inhibition of filopodium
formation and decreased substrate adhesion.
Taken together with the presence of FERM
domains in the myosin tail (which can bind to
transmembrane proteins), myosin VII has been
attributed a role in the assembly and disassembly
of adhesion proteins at the plasma membrane [81].
We suggest that some adhesion proteins are
incorporated into adhesion sites by first targeting
lamellipodia and filopodium tips, through myosin.
Subsequently, complex formation and linkage to
retrograde flow could transport these components to
the base of lamellipodia and filopodia, to initiate
adhesion. This route is suggested by the dual
localization of proteins such as VASP [10], talin
[92] and integrin α6β1 [93] at or towards [92] the
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