1. REVIEW
The many faces of actin: matching assembly factors
with cellular structures
Ekta Seth Chhabra and Henry N. Higgs
Actin filaments are major components of at least 15 distinct structures in metazoan cells. These filaments assemble from a
common pool of actin monomers, but do so at different times and places, and in response to different stimuli. All of these
structures require actin-filament assembly factors. To date, many assembly factors have been identified, including Arp2/3
complex, multiple formin isoforms and spire. Now, a major task is to figure out which factors assemble which actin-based
structures. Here, we focus on structures at the plasma membrane, including both sheet-like protrusive structures (such as
lamellipodia and ruffles) and finger-like protrusions (such as filopodia and microvilli). Insights gained from studies of adherens
junctions and the immunological synapse are also considered.
To the best of our knowledge, actin polymerizes into one type of struc- we divide these protrusive structures into those that are sheet-like (lamel-
ture in eukaryotic cells: helical filaments. However, these filaments can lipodia/lamella, ruffles, phagocytic cups, podosomes and invadopodia)
be assembled further into a wide variety of higher-order cellular struc- and those that are finger-like (filopodia and microvilli). We also discuss
tures ranging from lamellipodia to microvilli (Fig. 1), each of which two other structures, the immunological synapse and adherens junc-
performs specific functions. We ask one basic question in this review: tions, because of unique issues they raise, particularly concerning links
how are filaments assembled for these structures? to the microtubule cytoskeleton. Space limitations prevent us from dis-
Actin filament nucleation is unfavourable; therefore, nucleation cussing the many other metazoan actin-based structures (for example,
factors are required to initiate assembly of any actin-based structure1. stress fibres, endocytic pits, peri-organellar actin, the cytokinetic ring,
At present, there are three known classes of nucleation factor: Arp2/3 cortical spectrin-actin and nuclear actin).
complex, formin proteins and spire. As many of these factors function
in multiple steps during assembly of actin-based structures, we prefer Kickstarting polymerization: actin assembly factors
the term ‘assembly factor’. Identifying the assembly factor for a given Actin monomers self-assemble into helical filaments (Fig. 2a)1,3. The
actin-based structure is a key step in determining assembly mechanisms, initial ‘nucleation phase’ (assembly of dimeric and trimeric complexes)
and has been most thoroughly investigated in yeast2. Here, we focus on is highly unfavourable and slow but, once overcome, filament elonga-
actin-based structures in metazoan cells, which are more complex in tion is much more rapid. As all actin subunits face the same direction,
several respects: first, they contain more known actin-based structures; the filament is polar, with a ‘barbed’ and a ‘pointed’ end. Monomers can
second, these structures often overlap spatially, making isolated analysis add to or depolymerize from either end, but both processes are more
more difficult; and third, metazoans have more known assembly factor than tenfold faster at the barbed end. In metazoan non-muscle cells,
isoforms (18 in mammals versus three or four in yeasts). the action of profilin – which adds actin monomers to the barbed end
Once nucleated, actin filaments can have several fates. It is the pres- only — enhances this effect and effectively limits elongation to barbed
ence of additional regulators that determines which type of actin struc- ends. Most actin-based structures maintain appreciable flux of actin
ture will form. In the absence of other factors, filaments are capped by monomers through the filament, with monomers adding at barbed ends
abundant barbed-end capping proteins, preventing further elongation. and depolymerizing at pointed ends4–7.
Inhibition of capping is essential for structures requiring longer filaments. The first actin assembly factor to be identified was the Arp2/3 com-
Furthermore, mature structures often require filament crosslinking into plex, composed of seven polypeptides (ARPC 1–5, Arp2 and Arp3).
networks, parallel bundling or interaction with membranes. Although Arp2 and Arp3 are actin-related proteins, and are postulated to mimic an
we do not discuss in detail the proteins mediating these processes, both actin dimer that can elongate towards the barbed end. The Arp2–Arp3
Arp2/3 complex and formins can perform these functions (Fig. 2). dimer thereby serves as a nucleation site, and the complex remains at
Instead, we focus on the relationship between specific assembly factors the pointed end of the filament8,9. In addition, the Arp2/3 complex binds
and protrusive actin-based structures at the plasma membrane. Broadly, to the side of pre-existing actin filaments, and its nucleation activity
Ekta Seth Chhabra and Henry N. Higgs are in the Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
e-mail: henry.n.higgs@dartmouth.edu
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2. REVIEW
a
Filopodium
Lamellipodium
Ruffle
Lamellum
Microvillus
Cortical actin
Podosome
Endosome
Phagocytic cup Stress fibre
Endocytic pit
Cadherin-based
Golgi-associated actin
adherens junction
Nuclear actin
b Dorsal ruffle
N
Peripheral ruffle
Lamellipodium Podosome
N
Substratum Invadopodium
Lamellum
Figure 1 Actin-based structures in metazoan cells. The helical actin ruffles are distinct structures; podosomes and invadopodia are ventral;
filament is used in a myriad of cellular processes. (a) A hypothetical lamellipodia are weakly adherent to the substratum. As far as we know,
metazoan cell, migrating upwards and attached to a second cell on all these structures use the same basic helical actin filament. This is by
the right. Cellular structures known or strongly suspected to contain no means a complete inventory of metazoan actin-based structures, and
actin filaments are indicated. (b) A side view of a cell, emphasizing we strongly suspect filaments to be involved in other, as yet unidentified
several points: ruffles are dorsal structures; peripheral ruffles and dorsal processes. N, nucleus.
thereby produces a branched filament structure with an angle of 70° the association of profilin with the FH1 domain of formin15. Although
between the filaments. Repeated branching leads to a ‘dendritic net- all formins have these basic properties, they vary significantly in their
work’ (Fig. 2b). In this manner, the Arp2/3 complex functions not only potency14. In addition, some formins also bundle, sever or depolymer-
as a nucleation factor, but also provides structure to the network. Most ize actin filaments16–20. Most organisms have multiple formin isoforms
of the Arp2/3 complex subunits are single isoforms in all organisms, (15 in mammals, six each in Drosophila and Caenorhabditis elegans)21.
with a few exceptions in mammals10–12 and Drosophila13. The Arp2/3 It is becoming increasingly clear that formins have additional roles in
complex requires activation by one of several nucleation promoting fac- microtubule dynamics (see below)22,23. At present, the best understood
tors (NPFs), including WASp, N-WASP, Scar/WAVE1, Scar/WAVE2 and mechanism of formin regulation is through direct activation by Rho
Scar/WAVE3 in mammals8. family GTPases24.
Formins were identified more recently as a second family of actin Spire proteins are a third class of actin nucleators that were initially
assembly factors. In contrast with the Arp2/3 complex, formins are identified in Drosophila25. Spire proteins are single polypeptides charac-
single polypeptide, multidomain proteins14. All formins studied to date terized by the presence of four Wasp homology 2 (WH2) motifs, which
are dimeric, due to dimerization of their formin homology 2 (FH2) are responsible for actin nucleation26. As with formins, spire-nucleated
domain. The FH2 domain is responsible for driving actin nucleation filaments are not branched (Fig. 2b). Spire can crosslink microtubules
and formin-nucleated filaments are not branched. After nucleation, the and actin filaments27, and also inhibits actin nucleation by the formin
FH2 domain remains bound at the barbed end and moves processively cappuccino in Drosophila and its mammalian homologue, formin1 (M.
as it elongates, promoting elongation by preventing the access of capping Quinlan, University of California at San Francisco, San Francisco, CA,
proteins (Fig. 2b). Formin-mediated elongation is further enhanced by and R. D. Mullins, University of California at Los Angeles, Los Angeles,
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3. REVIEW
CA, unpublished observations). This inhibition is through bind- a
ing between KIND domain of spire and the FH2 domain of formin.
B P
Metazoans have two spire genes, whereas yeast have none.
Nucleation factor
Assembling filaments for diverse cellular functions
A schematic overview of metazoan actin-based structures is shown in Fig. 1.
b
In this section, we review what is known of the actin-filament architectures
underlying some of these structures, and how they are assembled.
Sheet-like protrusive structures. Cells extend a variety of sheet-like
structures, and we discuss three basic types: lamellipodia/lamella, ruf-
fles and phagocytic cups. The adhesive structures known as podosomes
and invadopodia are not strictly sheet-like, but require many of the same
assembly proteins as lamellipodia.
Arp 2/3 Formin Spire
Lamellipodium and lamellum. We define the lamellipodia/lamella
as surface-attached sheet-like membrane protrusions observed dur-
ing crawling cell motility and spreading. The first step in crawling
cell motility is the forward protrusion of the cell front, generally as
a thin sheet of membrane-enclosed cytoplasm1. In an elegant series
of papers, Abercrombie et al. defined the key characteristics of two
regions within this protrusive front, now known as the lamellipodium
and lamellum28–31 (see Timeline). The lamellipodium is more distal,
starting at the leading edge and extending several micrometres back.
The lamellum then takes over, extending from the lamellipodium to
the cell body. The lamellipodium is thinner (100–160 nm thick versus
>200 nm), more densely stained in electron microscopy images31, less Figure 2 Filament polymerization by actin assembly factors. (a) During actin-
adherent, seems devoid of organelles and is more dynamic. Although filament assembly from monomers, the dimerization and trimerization steps
these properties were revealed by Abercrombie’s landmark studies, are unfavourable, whereas subsequent monomer additions are much more
subsequent work better defined additional adhesive differences, with favourable. Nucleation factors overcome these unfavourable dimerization
and/or trimerization steps. Actin filaments are polar structures, with the
lamellipodia being weakly adherent, and strong adhesion beginning at barbed end (B) being the sole site of elongation in non-muscle cells. Thus,
the lamellipodia–lamella boundary32–34. physiologically relevant nucleation factors must allow barbed-end elongation.
Two breakthroughs have changed our conception of the lamellipo- (b) The Arp2/3 complex (multi-coloured) nucleates a new filament from the
side of an existing filament, causing filament branching at a 70° angle. The
dium and lamellum dramatically. The first was the discovery that den-
Arp2/3 complex remains at the branch point between the pointed end of
dritically branched filaments dominate the actin network at the extreme the new filament and the side of the existing filament. Repeated branching
leading edge35,36; the second was the observation that two distinct actin- results in the assembly of a dendritic network. The formin FH2 domain
based networks constitute the lamellipodium and lamellum37. The evo- (semi-circles) nucleates a filament, and then moves processively with
the barbed end as it elongates. Some formins can also bundle filaments.
lution of this field is a beautiful example of two basic facets of modern
Spire (circles with black connectors) nucleates a filament by stabilizing
biomedical research: the ability of technological advances to change a a longitudinal tetramer. The current model is that spire dissociates from
field; and the power of having multiple laboratories investigating over- filaments soon after nucleation.
lapping subjects.
The first finding, that dendritically branched filaments dominate lamellipodial speckles, is observed at the leading-edge plasma mem-
near the leading edge35,36, represented a major change in our view of cell brane and disappears abruptly about 1–3 µm back37, seemingly at the
motility. Before these data, the protrusive region of the cell was generally lamellipodial–lamellar boundary where the first stable adhesion to the
considered to contain orthogonally crosslinked filaments. The discov- substratum occurs34. A second population appears in a more graded
ery of the dendritic network coincided closely with the finding that the fashion, increasing in frequency with distance from the leading edge and
Arp2/3 complex could nucleate dendritically branched filaments38. Very persisting through the lamellum. The two populations differ in kinetics,
rapidly, the Arp2/3 complex was considered the dominant nucleator with lamellipodial speckles exhibiting rapid retrograde flow and lamel-
driving leading-edge protrusion. lar speckles moving more slowly; transitions between the two states are
The second finding, that two filament populations exist at the leading extremely rare. Taken together, these data suggest that two sets of actin
edge, was a direct result of technological advances and astute observa- filaments exist in the lamellipodia and lamella, which are independently
tion37. The technological advance was fluorescent speckle microscopy nucleated and disassembled.
(FSM) — the phenomenon of ‘speckle’ movement on cellular structures Similar results have been obtained using Drosophila S2 cells40, and
because of low levels of fluorophore incorporation39. Using FSM on actin further support for two independent actin networks comes from ret-
filaments in marsupial kidney epithelial cells, two speckle populations rospective examination of fixed cells. In highly motile fish or Xenopus
were observed in the protruding region. One population, referred to as keratocytes, the actin network is significantly more dense near the
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4. REVIEW
TIMELINE – History of the terms “Lamellipodia”, “Lamella” and “Ruffles”
LAMELLUM (plural, lamella) 1971
Coined in 1969 to replace In 1971, differences between lamellipodia
“ruffled membrane” for the and lamella were defined more fully by
protrusive region of a motile EM criteria, in terms of thickness, staining
fibroblast133. Also recognized density, presence of organelles, and
that only the first 3-5 microns of apparent adhesion31.
the lamellum had ruffling activity.
1958 1969 1970 1971 1980
RUFFLE LAMELLIPODIUM (plural, lamellipodia) 1980
Coined in 1958 to describe the Coined in 1970 to differentiate the highly First record of differentiation between
protruding leading edge of a motile protrusive, distal portion at the front of lamellipodia and ruffles. In Abercrombie’s
cell132. Used interchangeably the motile cell (first several microns) from much-cited review134, lamellipodia defined
with “ruffled membrane”. the rest of the lamellum22. No distinction as forward-protruding structures that adhere
made between protrusions parallel to the weakly to substratum, with ruffles being of
substratum and those that protruded similar morphology, but non-adhered and
vertically, although the term “ruffle” was often protruding dorsally.
used as well to describe these structures.
leading edge than it is farther back, and a large proportion of these dendritic branch points in these cells36, as well as to the leading edge in
filaments are labile to extraction35,36,41. Electron microscopy of fish multiple cell types10,45–48. Second, Arp2/3-complex dynamics are confined
keratocytes reveals that filaments within 1 µm of the leading edge are largely to lamellipodia in Xenopus49 and Drosophila40. In the latter case,
predominantly short and crosslinked in dendritic branches, whereas treatments that vary the width of the lamellipodium proportionally vary
most filaments farther back are longer and less dendritically branched35. the width of the Arp2/3 complex-rich region. Third, altering the activity
Electron microscopic analysis of the protrusive regions of mouse embryo of the Arp2/3 complex affects lamellipodial structures. Overexpression
fibroblasts suggests a similar organization of the two networks42. Where of central acidic (CA) or WH2 central acidic (WCA) constructs that
studied, both lamellipodial and lamellar filaments orient with barbed inhibit the Arp2/3 complex also block lamellipodia assembly10,48,50,
ends toward the leading edge plasma membrane35 and the movement although efficacy of these constructs is not universally accepted (Box 1).
of actin speckles supports this orientation37. Microinjection of antibodies that inhibit dendritic branching by the
It has also been suggested that the lamellipodium may lie on top of Arp2/3 complex inhibits lamellipodial protrusion51. Knockdown of an
the lamellum43. At the leading edge, a layer of densely packed filaments Arp2/3-complex subunit causes significant lamellipodial loss in mouse
seems to lie above a second network of less dense filaments that extends cells52 and strongly inhibits cell spreading in Drosophila cells53.
further back. The speculation is that the upper layer represents lamel- Debate continues, however, as to how universal the role of the
lipodial filaments, whereas the lower layer is lamellar (Fig. 3b). The Arp2/3 complex at the leading edge is. One RNA interference (RNAi)
rear of the lamellipodium is thought to be specified by myosin-II-based study in mouse embryonic fibroblasts argues that the Arp2/3 complex
contraction at adhesion sites, which is vital for lamellipodial disassem- is not required in lamellipodia54. Another suggests that the Arp2/3
bly. Although this model of the two networks being vertically separated complex does not localize to the leading edge in neuronal growth
needs to be tested further, there may also be support for this idea in the cones, and that overexpression of CA constructs does not alter growth
early electron microscopy work by Abercrombie31. cone morphology or slow growth cone motility48. Others have found
Based on the above findings, we conclude that two autonomous actin the Arp2/3 complex present in, and necessary for, growth-cone pro-
networks are present in the protrusive region, with the lamellipodial trusion (T. Svitkina, University of Pennsylvania, Philadelphia, PA,
filaments probably overlaying the lamellar filaments (Fig. 3a). Clearly, unpublished observations).
the proportions and characteristics of these networks might vary sub- One issue in these studies may be how efficiently the Arp2/3 complex
stantially between cell types, especially between slow-moving (for exam- is inhibited, given that it is highly abundant9. Mathematical models of
ple, fibroblasts) and fast-moving cells (for example, keratocytes). In this leading-edge protrusion suggest that the Arp2/3 complex may be in
respect, speckle analysis of actin networks in keratocytes would be highly large excess over that needed for ‘normal’ migration on experimental
informative, as so much ultrastructural information is available for these surfaces55. It would, therefore, be interesting to evaluate Arp2/3 com-
cells. The question also remains concerning the relative importance of plex-inhibited cells by FSM to determine whether the narrow band of
these networks in motility. lamellipodial speckles is completely ablated.
How are lamellipodia and lamella assembled? Most evidence suggests Less is known about the nucleators required for lamellar actin fila-
that lamellipodia are Arp2/3 complex dependent: first, there is a strong ments. The Arp2/3 complex seems largely absent from this region36,40,49,
correlation between Arp2/3-complex localization and lamellipodial and inhibiting the Arp2/3 complex or other lamellipodial proteins
dynamics. The lamellipodial network contains predominately dendritic does not alter lamellar properties40,50. The formin mDia2 is implicated
branches in several instances35,36,44 and the Arp2/3 complex localizes to in lamellar filament assembly, as well as in focal adhesion turnover
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5. REVIEW
a b
Lamellipodium Lamellum
Substratum
Weak adhesion site Stable adhesion site
Ruffle
c
Filopodium
Filopodium
Figure 3 Schematic representations of models of assembly for lamellipodia, Lamellipodial filaments may lie above lamellar filaments. At the leading
lamella, peripheral ruffles and filopodia. (a) Top view of lamellipodia and edge, lamellipodial filaments predominate, but lamellar filaments increase
lamella. Lamellipodial actin filaments (blue) are dendritically branched and in abundance further back. A lamellipodium maintaining weak attachment to
Arp2/3-complex-dependent. These filaments assemble at the leading edge the substratum is shown in the upper panel. Myosin II activity at the stable
plasma membrane (due to assembly factors bound there) and disassemble adhesion site causes retrograde flow of the lamellipodial actin network.
abruptly at a band corresponding to the first sites of stable adhesion (green), If the lamellipodial attachment with the substrate is broken, myosin II
generally 1–3 µm from the leading edge. Lamellar actin filaments (red) are activity causes this membrane sheet to move rearward as a peripheral ruffle,
in a less defined network, and are not Arp2/3 complex-dependent. They can which disassembles at the stable adhesion site (lower panel). (c) Filopodial
initiate throughout the lamellipodium and lamellum, but their frequency assembly from the lamellipodium or lamellum. Filopodial assembly from
increases as one moves back from the leading edge. Lamellar filaments the lamellipodial network through the convergent-elongation mechanism is
persist many micrometres behind the lamellipodium, and disassembly can shown on the left. Possible filopodial assembly from the lamellar network,
occur throughout this region. (b) Side views of lamellipodium and lamellum. through an as yet undefined mechanism, is shown on the right.
(C.Waterman-Storer, University of Washington, WA, unpublished imaging studies provide convincing evidence that peripheral ruffles
observations), suggesting a potential association between lamellar fila- and lamellipodia have common origins29, and recent results support this
ments and focal adhesions. However, mDia2 is not expressed in sev- hypothesis43. The Arp2/3 complex seems to be important for assembly
eral motile cell types, suggesting it may be functionally redundant with of both types of ruffle56,58. Most recently, the presence of dendritically
mDia1, mDia3 or other fomins. branched filaments in peripheral ruffles was observed59.
Formins may also function in peripheral-ruffle dynamics: first, active
Ruffles. We define ruffles as sheet-like membrane protrusions that RhoA — thought to directly activate the formins mDia1 and mDia2, but
do not attach at all to the substratum, as opposed to weakly adherent not the Arp2/3 complex — is enriched at the leading edge of multiple
lamellipodia. There are at least two distinct varieties of these transient mammalian cell types, both in lamellipodia and ruffles60–64, and is required
structures, both with half-lives of minutes. Peripheral ruffles assemble for ruffle assembly61; second, mDia1 is enriched at ruffle edges60,61,63; and
at the leading edge of motile cells, and move rearward29. Circular dorsal third, coexpression of constitutively active Rac1 and dominant-negative
ruffles — also called ‘waves’ or ‘actin ribbons’ — assemble on the dorsal RhoA allows spreading, but blocks ruffles61. This result hints at potential
surface and constrict into a circular structure before disappearing. These differences between lamellipodia and peripheral ruffles, by suggesting
seem to be distinct structures, as rearward-moving peripheral ruffles do that Rac1 activation of the Arp2/3 complex (through Scar or WAVE) is
not transition to dorsal ruffles56. Peripheral ruffles are associated with sufficient for lamellipodia, but not ruffle, generation.
crawling-cell motility only, whereas dorsal ruffles also affect receptor The proposal that the lamellipodial network lies above the lamel-
internalization and possibly macropinocytosis57. A distinction between lar network might also explain the role of mDia1 in peripheral
peripheral and dorsal ruffles was made by Abercrombie29. ruffles 43: according to this study, the rear of the lamellipodium
It is tempting to speculate that peripheral and dorsal ruffles arise is specified by assembly of a myosin II-based contractile com-
through the same mechanisms as lamellipodia. Indeed, early live-cell plex at sites of stable adhesion (Fig. 3a), and its contraction pulls
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BOX 1 TOOLS FOR REGULATING THE ARP2/3 COMPLEX: WCA AND CA CONSTRUCTS
WCA constructs refer to the minimum polypeptide sufficient for in vitro activation of the Arp2/3 complex, located at the carboxyl termini
of most nucleation-promoting factors9. WCA binds both the Arp2/3 complex and an actin monomer. Overexpression of WCA constructs
has been used to inhibit the Arp2/3 complex, on the basis that these constructs would activate the Arp2/3 complex in a mislocalized fashion,
preventing localized activation by endogenous nucleation-promoting factors.
CA constructs are shorter and only bind to the Arp2/3 complex. The CA construct of WASp competes with Arp2/3-complex activators in
vitro, blocking nucleation73. In vivo, CA constructs are predicted to sequester the Arp2/3 complex from endogenous nucleation-promoting
factors.
Reviews are mixed on the efficacy and specificity of these constructs for Arp2/3-complex inhibition in cells. Many researchers have reported
successful cellular inhibition of the Arp2/3 complex with these constructs (see main text). However, one concern with WCA is that high
expression might sequester significant amounts of actin monomer, causing additional effects that are not dependent on the Arp2/3 complex.
As for CA, some report lack of effect on cellular processes strongly believed to depend on Arp2/3 complex.
lamellipodial filaments rearward. If the extreme leading edge Podosomes and invadopodia. Podosomes are integrin-containing struc-
remains adhered to the substratum, contraction causes retrograde tures on the basal cell surface, containing an actin-rich core surrounded
flow. If, however, the connection is broken, the whole leading edge, by a ring of several actin-associated proteins and signalling proteins
including actin filaments and plasma membrane, moves rearward (Fig. 1)57,69,70. They have been studied in detail in a limited number of
as a peripheral ruffle (Fig. 3b). Support for this model can be found cells — Src-transformed fibroblasts and monocyte-derived cells (such
in the earlier studies of Abercrombie31. The lamellar network makes as macrophages, dendritic cells and osteoclasts). However, the evidence
up part of the substrate–adhesion complex34 and so may be essen- is mounting that podosomes are also present in other cell types71,72, and
tial for peripheral ruffles. Perhaps mDia formins mediate lamellar- may mediate integrin-based adhesion to extracellular matrix.
filament assembly in fibroblasts? Immunofluorescence microscopy Invadopodia have a similar architecture and protein composition to
illustrates that mDia1 localizes to the edge of spreading cells in podosomes, but are formed by cells migrating over thick extracellular
several cell types (refs 23, 63; K. Eisenmann and A. Alberts, Van matrix, and mediate matrix degradation69,70. Podosomes — which some
Andel Institute, Grand Rapids, MI, unpublished observations; have postulated to be ‘invadopodia precursors’ — are smaller (approxi-
H.N.H, unpublished observations). mately 0.5 µm diameter and 0.5 µm height), whereas invadopodia are
Our current conclusion is that lamellipodia and peripheral ruffles wider (2 µm diameter) and protrude several micrometres into the extra-
arise from the same assembly mechanism, and differ in substrate adhe- cellular matrix (Fig. 1). Both structures are ‘hot spots’ for membrane
sion. The assembly mechanism for dorsal ruffles is unclear, but probably dynamics, and invadopodia have extensive plasma membrane invagina-
involves Arp2/3 complex-generated dendritic networks. tions. Where examined, both podosomes and invadopodia are enriched
in a specific matrix metalloprotease57,69,70.
Phagocytic cups and pits. Phagocytosis is the cellular uptake of parti- All evidence points to the Arp2/3 complex as the key nucleator for
cles larger than 0.5 µm diameter. Although both types of phagocytosis both podosomes and invadopodia. The Arp2/3 complex and several of its
conducted by macrophages — Fc receptor-mediated (FcR) and com- regulators are enriched in macrophage podosomes57,69,70. Microinjection
plement receptor-mediated (CR) — require actin polymerization, they of CA constructs blocks podosome assembly in macrophages73,74. Human
differ substantially in morphology64. In FcR-mediated phagocytosis, Wiskott-Aldrich Syndrome patients, who lack WASp, do not have mac-
the macrophage membrane protrudes around the phagocytosed par- rophage and dendritic cell podosomes75,76. N-WASP is enriched at podo-
ticle. Following membrane fusion at the distal tip, the particle is then somes of non-haematopoietic cells, and is important for their assembly77.
pulled into the macrophage. In CR-mediated phagocytosis, the parti- N-WASP and the Arp2/3 complex are enriched in, and are required for,
cle ‘sinks’ into the macrophage and the macrophage does not extend invadopodia assembly78.
toward the particle. The ultrastructural details of the actin network are unclear for podo-
The morphology of the actin filaments is not known for either proc- somes and invadopodia. Clearly, the filaments are densely packed in
ess. Based on the protrusive, sheet-like nature of the phagocytic cups both, but it is uncertain whether they are dendritically branched, straight
in FcR-mediated phagocytosis, we speculate that these cups use lamel- or crosslinked into orthogonal networks. In view of the requirement for
lipodia-like or lamella-like filaments. One difference, however, is that the Arp2/3 complex in these structures, one would postulate dendritic
phagocytic-cup assembly requires extensive vesicle fusion65, which has branching is present, but it will be important to confirm this.
not been shown for lamellipodia or lamella.
The Arp2/3 complex seems to be required for both types of phago- Finger-like protrusive structures. The range of finger-like protru-
cytosis, based on its localization to these structures and on inhibition sions observed in metazoan cells is enormous, and our general view
of both processes by WCA constructs66. In addition, WASP–GFP local- is that multiple assembly mechanisms are likely. We define filopodia
izes to FcR-mediated phagosomes, and macrophages lacking WASP are and microvilli as thin (less than 200 nm diameter) protrusions, con-
compromised in FcR-mediated phagocytosis67. The formins mDia1 and taining parallel bundles of 10–30 actin filaments that run the length
mDia2 seem to function in CR-mediated phagocytosis68. of the protrusion and orient their barbed ends towards the membrane.
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7. REVIEW
a
Tip complex
Ena/Vasp,
Formin, myosin X
b
Electron-dense mass
Fascin (and/or formin?)
Myosin I
ERM protein
Villin
Fimbrin
Epsin
Terminal web
Figure 4 Schematic representations of models for assembly of filopodia and assembly is thought to occur through re-organization of the Arp2/3 complex-
microvilli. (a) A top view of a filopodium — a finger-like, actin-rich protrusion dependent lamellipodial network. (b) A side view of a microvillus, which
that is adhered to the substratum. An electron-dense complex at the tip has similar finger-like morphology to filopodia, but is not attached to the
seems to be enriched in Ena/Vasp proteins, myosin X and formins. Fascin substratum. The most well characterized microvilli on epithelial cells (brush-
seems to be the predominant bundling protein in the most well characterized border microvilli) contain parallel bundles of actin filaments, crosslinked by
filopodia systems. One possibility is that formins might contribute to villin, fimbrin and espin. Microvilli contain an electron-dense mass at their
bundling in some cases. Most often, the filopodial base is embedded in the tip. At their base, epithelial microvilli seem to be embedded in the terminal
lamellipodium and lamellum. The convergent-elongation model of filopodial web, which is composed of actin filaments and myosin II.
We define filopodia as finger-like protrusions that are adhered in some Electron microscopy analyses show that filopodia contain bundles
manner to a substratum or another cell. We define microvilli as finger- of long parallel filaments36,88–90. These extend deep into the lamellipo-
like protrusions that are not adhered. Of course, grey areas exist between dium or lamellum, where they eventually splay at their pointed ends.
these definitions, in particular when both adherent and non-adherent The sites and mechanisms for filopodia assembly are currently under
finger-like protrusions are experimentally induced79. In addition to these debate. One model is that filopodia assemble by ‘convergent elongation’
simple finger-like protrusions, a wide variety of larger and more complex of lamellipodial filaments44,91, through re-organization of the Arp2/3
protrusions (including Drosophila bristles and hair-cell stereocilia) use complex-assembled dendritic network into bundles. This re-organiza-
multiple modules of actin bundles, but these are not discussed here (for tion is initiated when a subset of filament barbed ends associate with a
a review, see ref. 80). filopodial-tip protein complex at the leading edge plasma membrane.
This complex, containing the Ena/VASP actin-binding proteins and pos-
Filopodia. Filopodia protrude from the leading edge of many motile cells, sibly formins, protects barbed ends from capping proteins, thus allowing
including fibroblasts and nerve growth cones (Fig. 4)81. In these situations, them to elongate preferentially in the network. Contacting filaments
filopodia emanate from a lamellipodial or lamellar sheet, and are believed would then be bundled by the bundling protein fascin to eventually pro-
to function as directional sensors82 (perhaps through enrichment of acti- duce a filopodium91.
vated integrins in filopodial tips83). Viruses often bind at filopodial tips, The convergent elongation model requires Arp2/3 complex-medi-
and are transported back to the cell body before being internalized84,85. A ated nucleation, followed by rearrangement of the dendritic network.
similar process occurs for activated epidermal growth factor (EGF) recep- However, recent studies depleting subunits of the Arp2/3 complex
tor on adenocarcinoma cells86. Finally, filopodia are abundant on neuronal by RNAi have provided conflicting results in terms of whether filo-
dendrites, and seem to be important for dendritic spine development87. podia or just lamellipodia were affected (T. Svitkina, unpublished
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8. REVIEW
whereas monomer addition rate varies. An interesting possibility is that
Formins
(mDia1, FRL1)? formins are at the barbed ends of filopodial filaments and their regulation
controls elongation rate.
The basic structure of filopodia, and how they relate to lamellipodia
and lamella, is still open to surprises, and recent results may impact
on possible assembly mechanisms. Although most electron microscopy
Nucleus APC studies show that filopodia consist of long, parallel filaments, recent
cryo-electron microscopy work in Dictyostelium suggests that shorter
filaments may also be present that are not necessarily parallel, with very
T cell short filaments in a meshwork pattern at the tip99. The suggestion from
this work is that these shorter, labile, filaments may have been destroyed
Arp2/3
by previous electron microscopy preparations. We reserve judgment on
this possibility until similar observations are made for other filopodia, as
these Dictyostelium filopodia elongate approximately twentyfold faster
Figure 5 Actin assembly factors and immune synapse. The Arp2/3 complex
localizes to the actin-rich contact site (red) at the immune synapse in T cells, than most other protrusions (about 1 µmicron s–1) and, thus, may use a
and is necessary for full actin accumulation at the immune synapse. mDia1 fundamentally different protrusive mechanism.
and FRL1 localize to both the actin-rich site and to regions surrounding The suggestion that the lamellipodium lies on top of the lamellum43
the MTOC (green). Knockdown of either mDia1 or FRL1 inhibits MTOC
has implications for filopodia assembly. If filopodia derive from the
re-orientation toward the immune synapse. Effects of formins on actin
polymerization at the immune synapse are controversial, with one report action of the Arp2/3 complex in the lamellipodium, do they transition
indicating that deletion of mDia1 ablates filament accumulation, and at some point to the lower, lamellar layer, as their roots often end up
another indicating no effect of either mDia1 or FRL1 knockdown on filament proximal to lamellipodium (Fig. 3c)? Or, do they assemble from the
accumulation. FRL1 and mDia1 seem to act independently at the MTOC,
dendritic network at the lamellipodium–lamellum interface? Could filo-
in view of their differential localization patterns around the MTOC, and the
observations that knockdown of either inhibits MTOC re-orientation. APC, podia-like protrusions derive from the lamellum? Clearly, many other
antigen-presenting cell. mechanisms are possible.
The definition of a filopodium can be quite subjective, if based solely on
observations)52. Both of these studies use B16F1 mouse melanoma cells, light microscopy of fixed cells. Care must be taken to ensure that the struc-
but with potentially key variations in the substrates on which the cells tures are protrusive, and not ‘retraction fibres’. Some protrusions defined
were plated and in the medium added to the cells. A third study reports as filopodia in the literature are less finger-like than others, being wider
a slight increase in filopodia number in mouse embryonic fibroblasts overall and splaying to widths of several microns at their intersection with
when Arp3 is depleted by RNAi54. Finally, N-WASP has been shown the lamellipodium–lamellum. In addition, fibroblasts can protrude filopo-
to trigger filopodia assembly downstream of Cdc42 (ref. 92), but cells dia-like structures from regions containing no obvious lamellipodium or
lacking N-WASP still possess filopodia93. lamellum (S. Nicholson-Dykstra, Dartmouth, Hanover, NH, and H.N.H.,
Regarding formins, mDia2 has been linked to filopodia assembly79,94,95 unpublished observations). We question whether all such structures are
(T. Svitkina, unpublished observations). In many of these cases, it is subject to a common assembly mechanism.
unclear whether the structures hold to our adhesion-based definitions
of filopodia, as both adherent and non-adherent finger-like protrusions Microvilli. Microvilli are most recognizable on the luminal surfaces of
are visible. It is possible that overexpression of formins or other proteins intestinal and kidney epithelial cells, where they are densely packed and
used in these studies causes assembly of both structures. A formin is of uniform length (around 2 µm; ref. 100). However, shorter (<0.5 µm)
also required for filopodia in Dictyostelium96, although the localization and more variable length microvilli are present on many cell types,
of a GFP-fusion of this formin to finger-like protrusions irrespective of including circulating lymphocytes and the dorsal surfaces of many
substrate-attachment in this study raises questions about our distinction cultured cells101,102. Although the function of epithelial microvilli is to
between microvilli and filopodia. increase absorptive surface area, lymphocyte microvilli may segregate
Our opinion is that there may be multiple potential mechanisms for cell surface proteins103,104 — a property that may aid extravasation from
assembling filopodia. Filopodial assembly requires three basic steps: fila- blood to the periphery.
ment nucleation, sustained barbed-end elongation and filament bundling. Both epithelial and lymphocyte microvilli contain long actin fila-
A variety of molecules may be capable of accomplishing each task. Moreover, ments arranged in parallel bundles100,102. Although epithelial microvilli
bundled structures can be nucleated by the Arp2/3 complex or formins in maintain constant length after initial assembly, their actin filaments are
vitro17,19,97,98. We believe that it is perfectly possible that different mechanisms still dynamic, with subunits adding at barbed ends and dissociating at
of filament nucleation are used in distinct cellular environments. pointed ends7,105. Shorter microvilli seem to have dynamic lengths, elon-
After assembling, leading-edge filopodia elongate at rates between gating and retracting on a timescale of minutes101,102.
1–5 µm min–1 in multiple cell types5,52. However, elongation dynam- The cellular environment from which microvilli grow seems to be fun-
ics vary, even during the lifetime of a single filopodium, with periods damentally different from that of most filopodia, and does not involve
of elongation and retraction. In growth-cone filopodia, the elongation a clear lamellipodial or lamellar surface. The apical surface of epithelial
rate depends on a balance between monomer addition at the tip, and cells contains an actin meshwork called the ‘terminal web’ (Fig. 4) that is
retrograde flow combined with depolymerization at the base5. Most rich in myosin II and spectrin, and the actin filaments do not seem to be
commonly, retrograde flow and depolymerization seems fairly constant, dendritically branched106–108. The small diameter of blood lymphocytes
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9. REVIEW
(<10 µm) allows about 2 µm of cytoplasm surrounding the nucleus, and WAVE2 in these cells; WAVE2 seems to be the Arp2/3-complex regula-
it is unclear how actin filaments here are organized102. It would be useful tor primarily responsible for actin assembly at the synapse112. Another
to characterize actin organization at the base of microvilli in more detail, consideration is the time allowed for synapse assembly in these different
in light of our new knowledge of the Arp2/3 complex and formins. model systems.
We feel that it is unlikely that microvilli require the Arp2/3 complex for The role for these formins in MTOC repositioning is not the first evi-
assembly. RNAi-mediated knockdown of Arp2 has no effect on micro- dence of association between formins and microtubule-based structures.
villar morphology in a lymphocyte culture line (S. Nicholson-Dykstra, mDia regulates microtubule stabilization in fibroblasts22, and mDia2 can
Dartmouth, Hanover, NH, and H.N.H., unpublished observations). interact with microtubule-binding proteins113. In addition, there is evi-
However, formins affect microvillar assembly in both Dictyostelium96 and dence that mDia1 effects MTOC positioning in migrating fibroblasts,
NIH3T3 cells79. It is unclear how the latter structures relate to Cdc42- although this result is not universally accepted22,114.
dependent filopodia in NIH3T3 cells, which also require mDia2 (ref. 94). Are the effects of mDia1 and FRL1 on MTOC re-orientation in T cells
In addition, all of these structures deviate from our definition of micro- related to their effects on actin dynamics? Suppression of either formin
villi, as they include both attached and unattached protrusions. alone inhibits re-orientation23, and the formins localize in different pat-
terns at the MTOC, suggesting distinct roles. One study suggests that the
Novel insights from adhesion structures FH2 domain is required for mDia1-mediated effects on microtubules115,
Recent studies of adhesion structures have revealed an intricate interplay which suggests a link to actin. However, the FH2 domain is also sufficient
between multiple actin-based structures, as well as a role for formins in for the interaction of mDia2 with microtubule-binding proteins113, and
controlling microtubule-based structures. the FH2 domain of mDia1 can interact directly with microtubules (E.S.C.
and H.N.H., unpublished observations; F. Bartolini and G. Gundersen,
Immunological synapse. The term ‘immunological synapse’ refers Columbia University, New York, NY, unpublished observations). It is not
to the extensive interaction surface between a T lymphocyte and an currently known whether actin filaments colocalize with mDia1 or FRL1
antigen-presenting cell bound to specific antigen, mediated by both at the MTOC in T cells, although these may be difficult to detect. One
integrin-based and T-cell receptor-based interactions109. This structure final point is that the two effects of formins on microtubules (stabiliza-
is important in establishing cell–cell adhesion and T-cell polarity, and tion and MTOC repositioning) may involve distinct mechanisms, as they
is necessary for T-cell activation. An early and requisite step in syn- presumably occur at different ends of microtubules.
apse assembly is massive actin polymerization on the T-cell side, which
expands the interaction surface. Following this event, the microtubule Adherens junctions. Adherens junctions are cell–cell adhesions mediated
organizing centre (MTOC) (and also the Golgi) re-orients towards the by homophilic interaction of cadherins: their core components are the
synapse, and a variety of substances are secreted towards the antigen- transmembrane cadherin and two cytoplasmic proteins, β-catenin and α-
presenting cell. catenin116,117. The long-accepted model is that mature adherens junctions
The actin-based structure at the synapse is widely considered to be in epithelial cells are associated with a circumferential band of actin and
a lamellipodium110, and this is supported by recent RNAi studies23. myosin II that is contractile and runs parallel to the membrane118. This
Knockdown of either Arp2 or Arp3 in Jurkat T cells inhibits spreading band of actin filaments is relatively stable, and the actin–adherens junc-
or polymerization of a tight F-actin band at the synapse, depending tions interaction is mediated by α-catenin, bound to β-catenin, which is
on the experimental setting. However, polarized actin-rich finger-like bound to cadherin. However, new results call this model into question:
protrusions persist at the synapse interface in the near absence of the first, α-catenin cannot bind β-catenin and actin filaments simultane-
Arp2/3 complex (Fig. 5). Interestingly, although two formins (mDia1 ously in vitro119; second, actin filaments are much more dynamic than
and FRL1) colocalize with F-actin at the synapse, suppression of either cadherins or catenins at the adherens junctions119; and third, factors
formin alone or in combination affects neither spreading nor the fin- other than core adherens junction components seem to link junctions
ger-like protrusions. Instead, mDia1 and FRL1 also localize near the to actin120,121.
MTOC and are important for MTOC repositioning towards the synapse. Therefore, our image of the mature adherens junction may well change
Another study, using splenic T cells from mDia1 knockout mice, also over the next few years. However, it is clear that extensive actin dynamics
results in disrupted MTOC polarization111. However, these cells also have are required to assemble the mature structure. Cells approach each other
an almost complete loss of polymerized actin staining at the synapse, in by extension of sheet-like membrane protrusions122. On initial contact,
contrast with the Jurkat cell study. the two interacting cells extend actin-rich finger-like protrusions toward
Clearly, differences between the experiments could explain these each other, with cadherin concentrated at the tips123–125. These protru-
inconsistencies in the role of mDia1 in actin assembly at the immuno- sions interdigitate and then shorten as the junction matures, bringing the
logical synapse. One possibility is that the residual mDia1 remaining two cell membranes closer. The initial sheet-like protrusions are likely to
after RNAi is sufficient for the actin response, but not for the MTOC be lamellipodia or lamella, although it is less clear whether the finger-like
response. Using quantitative immunoblotting, we found that Jurkat protrusions are filopodia-like124 or more like an acto-myosin contractile
cells express 170,000 molecules of mDia1 per cell (H.N.H., unpublished structure126. Clearly, myosin II is important for adherens junction assem-
observations), so that >90% suppression (estimated from westerns in bly, but its localization has not been determined at high resolution.
ref. 23), would result in 10,000–20,000 remaining molecules. Another There is evidence that both the Arp2/3 complex and formins partici-
possibility is that mDia1 knockout in T lymphocytes results in second- pate in adherens junctions assembly. The Arp2/3 complex localizes to
ary effects on Arp2/3-complex levels or activity. It would be interest- sites of junction assembly127 and its inhibition disrupts this process127,128,
ing to probe the levels of Arp2/3-complex subunits, or the WASp and although it is unclear whether this may be due to general inhibition of
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