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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02849
1 Lineberger Comprehensive Cancer Center and Department of Biology, University
of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
2 Department of Biology, Bucknell University, Lewisburg, PA 17837, USA.
3 Department of Cell Biology and Program in Neuroscience, Harvard Medical
School, Boston, MA 02115, USA.
4 Massachusetts Institute of Technology, Department of Biology, Cambridge, MA
02139, USA.
* Author for correspondence (e-mail: peifer{at}unc.edu)
Accepted 12 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Ena/VASP, Epithelial morphogenesis, Cytoplasmic transport, Adhesion, Drosophila melanogaster
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Roles of many actin regulators were defined through studies in vitro, or in
relatively simple systems such as budding yeast or fibroblasts. Even single
cells deploy different actin modulators in different contexts, to construct
actin structures required for each event: for example, one subset mediating
cytokinesis while an overlapping but non-identical set regulates cell
migration.
One key choice is whether to continue actin polymerization or to cap barbed
ends with Capping protein. Enabled (Ena)/VASP family proteins bind barbed ends
and prevent Capping protein binding, allowing continued filament elongation
(Barzik et al., 2005;
Bear et al., 2002) (see also
Samarin et al., 2003).
Ena/VASP proteins regulate cell migration in cultured mammalian fibroblasts,
which predominantly produce lamellipodia
(Bear et al., 2000;
Bear et al., 2002).
Inactivating Ena/VASP proteins triggered production of short, highly branched
actin filaments within lamellipodia, driving more persistent lamellipodial
protrusion that allowed cells to migrate faster than controls. Concentrating
Ena/VASP proteins at the plasma membrane triggered production of long,
sparsely branched actin filaments presumably lacking the strength to provide
sustained force. Cells extended rapid but unstable lamellipodia and migrated
more slowly than controls.
Ena/VASP proteins also influence filopodia
(Mejillano et al., 2004), as
was extensively studied in growth cones in culture
(Lebrand et al., 2004),
Caenorhabditis elegans axons
(Chang et al., 2006) and
Dictyostelium (Han et al.,
2002). Growth cone motility shares features with cell migration
(Dent and Gertler, 2003).
Inactivating Ena/VASP in cultured neurons reduces growth cone filopodia,
whereas concentrating Ena/VASP at the plasma membrane promotes filopodia
(Lebrand et al., 2004).
Interestingly, axon guidance defects are seen in zygotic loss-of-function
Ena/VASP mutants in Drosophila
(Bashaw et al., 2000;
Gertler et al., 1995;
Wills et al., 1999), C.
elegans (Chang et al.,
2006; Gitai et al.,
2003; Shakir et al.,
2006; Withee et al.,
2004; Yu et al.,
2002) and mice (Lanier et al.,
1999; Menzies et al.,
2004). Dictyostelium DdVASP also promotes filopodia
(Han et al., 2002).
Ena/VASP proteins also localize to cell-cell adherens junctions (AJs) in
flies (Baum and Perrimon, 2001;
Grevengoed et al., 2001) and
mammals, and are implicated in AJ formation in cultured keratinocytes
(Vasioukhin et al., 2000) and
mammary cells (Scott et al.,
2006). This suggests that Ena/VASP proteins may play essential
roles in cell adhesion. Finally, Ena/VASP proteins localize to cell-matrix
junctions, perhaps underlying the role of VASP in platelet adhesion (reviewed
by Krause et al., 2003).
As yet, no one has assessed the consequences of completely eliminating
Ena/VASP function in mammals or flies. The three mammalian proteins (Mena,
VASP and Evl) complicate analysis. Mena mutants have relatively
subtle defects in forebrain commissures whereas VASP mutants have
altered platelet aggregation (reviewed by
Krause et al., 2003).
Mena;VASP double mutants have more severe central nervous system
(CNS) and craniofacial defects (Menzies et
al., 2004). In Drosophila, loss of zygotic Ena disrupts
axon guidance, but no one has removed maternal and zygotic Ena. Surprisingly,
C. elegans Ena (unc-34)-null mutants are viable, without
obvious effects in morphogenesis, but embryos double mutant for
unc-34 and the actin-regulator WASP have severe
morphogenesis defects (Withee et al.,
2004), suggesting that C. elegans Ena/VASP is important
but alternate actin regulatory mechanisms can compensate.
Ena/VASP proteins share several regions important for actin regulation
(reviewed by Krause et al.,
2003). The N-terminal EVH1 domain recognizes the sequence
D/EFPPPPXD/E (FP4) (Ball et al.,
2002) in Ena/VASP partners including zyxin, Robo receptors and
Lamellipodin/RIAM, positioning Ena/VASP proteins at sites where their
actin-regulatory activities are needed. The high affinity of EVH1 for its
ligands allowed the Gertler laboratory to design a novel strategy to
manipulate Ena/VASP function. They fused four FP4 motifs to a mitochondrial
localization signal (FP4mito). This recruits Ena/VASP proteins from their
normal cellular locations to mitochondria, effectively inactivating them and
mimicking phenotypes of Ena/VASP-deficient fibroblasts and neurons
(Bear et al., 2000;
Bear et al., 2002;
Lebrand et al., 2004). The
C-terminal EVH2 domain binds G- and F-actin and mediates tetramerization. The
central proline-rich region binds Profilin and SH3/WW-domain proteins,
including Src and Abelson (Abl) kinases. Two point mutations affecting the CNS
function of Ena in Drosophila were characterized
(Ahern-Djamali et al., 1998).
ena210 encodes a missense change in the EVH1 domain
(A97V), blocking interaction with zyxin in vitro. ena23
truncates Ena in the EVH2 domain, blocking tetramerization but not G- or
F-actin binding.
We seek to understand how cells use different tools in the actin regulatory toolkit to perform the diverse behaviors of development. We present the first assessment outside C. elegans of embryonic morphogenesis in the total absence of Ena/VASP function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) using
EcoRI and HindIII, subcloned into pSTBlue-1, cut out with
BamHI and XbaI and subcloned into the P-element vector pUASg
under the control of a Gal4-driven UAS promoter
(Brand and Perrimon, 1993).
The ena open reading frame (ORF) with an N-terminal green fluorescent
protein (GFP)-fusion was also subcloned into pUASg. Stocks to generate
ena germline clones and GAL4 drivers were from the Bloomington
Drosophila Stock Center. UAS-GFPactin or RFPactin were from P. Martin
(University College London, UK). ena germline clones were generated
by heat-shocking 48-72 hour hsflp12; FRT42B
ena/FRT42B ovoD1 larvae for 3 hours at 37°C.
Cuticle preparations were as in Wieschaus and Nüsslein-Volhard
(Wieschaus and Nüsslein-Volhard,
1986).
Immunolocalization and microscopy
S2 and D16-C3 cells were cultured and plated as in Rogers et al.
(Rogers et al., 2002) (with 10
µg/mL human insulin added for D16-C3), transfected, fixed and stained by
the actin protocol in Rogers et al.
(Rogers et al., 2004). For RNA
interference (RNAi), D16-C3 cells were plated in six-well plates to 75%
confluency and treated with 0.5 mL 20 µg/mL dsRNA for 30 minutes, followed
by 1.5 mL growth medium on days 1 and 4; on day 7 cells were resuspended by
pipetting and applied to acid-washed coverslips for 5 minutes. Embryos were
fixed as in Grevengoed et al. (Grevengoed
et al., 2001). Wing discs were fixed for 20 minutes in 4%
formaldehyde on ice. All were blocked/stained in PBS/1% goat serum/0.1% Triton
X-100 and mounted in Aqua-Polymount (Polysciences). Antibodies: mouse
anti-Ena5G2 (1:500), anti-BP102 (1:200); anti-ArmN27A1 (1:200) and rat
anti-DE-cadherin (DE-cad) (DCAD2, 1:200; all from the Developmental Studies
Hybridoma Bank). Mitochondria were visualized by 45 minutes of incubation in
100 nm MitoTracker® Deep Red 633 (Invitrogen), and actin with Alexa
Fluor-568-phalloidin. Secondary antibodies: Alexa Fluor-488, Alexa Fluor-568
and Alexa Fluor-647 (Invitrogen). Most images were acquired with an LSM510
confocal microscope (Carl Zeiss MicroImaging), LSM510AIM acquisition software
and 40x (1.3 NA) or 63x (1.4 NA) objectives. D16-C3 cell images
were acquired using a 100x (1.45 NA) objective, a TE2000-E inverted
microscope (Nikon) and a CoolsnapHQ CCD camera (Roper Scientific). Brightness
and contrast were adjusted in Adobe Photoshop® 7.0; alterations were
performed identically on comparable wild-type and mutant images. Time-lapse
imaging was as in Grevengoed et al.
(Grevengoed et al., 2001).
Images were captured every 5 seconds or 15 seconds. MetaMorph 6.1 (Molecular
Devices, Union City, CA) was used to enhance brightness and contrast.
Modifications were made on entire panels.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a specificity control we generated flies expressing a mito construct
with the conserved phenylalanine in each FP4 motif replaced by alanine
(AP4mito). AP4mito recruited significantly less Ena
(Fig. 2M,M'), although we
saw occasional weak Ena recruitment (data not shown). As a second specificity
control we expressed FP4mito throughout the epidermis in ena
loss-of-function mutants. This did not change the severity or frequency of
embryonic phenotypes (data not shown), suggesting that FP4mito does not have
substantial off-target effects. Finally, we examined whether FP4mito alters
WASP localization (WASP has an EVH1-like WH1 domain) - as expected, it does
not (Fig. 1C-C''),
consistent with known differences in binding specificities of WASP WH1 and
Ena/VASP EVH1 (Volkman et al.,
2002; Zettl and Way,
2002). Other EVH1-domain proteins such as Homer/VESL are similarly
unlikely to bind as they recognize sequences in which the aromatic residue
follows the prolines (Beneken et al.,
2000; Barzik et al.,
2001).
To verify that FP4mito inactivates Drosophila Ena, we expressed
FP4mito in the CNS and peripheral nervous system (PNS) where the function of
Ena is clear (reviewed by Krause et al.,
2003). CNS longitudinal axons extend parallel to the midline
(Fig. 1D, arrowhead) and
commissural axons cross it (Fig.
1D, arrow). FP4mito-expressing embryos have reduced longitudinal
axons (Fig. 1E,G, arrowheads),
resembling zygotic ena mutants in the range and severity of defects
(Fig. 1F,H; see Table S1 in the
supplementary material) (Bashaw et al.,
2000). To further assess FP4mito specificity we expressed it
zygotically in ena zygotic mutants - this did not increase severity
of CNS defects (see Table S1 in the supplementary material), consistent with
FP4mito specifically affecting Ena. We also sequestered Ena at mitochondria in
the PNS. Motor axon projections of intersegmental nerve b (ISNb) normally turn
toward their muscle targets (Fig.
1I, left; Fig. 1J).
In zygotic ena mutants ISNb axons fail to turn, continuing toward
inappropriate targets (Fig. 1I,
right) (Wills et al., 1999).
This bypass phenotype is seen in 88% of segments when FP4mito is expressed
using elav-Gal4 (n=86;
Fig. 1K,L); this is
quantitatively comparable to zygotic ena loss-of-function (79-96%)
(Wills et al., 1999). AP4mito
expression using elav-Gal4 (Fig.
1J) did not cause significant ISNb bypass (2% of segments,
n=150; like wild type). Thus, removing Ena from its normal location
and sequestering it at mitochondria effectively blocks Ena function in intact
animals. By contrast, FP4CAAX, which in cultured fibroblasts activates Ena by
recruitment to the plasma membrane (Bear et
al., 2000), had no effect on CNS axons (data not shown) or ISNb
guidance (Fig. 1N), consistent
with previous analysis of Ena overexpression
(Wills et al., 1999).
Expressing the control AP4CAAX in the PNS also had no effect
(Fig. 1M).
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)
(Fig. 2F,G, arrows) and in
cells at the anterior edge of the dorsal-fold
(Fig. 2H,I, arrows) that lead
during head involution.
Ena plays several roles during morphogenesis
To determine whether Ena is required for epithelial architecture or
morphogenesis, we inactivated maternal and zygotic (M/Z) Ena using two
approaches that each should cause strong loss-of-function. First, we expressed
FP4mito in the germline using mat
4-Gal4-VP16 (matGal4; mat-FP4mito), so
that FP4mito was loaded into eggs [only 8% of mat-FP4mito eggs are
fertilizable (n=400), because of the roles of Ena during oogenesis;
our unpublished data]. Although no zygotic Gal4 is produced, maternal FP4mito
remained throughout embryogenesis, effectively sequestering zygotic and
maternal Ena from cellularization (see Fig. S1J,K in the supplementary
material) through dorsal closure and later
(Fig. 3A versus
Fig. 3B,C). No Ena was
detectable at its usual locations, even in places in which it accumulates to
high levels, such as leading-edge cell AJs
(Fig. 3A versus
Fig. 3C, arrows), so this
should be a very strong to complete loss of maternal and zygotic function.
Second, we generated females with germlines mutant for the very strong
loss-of-function ena23 allele or the strong
ena210 allele (allele strength assessed in the PNS)
(Wills et al., 1999), and
crossed them to males heterozygous for the null allele
enaGC1; half the embryos are M/Zena mutant. In
M/Zena23 mutants selected using a GFP-marked balancer, Ena
protein levels were significantly reduced and no localized protein was seen
(Fig. 3D'' versus
Fig. 3E''). Neither
ena23 nor ena210 is protein null
(Ahern-Djamali et al., 1998),
but attempts to generate germline clones with the protein-null allele
enaGC1 failed, perhaps because it results from a
chromosomal inversion.
The cuticle secreted by epidermal cells provides a simple means to assess
morphogenesis defects. More than 70% of zygotic ena mutants are wild
type, whereas 
20% display misalignment/puckering along the dorsal
midline, indicating late dorsal closure defects. Ubiquitous zygotic FP4mito
expression has similar effects (Fig.
4A,J versus Fig.
4B,K, arrowheads). By contrast, 79% of mat-FP4mito embryos die,
and both mat-FP4mito embryos and M/Zena23 mutants disrupt
several morphogenetic processes [the control matGal4-AP4mito is not embryonic
lethal (1%, n=586)]. Eighty to ninety per cent of mat-FP4mito or
M/Zena23 mutants have defects in head involution,
disrupting head cuticle (Fig.
4D, arrow). To our surprise, dorsal closure was completed, but
embryos have dorsal midline defects like those of ena zygotic nulls
(Fig. 4B,D,G, arrowheads). A
subset of embryos exhibited ventral epidermal defects
(Fig. 4H). Two differences were
apparent between mat-FP4mito and M/Zena23.
M/Zena23 mutants did not exhibit germband retraction
defects, but their ventral epidermal defects were more frequent.
M/Zena210 mutants were similar to, but significantly less
severe, than M/Zena23
(Fig. 4).
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).
By contrast, maternal ena23 mutants form a normal
cellular blastoderm without large-scale defects in pseudocleavage furrows or
cellularization (see Fig. S1C,D,L in the supplementary material). Many
mat-FP4mito early embryos were also roughly normal (see Fig. S1E,F in the
supplementary material), although some embryos had elevated nuclear loss (see
Fig. S1G,H, arrows, in the supplementary material), a defect we also saw in
some maternal ena23 mutants. This may be because of loss
of maternal Ena, or may result from oogenesis defects. A subset of mat-FP4mito
embryos had major defects in nuclear distribution and actin at the anterior
end (see Fig. S1I in the supplementary material). We observed similar defects
in armXP33 maternal mutants that disrupt nurse cell
dumping during oogenesis (Cox et al.,
1996). We thus think it likely that these defects are indirect
consequences of oogenesis defects in mat-FP4mito mothers. mat-FP4mito embryos
are also smaller than wild type, probably because of defects in nurse cell
dumping (data not shown).
Ena plays an important role in adhesion and cortical actin in cultured
keratinocytes, mammary cells and Drosophila follicle cells
(Baum and Perrimon, 2001;
Vasioukhin et al., 2000;
Scott et al., 2006). We thus
examined epithelial integrity in M/Zena23 and mat-FP4mito
embryos. Both proceed through gastrulation normally
(Fig. 5A-D). Epithelial
integrity is normal with no detectable change in Drosophila
E-cadherin (DE-cad), Armadillo, alpha-catenin
(Fig. 2O;
Fig. 5I versus
Fig. 5J; data not shown) or
cortical actin (Fig. 2N,
Fig. 5E-H). Thus, disrupting
Ena function does not disrupt AJs or epithelial integrity. To assess whether
embryonic epithelia are the exception, we disrupted Ena function in posterior
cells of larval imaginal discs (FP4mito x engrailed-Gal4), a
polarized, folded epithelium with actin and DE-cad at apical AJs (see Fig.
S1N,O in the supplementary material). FP4mito relocalized Ena to presumptive
mitochondria (see Fig. S1M, arrowheads, in the supplementary material), with
no detectable effect on epithelial architecture, cortical actin or DE-cad (see
Fig. S1N,O in the supplementary material). We also generated clones of
ena23 mutant cells, which did not affect actin or
epithelial architecture (data not shown). Thus, Ena is not essential for
epithelial architecture or actin organization in embryos or imaginal
discs.
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).
This requires stable connections between amnioserosa and epidermis, achieved
in part by large amnioserosal lamellipodia extending over the posterior-most
epidermal cells. ßPS-integrin mutants fail to form stable lamellipodia;
when amnioserosal cells change shape, they detach from posterior epidermal
cells, disrupting germband retraction
(Schock and Perrimon, 2003).
Approximately 40% of mat-FP4mito embryos failed to complete germband
retraction (Fig. 4E), and had
defects in attachment of amnioserosa and epidermal cells. Although
amnioserosal detachment is most obvious after retraction begins
(Fig. 5O,P versus
Fig. 5M,N, arrows), prior to
germband retraction there is less overlap of amnioserosal cells and posterior
epidermis (Fig. 5K versus
Fig. 5L, brackets). As a
result, while amnioserosal cells change shape, germband retraction fails
(Fig. 5O,P).
M/Zena23 and mat-FP4mito embryos also have major
defects in segmental furrow retraction. Ena has a striking localization in
segmental groove cells (Fig.
2F,G), which apically constrict at the onset of germband
retraction and migrate inwards, pulling in neighbors on either side
(Larsen et al., 2003).
Segmental grooves persist ventrally and dorsally through early dorsal closure,
regressing by its completion; grooves remain longest laterally
(Fig. 6E, arrowheads).
M/Zena23 (Fig.
6B,D,H, arrowheads) and FP4mito segmental grooves
(Fig. 6F,M, arrowheads) are
deeper than wild type and persist long after they should have regressed
(Fig. 6T,U, arrowheads;
Fig. 6S). The leading edge
during dorsal closure is often uneven in M/Zena23 and
FP4mito (Fig. 6B,M), perhaps in
part because of overly deep grooves.
Shortly after germband retraction, head involution begins, with ectoderm migrating anteriorly driven by coordinated cell shape changes. Most M/Zena23 mutants and mat-FP4mito embryos fail in this process (Fig. 6S,T,X, brackets), leading to the head holes seen in cuticles (Fig. 4C,D, arrows). Cells that should lead head involution appear to constrict far more than in wild type, nearly severing the head from the thorax (Fig. 6S,X).
Ena localization in filopodia
Among the most dramatic morphogenetic events is dorsal closure, in which
lateral epidermal sheets move toward one another, meet and fuse at the dorsal
midline, enclosing embryos in epidermis. This is driven by several forces,
including leading-edge actin/myosin cable contraction, amnioserosal cell
apical constriction and dorsal/ventral elongation of epidermal cells
(Kiehart et al., 2000;
Hutson et al., 2003). Ena is
concentrated at leading-edge AJs (Fig.
2E-G, arrowheads), where the actin cable is anchored, and
leading-edge cells exhibit highly dynamic actin protrusions while migrating
(Jacinto et al., 2000). This
suggested that Ena might play a key role in dorsal closure.
As dorsal closure begins, leading-edge cells produce lamellipodia and
filopodia (Jacinto et al.,
2000). In live embryos, GFP-Ena concentrated at tips of
leading-edge filopodia (Fig.
2J, arrowhead) and at leading-edge AJs
(Fig. 2K, arrowhead). Although
filopodia are not generally preserved after fixation, they were easily
visualized in fixed embryos overexpressing Ena
(Fig. 2P). We did not observe
GFP-Ena concentration at the lamellipodial leading edge, but high levels of
GFP-Ena in AJs may obscure this. GFP-Ena also accumulated in intracellular
puncta that may be overexpression aggregates, but these do not substantially
disrupt cell function as pan-epidermal GFP-Ena expression is not embryonic
lethal (e22c-GAL4; 1% lethality, n=294).
GFP-Ena allowed us to examine the localization of Ena within leading-edge filopodia as they extend and retract. Consistent with the anti-capping role of Ena, GFP-Ena concentrated at tips of extending filopodia (Fig. 7A, arrow). Remarkably, GFP-Ena is retained at filopodial tips as they retract (Fig. 7B, arrow; see Movie 1 in the supplementary material). GFP-Ena particles move away from filopodial tips before they retract (Fig. 7B, arrowhead; see Movie 1 and Fig. S2 in the supplementary material), with some GFP-Ena remaining at tips during initial retraction. Rearward movement of GFP-Ena continues as retraction proceeds and tip-associated GFP-Ena usually eventually disappears.
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Inactivating Ena slows epithelial zippering and impedes cell matching
We hypothesized that Ena would play a key role in dorsal closure,
modulating actin cables or helping drive migration. However, both mat-FP4mito
and M/Zena23 mutants complete dorsal closure with levels
and localization of DE-cad (data not shown) and cortical actin
(Fig. 6I-L) in amnioserosa and
epidermis indistinguishable from wild type, and with a largely normal
actin/myosin cable (Fig. 6I
versus Fig. 6J,L, arrows).
Amnioserosal apical constriction and epidermal cell elongation
(Fig. 6J-L, arrowheads;
Fig. 6O versus
Fig. 6P) are usually normal,
although occasional cells have splayed open
(Fig. 6J,L, brackets) or
hyperconstricted leading edges. In a few embryos many epidermal cells fail to
change shape (Fig. 6N,
arrowheads). These defects do not block completion of dorsal closure, but Ena
inactivation disrupts cell matching along the dorsal midline
(Fig. 6W versus
Fig. 6V). FP4mito expression in
stripes in each segment (engrailed- or paired-Gal4) also did
not affect cortical actin or the actin/myosin cable
(Fig. 2N', between
arrowheads).
To examine the role of Ena during dorsal closure more closely, we inactivated it throughout the epidermis and amnioserosa (FP4-mito x e22c-Gal4). This caused embryonic lethality (85%, n=163; versus AP4mito lethality=1%, n=147), and disrupted cell matching at the midline (Fig. 4A,J versus Fig. 4B,K, arrowheads), similar to ena zygotic mutants. In fixed embryos we observed deep segmental grooves and an uneven leading edge (data not shown), as in M/Zena23 mutants. We examined dorsal closure in real time in embryos expressing FP4mito and GFP-actin using e22c-Gal4. This revealed severe abnormalities in epithelial zippering; dorsal openings were oval rather than almond-shaped (Fig. 8A versus Fig. 8B) and leading edges sometimes met in the middle before zippering occurred (Fig. 8B, 62:42, arrow). As a result, migration was 2.2-fold slower than wild type (P=2x10-7), with much of the delay in late epithelial zippering (Fig. 8; see Movies 3, 4 in the supplementary material).
Inactivating Ena disrupts filopodia
We next examined how Ena inactivation affects actin-based protrusions.
Wild-type leading-edge cells produce protrusions resembling growth cones, with
filopodia arising from lamellipodia (Fig.
9A, arrows; see Movie 5 in the supplementary material). We
expressed GFP-actin and FP4mito together, using engrailed-Gal4,
allowing us to monitor leading-edge protrusions when Ena is inactivated. The
results were striking. FP4mito-expressing leading-edge cells produce
lamellipodia (Fig. 9B; see
Movie 6 in the supplementary material), but produce only rare, very short
filopodia (Fig. 9B, 4:00,
arrow). We measured the number and maximum length of filopodia (any thin
protrusion extending beyond the lamellipodium or leading edge within one-half
of an engrailed stripe in 3-4 embryos/genotype as leading edges moved
from 23.6 µm to 10.9 µm apart, using a defined distance rather than a
defined time as FP4mito-expressing embryos close more slowly). Wild-type cells
produce an average of 58 filopodia, with maximum length averaging 2.18 µm.
FP4mito expression reduces both filopodial number and length (average
filopodia number reduced from 58 to 20; average maximum length reduced from
2.18 µm to 1.16 µm; P<0.0001 for length and P=0.013
for number; these and other P values are via Student's
t-test; Table 1;
Fig. 9F), paralleling effects
of inactivating Ena/VASP proteins in cultured neurons
(Lebrand et al., 2004) or
Dictyostelium (Han et al.,
2002).
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|
).
We did not observe obvious effects of Ena inactivation on lamellipodia.
Because wild-type protrusions combine lamellipodia and filopodia and new
protrusions often form adjacent to or on top of existing ones, it is difficult
to accurately follow a single lamellipodium through its lifecycle. Although
lamellipodia of FP4mito-expressing cells can remain extended for several
minutes (Fig. 9C), this is not
substantially different from wild type.
Ena promotes filopodial formation and elongation
Given the ability of Ena to promote filament elongation, we hypothesized
that increasing Ena activity would increase filopodial number and/or length.
Embryos overexpressing Ena produce longer filopodia than wild type
(Fig. 9D; see Movie 7 in the
supplementary material; average maximum length=2.52 µm versus 2.18 µm
for wild type; P=0.0024; Table
1; Fig. 9F).
Overexpressing Ena may also increase filopodial number: it increased to 91
filopodia versus 58 filopodia in wild type
(Table 1), although this
difference did not reach statistical significance in the sample size we
quantitated. FP4CAAX, which should increase plasma membrane Ena activity, also
promoted longer filopodia (average maximum length=3.00 µm versus 2.18 µm
for wild type; P=0.0014; Table
1; Fig. 9E,F).
Normally, filopodia are only produced by leading-edge cells, whereas lateral
epithelial cells do not produce obvious protrusions. Interestingly, lateral
epithelial cells overexpressing Ena produce many filopodia
(Fig. 9G) on their lateral and
apical surfaces. Thus, overexpressing Ena or concentrating it at the membrane
both trigger longer filopodia and may promote de novo filopodial formation.
Despite the dramatic effect on filopodia, dorsal closure is completed
normally, and animals overexpressing Ena or expressing FP4CAAX ubiquitously
(e22c-Gal4) complete embryogenesis and survive to adulthood.
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). Ena depletion dramatically altered D16-C3 cell morphology,
with most cells (94.5%, n=540 cells) lacking any detectable
filopodia. Instead, Ena RNAi-treated cells exhibited a morphology dominated by
lamellipodial protrusions (Fig.
10B). By contrast, CPB RNAi produced D16-C3 cells (91.3%,
n=470 cells) with more numerous and longer filopodia
(Fig. 10C). In addition,
fluorimetric analysis revealed that, relative to control-treated cells
(n>5000 cells), CPB RNAi increased mean F-actin levels by >160%
(n>5000 cells) whereas in Ena-depleted cells F-actin remained
unchanged. Therefore, CPB is a key regulator of actin polymer levels in these
cells. To test the role of Abl in filopodia, we performed Abl RNAi. Consistent
with a role for Abl as an Ena negative regulator
(Gertler et al., 1995), Abl
depletion resulted in a dramatic increase in filopodial length and number
(Fig. 10D), and increased mean
F-actin levels to 120% (n>5000 cells) over controls. We next
assessed whether this occurred via effects on Ena localization. Filopodia in
Abl RNAi-treated cells accumulated qualitatively higher Ena levels at
filopodial tips than did control RNAi cells
(Fig. 10E,F). We next turned to embryos to examine whether Abl regulation was relevant to filopodia during dorsal closure. Wild-type filopodia are poorly preserved after fixation, and thus we are not often able to visualize them in wild-type embryos. However, Ena overexpression produced filopodia that were robust to fixation (Fig. 2P). To assess the role of Abl in filopodial regulation, we examined embryos maternally and zygotically mutant for the null allele abl4 (M/Zabl), focusing on the leading edge during dorsal closure. In wild-type controls we saw strong Ena localization to leading-edge AJs as well as the amnioserosal cell cortex, but did not see obvious Ena localization at filopodial tips (Fig. 10I,J). By contrast, in M/Zabl mutants we saw numerous filopodia at the leading-edge terminating in prominent Ena dots (Fig. 10K,L, arrows). Furthermore, reducing Abl levels via abl heterozygosity reduced the phenotypic severity of zygotic Ena inactivation (FP4mito x e22c-Gal4; see Table S2 in the supplementary material). These data suggest that Abl negatively regulates filopodial formation by preventing Ena recruitment to forming filopodia. This is consistent with effects of overexpressing Abl in embryos, in which filopodia are significantly reduced (T. Stevens and M.P., unpublished).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and Ena/VASP proteins help establish cell
adhesion in keratinocytes (Vasioukhin et
al., 2000) and mammary epithelial cells
(Scott et al., 2006). We first
tested the hypothesis that Ena/VASP proteins play key roles in cell-cell
adhesion. We were surprised to find that Ena inactivation or mutation did not
disrupt AJs or epithelial integrity in embryos or imaginal discs, suggesting
that they do not play general roles in these processes. Of course, Ena/VASP
proteins may play cell type-specific roles; e.g. filopodial zippers initiating
keratinocyte adhesion may require Ena, whereas other mechanisms of
establishing epithelial architecture may not. Our data also reveal
morphogenetic events in which Ena is nonessential; e.g. there were no apparent
defects in ventral furrows or germband extension. Consistent with this,
Mena/VASP/Evl triple mutant mice complete many morphogenetic processes without
major defects (F.B.G., unpublished).
|
) and actin regulation in follicle cells
(Baum and Perrimon, 2001),
providing a view of the spectrum of biological events requiring Ena. We
hypothesize that Ena acts in a mechanistically similar manner in each process,
as an actin anti-Capping protein promoting filament elongation. Flies use this
tool in many different ways, promoting distinct cell behaviors.
Some events can be linked fairly directly to known Ena/VASP functions. The
role of Ena in axon guidance fits its actin regulatory role well. During
dorsal closure, Ena inactivation reduces filopodia, consistent with its
biochemical function. Our data reveal that this slows epithelial zippering and
disrupts precise alignment between the two sheets. Leading-edge filopodia were
proposed to function both as sensors directing proper cell matching, and to
facilitate adhesion of cells from opposing edges in Drosophila
(Jacinto et al., 2000) and
C. elegans (Williams-Masson et
al., 1997). Although our data support a sensory role for
filopodia, we did not observe disrupted epithelial adhesion/fusion. Our data
support and contrast with work on embryos expressing dominant-negative Cdc42
(Cdc42DN), which produce rudimentary protrusions
(Jacinto et al., 2000). Like
ena mutants, Cdc42DN embryos display mismatching of cells from
opposing edges, but, unlike ena mutants, Cdc42DN embryos have gaps
between cells along the midline. Although Cdc42DN-expressing leading-edge
cells produce only rudimentary protrusions, FP4mito-expressing cells produce
robust lamellipodia. Because AJ formation in cultured mammalian cells can be
mediated either by filopodia (Vasioukhin
et al., 2000) or lamellipodia
(Ehrlich et al., 2002),
lamellipodia may mediate adhesion in the absence of filopodia.
Ena is also essential for morphogenetic events in which its cell biological
role is more speculative. Germband retraction requires integrin-mediated
adhesion of amnioserosa and epidermis to couple cell-shape changes in the two
tissues (Schock and Perrimon,
2002; Schock and Perrimon,
2003). Ena inactivation mimics integrin loss. Ena/VASP proteins
localize to focal adhesions, and Drosophila Ena colocalizes with
integrins at ends of planar-polarized actin bundles in follicle cells
(Bateman et al., 2001).
Ena/VASP inactivation does not disrupt focal adhesions
(Bear et al., 2000), but may
modulate their size and stress fiber robustness under mechanical stress
(Galler et al., 2005;
Yoshigi et al., 2005).
Drosophila Ena may strengthen the cytoskeleton during germband
retraction, promote amnioserosal lamellipodia or regulate extracellular matrix
(ECM) adhesion more directly, as VASP does in platelets (reviewed by
Krause et al., 2003) and as
Ena/VASP proteins may do in Xenopus somitogenesis
(Kragtorp and Miller, 2006).
Less is known about mechanisms by which segmental grooves form and retract
(Larsen et al., 2003).
However, Ena is planar-polarized to dorsal-ventral cell boundaries in these
cells; perhaps it stabilizes actin attachment at borders where it is enriched.
Defects in head involution result from alterations in dorsal-fold cell shape
change, which may share mechanistic similarities with segmental groove
formation.
Ena and filopodia in vivo
One key challenge is to identify machinery required to generate filopodia
and lamellipodia. We found a striking correlation between Ena activity and
filopodial length and number. Inactivating Ena significantly decreased
filopodial length and number, whereas increasing Ena activity increased
filopodial length. Interestingly, maximum filopodial length was not
substantially altered, and thus is probably not limited by Ena levels.
However, Ena can be rate-limiting in filopodial formation as Ena
overexpression generated filopodia on lateral epithelial cells that normally
do not produce them. Together, these data suggest that Ena promotes both
initiation and elongation of leading-edge filopodia.
Ena is concentrated at the tips of elongating filopodia, consistent with its influence on filopodial length and its biochemical function. Interestingly, GFP-Ena particles move rearward prior to retraction, presumably by retrograde flow, and some GFP-Ena is retained at filopodial tips as they retract. We had not expected this, as the anti-capping function of Ena/VASP suggested that Ena at filopodial tips would promote extension. Although this could be an artifact of GFP-Ena, it may indicate complexity in the control of filopodial dynamics. For example, whether filopodia continue extending or retract may be determined not only by actin polymerization rates at the tip, but also by depolymerization and/or retrograde flow rates at its base. In addition, filopodial dynamics may be regulated at individual filaments within filopodia rather than the structure as a whole. GFP-Ena particles may be locally inactivated Ena on individual filaments moving away from the tip by retrograde flow.
One unanswered question is whether different filopodial regulators act
additively or in series. Mammalian Ena/VASP can act downstream of Cdc42
together with IRSp53 (Krugmann et al.,
2001), but IRSp53 can promote Ena/VASP-independent filopodia
(Nakagawa et al., 2003).
Formins also promote filopodia, but whereas Dictyostelium dDia2 and
VASP directly interact, Ena/VASP:formin relationships remain unclear (reviewed
by Faix and Rottner, 2006).
The reduced number of short filopodia formed when Ena is inactivated is
consistent with multiple mechanisms acting additively/synergistically to
produce the appropriate filopodial number/length.
Our data also test in vivo one aspect of the convergent elongation model
(Svitkina et al., 2003). This
proposes that tip complex proteins bind filaments and protect them from
capping, allowing continued elongation, and then interact laterally, bundling
filaments and forming filopodia. Ena/VASP proteins may supply anti-capping
activity, and could also help mediate lateral association via tetramerization.
GFP-Ena overexpression promoted large lamellipodia containing numerous actin
microspikes; these often converged at their distal ends to form filopodia,
supporting the convergent elongation model.
We also examined filopodial regulation. Our data demonstrate that Abl is a key negative regulator of filopodial extension in cultured cells and in vivo, inhibiting Ena accumulation at nascent filopodial tips. This idea is further supported by our parallel analysis of embryos expressing activated Bcr-Abl or excess wild-type Abl; both reduce filopodia on leading-edge and amnioserosal cells (T. Stevens and M.P., unpublished). This provides a means for signal transduction pathways to regulate cell behavior.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2027/DC1
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ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ahern-Djamali, S. M., Comer, A. R., Bachmann, C., Kastenmeier,
A. S., Reddy, S. K., Beckerle, M. C., Walter, U. and Hoffmann, F. M.
(1998). Mutations in Drosophila enabled and rescue by human
vasodilator-stimulated phosphoprotein (VASP) indicate important functional
roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains. Mol.
Biol. Cell 9,2157
-2171.
Ball, L. J., Jarchau, T., Oschkinat, H. and Walter, U. (2002). EVH1 domains: structure, function and interactions. FEBS Lett. 513,45 -52.[CrossRef][Medline]
Barzik, M., Carl, U. D., Schubert, W. D., Frank, R., Wehland, J. and Heinz, D. W. (2001). The N-terminal domain of Homer/Vesl is a new class II EVH1 domain. J. Mol. Biol. 309,155 -169.[CrossRef][Medline]
Barzik, M., Kotova, T. I., Higgs, H. N., Hazelwood, L., Hanein,
D., Gertler, F. B. and Schafer, D. A. (2005). Ena/VASP
proteins enhance actin polymerization in the presence of barbed end capping
proteins. J. Biol. Chem.
280,28653
-28662.
Bashaw, G. J., Kidd, T., Murray, D., Pawson, T. and Goodman, C. S. (2000). Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101,703 -715.[CrossRef][Medline]
Bateman, J., Reddy, R. S., Saito, H. and Van Vactor, D. (2001). The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium. Curr. Biol. 11,1317 -1327.[CrossRef][Medline]
Baum, B. and Perrimon, N. (2001). Spatial control of the actin cytoskeleton in Drosophila epithelial cells. Nat. Cell Biol. 3,883 -890.[CrossRef][Medline]
Bear, J. E., Loureiro, J. J., Libova, I., Fassler, R., Wehland, J. and Gertler, F. B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101,717 -728.[CrossRef][Medline]
Bear, J. E., Svitkina, T. M., Krause, M., Schafer, D. A., Loureiro, J. J., Strasser, G. A., Maly, I. V., Chaga, O. Y., Cooper, J. A., Borisy, G. G. et al. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109,509 -521.[CrossRef][Medline]
Beneken, J., Tu, J. C., Xiao, B., Nuriya, M., Yuan, J. P., Worley, P. F. and Leahy, D. J. (2000). Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition. Neuron 26,143 -154.[CrossRef][Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Chang, C., Adler, C. E., Krause, M., Clark, S. G., Gertler, F. B., Tessier-Lavigne, M. and Bargmann, C. I. (2006). MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin. Curr. Biol. 16,854 -862.[CrossRef][Medline]
Cox, R. T., Kirkpatrick, C. and Peifer, M.
(1996). Armadillo is required for adherens junction assembly,
cell polarity, and morphogenesis during Drosophila embryogenesis.
J. Cell Biol. 134,133
-148.
Dent, E. W. and Gertler, F. B. (2003). Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron 40,209 -227.[CrossRef][Medline]
Ehrlich, J. S., Hansen, M. D. and Nelson, W. J. (2002). Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion. Dev. Cell 3,259 -270.[CrossRef][Medline]
Faix, J. and Rottner, K. (2006). The making of filopodia. Curr. Opin. Cell Biol. 18, 18-25.[CrossRef][Medline]
Galler, A. B., Garcia Arguinzonis, M. I., Baumgartner, W., Kuhn, M., Smolenski, A., Simm, A. and Reinhard, M. (2005). VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment. Histochem. Cell Biol. 115,457 -474.
Gertler, F. B., Comer, A. R., Juang, J., Ahern, S. M., Clark, M.
J., Liebl, E. C. and Hoffmann, F. M. (1995).
enabled, a dosage-sensitive suppressor of mutations in the
Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3
domain-binding properties. Genes Dev.
9, 521-533.
Gitai, Z., Yu, T. W., Lundquist, E. A., Tessier-Lavigne, M. and Bargmann, C. I.. (2003). The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37, 53-65.[CrossRef][Medline]
Grevengoed, E. E., Loureiro, J. J., Jesse, T. L. and Peifer,
M. (2001). Abelson kinase regulates epithelial morphogenesis
in Drosophila. J. Cell Biol.
155,1185
-1198.
Grevengoed, E. E., Fox, D. T., Gates, J. and Peifer, M.
(2003). Balancing different types of actin polymerization at
distinct sites: roles for Abelson kinase and Enabled. J. Cell
Biol. 163,1267
-1279.
Han, Y. H., Chung, C. Y., Wessels, D., Stephens, S., Titus, M.
A., Soll, D. R. and Firtel, R. A. (2002). Requirement of a
vasodilator-stimulated phosphoprotein family member for cell adhesion, the
formation of filopodia, and chemotaxis in dictyostelium. J. Biol.
Chem. 277,49877
-49887.
Hutson, M. S., Tokutake, Y., Chang, M. S., Bloor, J. W.,
Venakides, S., Kiehart, D. P. and Edwards, G. S. (2003).
Forces for morphogenesis investigated with laser microsurgery and quantitative
modeling. Science 300,145
-149.
Jacinto, A., Wood, W., Balayo, T., Turmaine, M., Martinez-Arias, A. and Martin, P. (2000). Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure. Curr. Biol. 10,1420 -1426.[CrossRef][Medline]
Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L.
and Montague, R. A. (2000). Multiple forces contribute to
cell sheet morphogenesis for dorsal closure in Drosophila. J. Cell
Biol. 149,471
-490.
Kragtorp, K. A. and Miller, J. R. (2006).
Regulation of somitogenesis by Ena/VASP proteins and FAK during Xenopus
development. Development
133,685
-695.
Krause, M., Dent, E. W., Bear, J. E., Loureiro, J. J. and Gertler, F. B. (2003). Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu. Rev. Cell Dev. Biol. 19,541 -564.[CrossRef][Medline]
Krugmann, S., Jordens, I., Gevaert, K., Driessens, M., Vandekerckhove, J. and Hall, A. (2001). Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex. Curr. Biol. 11,1645 -1655.[CrossRef][Medline]
Lanier, L. M., Gates, M. A., Witke, W., Menzies, A. S., Wehman, A. M., Macklis, J. D., Kwiatkowski, D., Soriano, P. and Gertler, F. B. (1999). Mena is required for neurulation and commissure formation. Neuron 22,313 -325.[CrossRef][Medline]
Larsen, C. W., Hirst, E., Alexandre, C. and Vincent, J. P.
(2003). Segment boundary formation in Drosophila embryos.
Development 130,5625
-5635.
Lebrand, C., Dent, E. W., Strasser, G. A., Lanier, L. M., Krause, M., Svitkina, T. M., Borisy, G. G. and Gertler, F. B. (2004). Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 42,37 -49.[CrossRef][Medline]
Mejillano, M. R., Kojima, S., Applewhite, D. A., Gertler, F. B., Svitkina,T. M. and Borisy, G. G. (2004). Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end. Cell 118,363 -373.[CrossRef][Medline]
Menzies, A. S., Aszodi, A., Williams, S. E., Pfeifer, A.,
Wehman, A. M., Goh, K. L., Mason, C. A., Fassler, R. and Gertler, F. B.
(2004). Mena and vasodilator-stimulated phosphoprotein are
required for multiple actin-dependent processes that shape the vertebrate
nervous system. J. Neurosci.
24,8029
-8038.
Nakagawa, H., Miki, H., Nozumi, M., Takenawa, T., Miyamoto, S.,
Wehland, J. and Small, J. V. (2003). IRSp53 is colocalised
with WAVE2 at the tips of protruding lamellipodia and filopodia independently
of Mena. J. Cell Sci.
116,2577
-2583.
Pollard, T. D. and Borisy, G. G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112,453 -465.[CrossRef][Medline]
Rogers, S. L., Rogers, G. C., Sharp, D. J. and Vale, R. D.
(2002). Drosophila EB1 is important for proper assembly,
dynamics, and positioning of the mitotic spindle. J. Cell
Biol. 158,873
-884.
Rogers, S. L., Wiedemann, U., Stuurman, N. and Vale, R. D.
(2003). Molecular requirements for actin-based lamella formation
in Drosophila S2 cells. J. Cell Biol.
162,1079
-1088.
Rogers, S. L., Wiedemann, U., Hacker, U., Turck, C. and Vale, R. D. (2004). Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr. Biol. 14,1827 -1833.[CrossRef][Medline]
Samarin, S., Romero, S., Kocks, C., Didry, D., Pantaloni, D. and
Carlier, M. F. (2003). How VASP enhances actin-based
motility. J. Cell Biol.
163,131
-142.
Schock, F. and Perrimon, N. (2002). Cellular processes associated with germ band retraction in Drosophila. Dev. Biol. 248,29 -39.[CrossRef][Medline]
Schock, F. and Perrimon, N. (2003). Retraction
of the Drosophila germ band requires cell-matrix interaction. Genes
Dev. 17,597
-602.
Scott, J. A., Shewan, A. M., den Elzen, N. R., Loureiro, J. J.,
Gertler, F. B. and Yap, A. S. (2006). Ena/VASP
proteins can regulate distinct modes of actin organization at
cadherin-adhesive contacts. Mol. Biol. Cell
17,1085
-1095.
Shakir, M. A., Gill, J. S. and Lundquist, E. A.
(2006). Interactions of UNC-34 Enabled with Rac GTPases and the
NIK kinase MIG-15 in C. elegans axon pathfinding and neuronal migration.
Genetics 172,893
-913.
Svitkina, T. M., Bulanova, E. A., Chaga, O. Y., Vignjevic, D.
M., Kojima, S., Vasiliev, J. M. and Borisy, G. G. (2003).
Mechanism of filopodia initiation by reorganization of a dendritic network.
J. Cell Biol. 160,409
-421.
Vasioukhin, V., Bauer, C., Yin, M. and Fuchs, E. (2000). Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100,209 -219.[CrossRef][Medline]
Volkman, B. F., Prehoda, K. E., Scott, J. A., Peterson, F. C. and Lim, W. A. (2002). Structure of the N-WASP EVH1 domain-WIP complex: insight into the molecular basis of Wiskott-Aldrich Syndrome. Cell 111,565 -576.[CrossRef][Medline]
Wieschaus, E. and Nüsslein-Volhard, C. (1986). Looking at embryos. In Drosophila, A Practical Approach (ed. D. B. Roberts), pp.199 -228. Oxford: IRL Press.
Williams-Masson, E. M., Malik, A. N. and Hardin, J. (1997). An actin-mediated two-step mechanism is required for ventral enclosure of the C. elegans hypodermis. Development 124,2889 -2901.[Abstract]
Wills, Z., Bateman, J., Korey, C. A., Comer, A. and Van Vactor, D. (1999). The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance. Neuron 22,301 -312.[CrossRef][Medline]
Withee, J., Galligan, B., Hawkins, N. and Garriga, G.
(2004). Caenorhabditis elegans WASP and Ena/VASP proteins play
compensatory roles in morphogenesis and neuronal cell migration.
Genetics 167,1165
-1176.
Yoshigi, M., Hoffman, L. M., Jensen, C. C., Yost, H. J. and
Beckerle, M. C. (2005). Mechanical force mobilizes zyxin from
focal adhesions to actin filaments and regulates cytoskeletal reinforcement.
J. Cell Biol. 171,209
-215.
Yu, T. W., Hao, J. C., Lim, W., Tessier-Lavigne, M. and Bargmann, C. I. (2002). Shared receptors in axon guidance: SAX-3/Robo signals via UNC-34/Enabled and a Netrin-independent UNC-40/DCC function. Nat. Neurosci. 5,1147 -1154.[CrossRef][Medline]
Zettl, M. and Way, M. (2002). The WH1 and EVH1 domains of WASP and Ena/VASP family members bind distinct sequence motifs. Curr. Biol. 12,1617 -1622.[CrossRef][Medline]
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