Research Colloquia
What is in a filopodium? Starfish versus hedgehogs
S. Passey1 , S. Pellegrin and H. Mellor
Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K.
Abstract
Many cell types can generate thin actin-based protrusive structures, which are often classified under the
general term of ‘filopodia’. However, a range of filopodia-like structures exists that differ both morphologically and functionally. In this brief review, we discuss the different types of filopodial structures, together
with the actin-binding proteins and signalling pathways involved in their formation. Specifically, we highlight
the differences between the filopodial extensions induced by the Rho GTPases Cdc42 and Rif.
What and where are filopodia?
Filopodia are highly dynamic protrusions made of actin
filaments aligned in tight parallel bundles with their fastgrowing barbed end pushing against the plasma membrane
[1,2]. A distinction is sometimes made between ‘microspikes’,
which are short and appear to be embedded deep within the
cell cortex, and filopodia, which are longer and extend out
from the cell surface [2]. The most extensively studied are the
filopodia arising from the sheet-like membrane projections
(lamellipodia) at the leading edge of migrating fibroblasts
and mouse melanoma B16F1 cells (Figure 1a) [2,3]. Neurite
growth cones also extend lamellipodia and filopodia [4], as
shown in Figure 1(b). In both instances, filopodia have been
proposed to function in sensing environmental cues to guide
cell migration or axon extension [1,4].
Filopodia have also been observed in epithelial cells during
wound healing and morphogenetic processes such as dorsal
closure in Drosophila [1,5]. Filopodia extend beyond the
actin–myosin cable at the leading edge of sheets of migrating
epithelial cells (Figure 1c) and are proposed to play a role in
helping opposing epithelial cells find their partner cell and
form adherens junctions. As opposing epithelial sheets meet,
the filopodia interdigitate allowing E-cadherins to interact at
sites of contact. These sites then mature into adherens junctions through recruitment of catenins and alignment of the
plasma membranes, thereby ‘zipping’ the two sheets together
[1,5].
Small highly dynamic actin-rich projections can also be
seen on the apical surface of a variety of cells in culture [6].
Although the function of these structures is unknown, apical filopodia of certain cell types have been proposed to be
precursors of more specialized protrusions such as brushborder microvilli (Figure 1d) or the sensory stereocilia of
the inner ear hair cells (Figure 1e) [7]. Thus cells produce a
range of thin projections from their surfaces with seeming
morphological similarities, but also apparent differences that
may be linked to specialized functions. How these different
Key words: actin, Cdc42, filopodium, Rif.
Abbreviations used: Ena/VASP, enabled/vasodilator-stimulated phosphoprotein; IRSp53, insulin
receptor tyrosine kinase substrate p53; WASP, Wiskott–Aldrich syndrome protein; N-WASP,
neuronal WASP.
1
To whom correspondence should be addressed (email [email protected]).
types of filopodia relate to one another is a major question in
this research area.
Mechanisms of filopodia formation
The regulation of filopodia formation is complex and involves
a large number of actin-binding and signalling proteins.
Nucleation of new actin filaments is required for filopodia
formation and one way by which this occurs is through
activation of the Arp2/Arp3 complex. The Arp2/Arp3 actin
nucleation complex generates new actin filaments by binding
to their slow-growing pointed ends. It does so by attaching to the sides of existing actin filaments and nucleating new
filaments at an angle, thereby generating a branched actin
network [2]. Branched networks can be reorganized to form
filopodial bundles and a model describing how this might
occur has been proposed by Svitkina et al. [3]. The Rho
GTPase Cdc42 was the first signalling protein to be shown to
induce filopodia [8,9] and Cdc42 is important for filopodia
formation at the leading edge of migrating fibroblasts [10]
and Drosophila epithelial cells [11]. Cdc42 can generate
filopodia through Arp2/Arp3 by interacting with the WASP/
N-WASP (Wiskott–Aldrich syndrome protein/neuronal
WASP), which in turn recruits and activates Arp2/Arp3 [2].
Despite the existence of the Cdc42–WASP–Arp2/Arp3
pathway to filopodia formation, cells deficient in WASP/NWASP are still capable of forming filopodia [2], suggesting
that there are alternative pathways to filopodia formation
which do not require WASP/N-WASP.
Actin nucleation can also occur without Arp2/Arp3 involvement. Formins are known to nucleate straight actin filaments by binding to their fast-growing barbed ends [12]
and Cdc42 can also induce filopodia formation by interacting with and activating the Diaphanous-related formin
p134mDia2/Drf3 (mouse Diaphanous 2/Diaphanous related
formin 3) [12].
In addition to actin nucleation, actin-bundling proteins
such as fascin are needed for filopodia formation to cross-link
adjacent actin filaments into parallel bundles [3]. The adaptor
protein IRSp53 (insulin receptor tyrosine kinase substrate
p53) has recently been shown to contain an evolutionarily
conserved F-actin bundling region at its N-terminus, which
can induce filopodia formation [13]. Cdc42 can interact
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Figure 1 Filopodia and other thin, actin-rich cellular protrusions
Filopodia are seen at the leading edge of migrating fibroblasts
(a) and in neuronal growth cones (b). In both instances, filopodia are
associated with lamellipodia. Filopodia are also found at the leading
edge of epithelial sheets (c) during such processes as wound healing or
Drosophila dorsal closure. The radial actin bundles found in those ‘zipping’
filopodia are linked to an actin-myosin ‘purse string’ cable. Depending on
the cell type, apical filopodia or short microvilli seen on many cultured
cells might be precursors of more mature actin-rich structures such as
brush-border microvilli (d) and inner ear cell stereocilia (e).
with IRSp53 through a partial CRIB (Cdc42/Rac interactivebinding) motif, allowing induction of filopodia formation
through IRSp53 [13].
Actin filaments are normally capped at their fast-growing
barbed ends by capping proteins to prevent further elongation
[3]. However, the filopodial tip complex contains proteins
such as Ena/VASP (enabled/vasodilator-stimulated phosphoprotein), which bind to barbed ends, protecting them
from capping and allowing continued elongation of filopodial
actin filaments [3]. IRSp53 has been shown to interact with
Mena (mammalian enabled), a member of the Ena/VASP
family, and to act synergistically in the formation of filopodia,
and Cdc42 can regulate this process through its interaction
with IRSp53 [14].
It is thus clear that routes to filopodia formation are complex and highly regulated. The Rho GTPase Cdc42 is a key
regulator of filopodia formation and appears to be involved
in a number of steps along these pathways. However, given
the diversity of filopodial structures, it is still unclear whether
all are dependent on Cdc42 for their formation.
Cdc42 versus Rif: starfish versus hedgehog
Filopodia induced by active Cdc42 are short and thick, and
emanate from the cell periphery, making cells adopt a ‘starfish’
appearance (Figure 2). Cdc42 is predominantly cytoplasmic
Figure 2 Cdc42 and Rif-induced filopodia in HeLa cells
HeLa cells were transfected with constitutively active mutants of Cdc42 (upper panels) or Rif (lower panels). Cells were
stained for the relevant Rho GTPase (green) and with phalloidin for F-actin (red).
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Research Colloquia
and does not appear to localize to filopodia. Moreover,
Cdc42-dependent filopodia contain focal complexes at their
tips, which are rich in vinculin and paxillin [8]. The RhoGTPase Rif (Rho in filopodia) has also been shown to induce
filopodia when overexpressed in HeLa cells [15]. However,
Rif-induced filopodia are morphologically different from
those induced by Cdc42 (Figure 2). Rif induces numerous
long, thin apical filopodia more reminiscent of a ‘hedgehog’
than a ‘starfish’. Unlike Cdc42, Rif localizes to the plasma
membrane and along the lengths of filopodia, and Rif-induced filopodia lack focal complexes at their tips [15]. Rif
has been shown to induce filopodia independent of Cdc42,
WASP/N-WASP and Arp2/Arp3 ([15], and S. Pellegrin and
H. Mellor, unpublished work). The pathway linking Rif
to filopodia is yet to be elucidated, pointing to another
alternative route to the generation of filopodial protrusions.
Conclusion
Numerous cells possess thin actin-rich surface protrusions,
which differ in length, thickness, dynamics, localization (apical versus peripheral) and origin (lamellipodial or non-lamellipodial). One can imagine that by varying these properties,
the cell can produce a range of discrete tools to perform
specialized cellular functions. One obvious way to generate
this diversity would be through the recruitment of specific
sets of actin-binding and signalling proteins. Cdc42 and Rif
represent signalling proteins that control the formation of
protrusions with very different properties. It is by understanding the function of these and other key regulators of filopodia that we can begin to categorize the wide range of
specialized filopodia and to understand fully their cellular
functions.
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Received 23 July 2004
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