1 Department of Cell Biology, Institute of Molecular Biology, Austrian Academy
of Sciences, Billrothstraße 11, Salzburg A-5020, Austria
2 Department of Biochemical Engineering and Science, Kyushu Institute of
Technology, Iizuka, Fukuoka 820-8502, Japan
3 Division of Cancer Genomics, Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
4 Department of Biochemistry, Institute of Medical Science, University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
5 Gesellshaft für Biotechnologische Forschung (GBF), Mascheroder Weg 1,
38124 Braunschweig, Germany
* Author for correspondence (e-mail: jvsmall{at}imolbio.oeaw.ac.at)
Accepted 7 March 2003
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Summary |
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Key words: IRSp53, WAVE, Mena, Lamellipodia, Filopodia
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Introduction |
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Ena/VASP family proteins, including Mena, localise to the tips of
lamellipodia and filopodia (Lanier et al.,
1999; Nakagawa et al.,
2001
; Rottner et al., 1999a;
Rottner et al., 2001
), as well
as to the head of the actin comet tail of Listeria monocytogenes in
infected cytoplasm (Laurent et al.,
1999
), and are thought to participate in the regulation of actin
filament dynamics (Bear et al.,
2000
; Bear et al.,
2002
; Gertler et al.,
1996
; Loisel et al.,
1999
; Rottner et al., 1999a). The WASP-related, WAVE/Scar family
is represented by three isoforms in human tissues, WAVE1-WAVE3
(Suetsugu et al., 1999
). All
isoforms share a C-terminal acidic region that stimulates the actin filament
nucleation activity of the Arp2/3 complex
(Machesky et al., 1999
;
Miki et al., 2000
), and the
conserved N-terminal WAVE/Scar homology domain (WHD) possesses lamellipodia
localisation activity (Nakagawa et al.,
2001
; Nozumi et al.,
2003
). Profilin provides monomeric actin molecules to the
elongating actin filaments near the cell membrane (reviewed by
Miller, 2002
).
Recently, the insulin receptor tyrosine kinase substrate IRSp53
(Yeh et al., 1996) was
proposed as an adaptor molecule involved in events downstream of Rac and/or
Cdc42 (Govind et al., 2001
;
Krugmann et al., 2001
;
Miki et al., 2000
). IRSp53 was
originally identified as the substrate of the insulin receptor tyrosine
kinase, although a corresponding function remains outstanding
(Yeh et al., 1996
). Miki et
al. (Miki et al., 2000
) found
that IRSp53 linked Rac to WAVE2, the ubiquitous member of the WAVE family
(Suetsugu et al., 1999
).
IRSp53 binds to Rac through its N-terminal region named the Rac binding region
(RCB) (Miki et al., 2000
;
Miki and Takenawa, 2002
). In a
later study, Govind et al. (Govind et al.,
2001
) reported that IRS-58, a C-terminal alternative splicing
variant of IRSp53 (Alvarez et al.,
2002
), bound to Cdc42 at its centre part. Furthermore, Krugmann et
al. (Krugmann et al., 2001
)
identified IRSp53 as a Rac and Cdc42 binding protein using the yeast
two-hybrid method. They defined the Cdc42 binding region as that containing a
partial CRIB motif (Cdc42 and Rac binding motif) (reviewed by
Hoffman and Cerione, 2000
) at
the same position as reported by Govind et al.
(Govind et al., 2001
). IRSp53
binds to the proline-rich regions of both WAVE2 and Mena through its SH3
domain (Krugmann et al., 2001
;
Miki et al., 2000
). From their
recent studies, Krugmann et al. (Krugmann
et al., 2001
) concluded that IRSp53 acts synergistically with Mena
in filopodia induction, rather than as a link between Rac and WAVE 2 in
signalling lamellipodia formation (Miki et
al., 2000
). IRSp53 has thus been attributed with apparently
alternative roles in the formation of either lamellipodia or filopodia. To
gain more insight into IRSp53 function, we investigated the localisation and
dynamics of IRSp53 and its target proteins in living cells.
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Materials and Methods |
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Antibodies and immunofluorescence microscopy
Affinity purified polyclonal antibodies against WAVE were characterised as
described previously (Miki et al.,
1998b; Nakagawa et al.,
2001
). Rabbit aniserum against green fluorescence protein was
purchased from Molecular Probes (Eugene, OR). Monoclonal antibody against
myc-tag (clone no. 9E10), FITC-conjugated anti-mouse immunoglobulin G (IgG)
goat IgG and rhodamine-phalloidin were obtained from Sigma-Aldrich Japan K.K.
(Tokyo, Japan). The immunofluorescence microscopy was performed using a
confocal laser scaning microscope (LSM510, Zeiss, Japan) as previously
described (Nakagawa et al.,
2001
). The pinhole of LSM510 was set to adjust the thickness of
focal planes at 0.7 µm for immunofluorescence microscopy.
Video microscopy of living cells
Transfected cells cultured on laminin-coated cover glass were mounted in an
open heating chamber (Warner Instruments, Hamden, CT) operating at 32°C
and 37°C for MVD7 cells and B16 cells, respectively. The
treatment with aluminium fluoride was carried out according to the method of
Hahne et al. (Hahne et al.,
2001). The chamber was mounted on an inverted microscope (Axiovert
S100TV, Zeiss, Austria) equipped for epifluorescence and phase contrast
microscopy. Data were acquired with a back-illuminated CCD camera (Roper
Scientific, Trenton, NJ). The interval time between video frames was set at 7
seconds (Supplemental movie) or 17 seconds. For the double transfections of
EGFP-tagged IRSp53 and DsRed1-tagged WAVE2, the time between sequential frames
in the two fluorescent channels was 1.5 seconds.
Measurement of fluorescence intensity
The fluorescence intensity of the tips of filopodia and lamellipodia were
measured using Scion Image 1.62c (Scion Corporation, Frederick, MD). The
fluorescence intensity profiles were obtained within a frame of 0.5 µm
width along filopodia or of 1 µm width across lamellipodia. The specific
fluorescence at the tip was obtained by subtracting the mean background
fluorescence on both sides of the tip (see
Fig. 7d).
|
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Results |
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To analyse how the localisation of IRSp53 was related to cell movement, we
observed the dynamics of IRSp53 tagged with EGFP (EGFP-IRSp53) in living B16
cells. In general, transfected cells were reluctant to spread, so cells were
cotransfected with dominant active Rac1 (Rac1V12) to promote cell spreading.
As shown in Fig. 2a-d,
EGFP-IRSp53 was concentrated at the lamellipodium tip similar to myc-IRSp53.
In cell edges undergoing retraction, EGFP-IRSp53 was absent but relocated to
the tips following extension of lamellipodia
(Fig. 2c,d, white lines), as
formerly found for other tip proteins
(Geese et al., 2000;
Hahne et al., 2001
; Rottner et
al., 1999a; Stradal et al.,
2001
). The same dynamics of IRSp53 were observed in B16 cells
stimulated with aluminium fluoride (Fig.
2e,f). In these cells, EGFP-IRSp53 localised at the protruding
tips of lamellipodia and was absent from retracting tips, as well as from
retraction fibres (Fig. 2e,f,
arrowheads). EGFP-IRSp53 was retained in membrane ruffles at the edge of
Rac1V12 cotransfected cells (Fig.
2c,d), as also earlier observed with EGFP-VASP (Rottner et al.,
1999a), and was also concentrated at the tips of elongating filopodia
(Fig. 6; Supplemental movie).
These observations suggest that IRSp53 could function downstream of both Rac
and Cdc42. The localisation of EGFP-IRSp53 in living cells was consistent with
the immunofluorescence localisation of myc-IRSp53.
|
|
IRSp53 binds to both Rac and Cdc42 through its RCB region and CRIB motif,
respectively, (Govind et al.,
2001; Krugmann et al.,
2001
; Miki et al.,
2000
; Miki and Takenawa,
2002
). The SH3 domain of IRSp53 is reported to interact with WAVE2
and Mena to form lamellipodia and/or filopodia
(Krugmann et al., 2001
;
Miki et al., 2000
). To clarify
which regions of IRSp53 regulate its localisation, we expressed deletion
constructs of IRSp53 in B16 melanoma cells tagged with myc, which does not
affect the interaction with Rac (Miki et
al., 2000
). myc
SH3 and myc-RCB fragments showed the
same localisation as the full-length IRSp53, but the SH3 domain alone
(myc-SH3) failed to localise (Fig.
3d,e). These results indicate that the localisation of IRSp53
could be regulated independently from the interaction with WAVE2 and/or Mena,
which are both mediated by the SH3 domain. They also suggest that the
N-terminal sequence in the RCB region could tether IRSp53 to the tips of both
lamellipodia and filopodia
|
IRSp53 is localised at the tips of lamellipodia and filopodia
protruding from Mena/VASP double-knockout cells
Because overexpression of IRSp53 induced filopodial extensions, it was
suggested that the complex of IRSp53 and Mena regulates filopodia elongation
(Krugmann et al., 2001). This
conclusion is inconsistent, however, with the recruitment of both IRSp53 (Figs
1,
2) and Mena (Gerlter et al.,
1996; Nakagawa et al., 2001
;
Rottner et al., 1999a; Rottner et al.,
2001
) to lamellipodia, as well as filopodia. To provide further
insight into the in vivo relationship between IRSp53 and Mena, we analysed the
localisation of EGFP-IRSp53 in MVD7, Ena/VASP family deficient
cells (Bear et al., 2000
). When
cotransfected with Rac1V12 and EGFP-IRSp53, MVD7 cells plated on
laminin extended lamellipodia and filopodia with EGFP-IRSp53 localised to
their tips (Fig. 4). This
result showed that the IRSp53:Mena complex is not essential for filopodia
elongation, and that IRSp53 may interact with WAVE2 rather than with Mena at
these tips.
|
WAVE2, but not WAVE1, localises to filopodia tips with IRSp53
Significantly, IRSp53 binds only WAVE2 of the three WAVE isoforms and
specifically enhances WAVE2 activity to induce Arp2/3 complex activation
(Miki et al, 2000). These
results suggested that WAVE2 might be localised at the tips of both
lamellipodia and filopodia, together with IRSp53. To investigate this
possibility, we determined the localisation of WAVE2 and compared its dynamics
with that of IRSp53 in B16 cells. Before the living cell observation, we
localised endogenous WAVE in B16 cell using an anti-WAVE antibody that
recognises all three WAVE isoforms. The antibody labelled the tips of both
lamellipodia and filopodia (Fig.
5a,b), in contrast to WAVE1, which localises only at the
lamellipodium tip (Hahne et al.,
2001
) when expressed in B16 cells. In the neural growth cone,
WAVE2 and 3 localise to both tips of lamellipodia and filopodia but only WAVE1
localises to the lamellipodium tip (Nozumi
et al., 2003
). Because WAVE1 and 3 are expressed mainly in neural
tissues (Suetsugu et al.,
1999
; Benachenhou et al.,
2002
), the tip label observed by antibody labelling in
untransfected B16 cells is most likely to be attributed to the ubiquitously
expressing WAVE2 isoform. The localisation of DsRed1-tagged WAVE2
(WAVE2-DsRed) confirmed this possibility
(Fig. 5c,d). WAVE2-DsRed-transfected B16 cells were stimulated with aluminium fluoride to
enhance lamellipodia formation (Hahne et
al., 2001
). As shown in Fig.
5c,d, WAVE2-DsRed localised to the edge of extending lamellipodium
and the tips of filopodia similar to the immunolabelling with anti-WAVE
antibody.
|
Different dynamics of IRSp53 and WAVE2 at the tip of retracting
filopodia
By observing the dynamics of IRSp53 and WAVE2 in the same cell, we could
compare the dynamics of these two proteins. B16 cells were cotransfected with
EGFP-IRSp53 and WAVE2-DsRed and were microinjected with constitutively active
Rac1L61 to promote cell spreading (Fig.
6). Consistent with the single label experiments, EGFP-IRSp53 and
WAVE2-DsRed were colocalised at the tips of lamellipodia.
The fluorescence intensity of EGFP-IRSp53 at the filopodium tip was decreased concomitant with retraction, whereas WAVE2-DsRed was retained at the tip during the early retraction phase (see fluorescent intensity profiles in Fig. 6). As shown in the bottom frame of Fig. 6, when the filopodium was fully retracted, the fluorescence intensity of both EGFP and DsRed at the tip was decreased to the same level. To analyse the difference of EGFP-IRSp53 and WAVE2-DsRed dynamics at the filopodia tips, we measured their fluorescence intensities during retraction. Because the expression time course was different between EGFP and DsRed1 vectors, it was difficult to find cells with moderate fluorescence of both EGFP and DsRed1. Fig. 6 shows an example of three filopodia whose tips could be traced over at least six video frames (35 seconds) sequentially before disappearing in lamellipodia. These filopodia elongated at a rate of 0.19±0.01 µm/min and retracted at a rate of 0.09±0.07 µm/min. The fluorescence intensity of EGFP-IRSp53 decreased to nearly half (IR=45.2±11.3%) within 35 seconds, whereas that of WAVE2-DsRed was preserved (IR=88.1±11.2%) in this period. The fluorescence at lamellipodia edges in the same video frames did not change in the same period; the IR values of EGFP-IRSp53 and WAVE2-DsRed were 85±16.3% and 95.6±18.1%, respectively. To compensate for photobleaching, we estimated the ratios between the IR of filopodia and lamellipodia. The ratios of EGFP-IRSp53 and WAVE2-DsRed were 53% and 92%, respectively. EGFP-IRSp53 and WAVE2-DsRed showed essentially the same dynamics in other filopodia. These results suggest that EGFP-IRSp53 delocalises from the filopodia tip before WAVE2-DsRed, during retraction.
![]() |
Discussion |
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The observation of Krugmann et al.
(Krugmann et al., 2001) that
the overexpression of IRSp53 induced the atypical retraction of cell edges,
and is dependent on incubation time, is consistent with the onset of
deleterious effects at high expression levels. We show here that the
N-terminal RCB region of IRSp53 is recruited to the tips of both lamellipodia
and filopodia, whereas the central region including the CRIB motif is not
responsible for the localisation. Although this N-terminal region has been
reported to be essential for Rac binding
(Miki et al., 2000
;
Miki and Takenawa, 2002
), it
also recruits IRSp53 to the tips of filopodia, suggesting the involvement of
other adaptors determining filopodia localisation. Alternatively, localisation
in filopodia could reflect the derivation of filopodia from lamellipodia
networks signalled via Rac (see Small et
al., 2002
). Further exploration of proteins interacting with the
IRSp53 N-terminal region should provide information about the additional
adaptors or complexes involved.
We show here that IRSp53 colocalises with WAVE2 at the tips of both
protruding lamellipodia and filopodia, independently of Mena. From these
results we propose a schematic model of IRSp53 function
(Fig. 7) that could explain the
two different models of its role in cell movement
(Govind et al., 2001;
Krugmann et al., 2001
;
Miki et al., 2000
). Like
WAVE1, WAVE2 is recruited to the tips of both lamellipodia and filopodia
through its WHD domain (Nakagawa et al.,
2001
; Nozumi et al.,
2003
). Mena localisation is regulated synergistically through both
the EVH (Ena VASP homology) 1 and EVH2 domains
(Bear et al., 2002
).
Considering the binding of IRSp53 with both WAVE2 and Mena through its SH3
domain (Fig. 7, solid
double-ended arrows) and their colocalisation at the tips of lamellipodia and
filopodia, IRSp53 could tie WAVE2 and Mena into a complex. This complex could
contribute to the regulation of actin polymerisation at the cell membrane, in
an alternative pathway, analogous to the complex of ActA and VASP at the
surface of L. monocytogenes
(Lanier et al., 1999
). As
previously reported (Miki et al.,
2000
), IRSp53 should also enhance WAVE2 activity to promote Arp2/3
complex-induced actin filament polymerisation in lamellipodia
(Fig. 7, solid arrow). Because
the Arp2/3 complex is absent from filopodia
(Svitkina and Borisy, 1999
),
WAVE2 may regulate actin polymerisation there through the recruitment of actin
molecules to filopodia tips by the interaction of its proline-rich region with
profilactin (Miki et al.,
1998b
). IRSp53 localisation is not dependent on WAVE2 or Mena.
This is indicated by the observation that IRSp53 disappeared from the
filopodia tips before WAVE2 (Fig.
6) and by the peripheral tip localisation of IRSp53 in the absence
of Mena. Therefore, IRSp53 may be involved in recruiting WAVE2 and Mena in
lamellipodia and filopodia via Rac and Cdc42, respectively.
The localisation of Ena/VASP family proteins to the lamellipodium tip is
highly correlated with the protrusion rate (Rottner et al., 1999a), but the
roles of these proteins are still controversial
(Arguinzonis et al., 2002;
Bear et al., 2000
;
Bear et al., 2002
;
Nakagawa et al., 2001
; Rottner
et al., 1999a). It remains to be shown whether the interaction of IRSp53 with
Mena influences actin polymerisation dynamics, either in vitro or in vivo.
Although N-WASP plays an important role in Cdc42-induced filopodia elongation
(Miki et al., 1998; Suetsugu et al.,
1998
), filopodia can also be induced in N-WASP-deficient
fibroblastic cells (Lommel et al.,
2001
; Snapper et al.,
2001
). These results suggest that, in the process of filopodia
formation, N-WASP functions as a modulator of actin cytoskeleton
reorganisation, but is not essential. From our present observation, WAVE2
represents an alternative regulator of actin polymerisation in filopodia.
Recent studies have shown that WAVE1 exists in an inactive form in a
heterotetrameric protein complex, and becomes accessible to Rac activation in
the presence of Nck, whereby both Rac and Nck become involved in complex
disassembly (Eden et al., 2002
;
Takenawa and Miki, 2001
;
Westphal et al., 2002). Because neither Nck nor Rac are present at
lamelliopodia tips, this activation may precede the engagement of WAVE with
IRSp53. Further analysis of the temporal localisation of WAVE-interacting
proteins should shed light on the differential regulation of lamellipodia and
filopodia protrusion.
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Acknowledgments |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alvarez, C. E., Sutcliffe, J. G. and Thomas, E. A.
(2002). Novel isoform of insulin receptor substrate p53/p58 is
generated by alternative splicing in the CRIB/SH3-binding region.
J. Biol. Chem. 277,24728
-24734.
Arguinzonis, M. I. G., Galler, A. B., Walter, U., Reinhard, M. and Simm, A. (2002). Increased spreading, Rac/p21-activated kinase (PAK) activity, and compromised cell motility in cells deficient in vasodilator stimulated phosphoprotein (VASP). J. Biol. Chem. published online M202873200.
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. and Gertler, F. B. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109,509 -521.[Medline]
Bear, J. E., Loureiro, J. J., Libova, I., Fässler, R., Wehland J. and Gertler, F. B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101,717 -728.[Medline]
Benachenhou, N., Massy, Y. and Vacher, J. (2002). Characterization and expression analyses of the mouse Wiskott-Aldrich syndrome protein (WASP) family member Wave1/Scar. Gene 290,131 -140.[CrossRef][Medline]
Eden, F., Rohatgi, R., Podtelejnikov, A. V., Mann, M. and Kirschner, M. W. (2002). Mechanism of regulation of WAVE-1-induced actin nucleation by Rac1 and Nck. Nature 418,790 -793.[CrossRef][Medline]
Frischknecht, F. and Way, M. (2001). Surfing pathogens and the lessons learned for actin polymerization. Trends Cell Biol. 11,30 -38.[CrossRef][Medline]
Geese, M, Schluter, K., Rothkegel, M., Jockusch, B. M., Wehland,
J. and Sechi, A. S. (2000). Accumulation of profilin II at
the surface of Listeria is concomitant with the onset of motility and
correlates with bacterial speed. J. Cell Sci.
113,1415
-1426.
Gertler, F. B., Niebuhr, K., Reinhard, M., Wehland, J. and Soriano, P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87,227 -239.[Medline]
Govind, S., Kozma, R., Monfries, C., Lim, L. and Ahmed, S.
(2001). Cdc42Hs facilitates cytoskeletal reorganization and
neurite outgrowth by localizing the 58-kD insulin receptor substrate to
filamentous actin. J. Cell Biol.
152,579
-594.
Hahne, P., Sechi, A., Benesch, S. and Small, J. V. (2001). Scar/WAVE is localised at the tips of protruding lamellipodia in living cells. FEBS Lett. 492,215 -220.[CrossRef][Medline]
Higgs, H. N. and Pollard, T. D. (2001). Regulation of actin filament network formation through Arp2/3 complex: activation by a diverse array of proteins. Annu. Rev. Biochem. 70,649 -676.[CrossRef][Medline]
Hoffman, G. R. and Cerione, R. A. (2000). Flipping the switch: the structural basis for signaling through the CRIB motif. Cell 102,403 -406.[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.[Medline]
Laurent, V., Loisel, T. P., Harbeck, B., Wehman, A., Grobe, L.,
Jockusch, B. M., Wehland, J., Gertler, F. B. and Carlier, M. F.
(1999). Role of proteins of the Ena/VASP family in actin-based
motility of Listeria monocytogenes. J. Cell Biol.
144,1245
-1258.
Loisel, T. P., Boujemaa, R., Pantaloni, D. and Carlier, M. F. (1999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 401,613 -616.[CrossRef][Medline]
Lommel, S., Benesch, S., Rottner, K., Franz, T., Wehland, J. and
Kühn, R. (2001). Actin pedestal formation by
enteropathogenic Escherichia coli and intracellular motility of
Shigella flexneri are abolished in N-WASP-defective cells.
EMBO Rep. 2,850
-875.
Machesky, L. M. and Hall, A. (1997). Role of
actin polymerization and adhesion to extracellular matrix in Rac- and
Rho-induced cytoskeletal reorganization. J. Cell Biol.
138,913
-926.
Machesky, L. M., Mullins, R. D., Higgs, H. N., Kaiser, D. A.,
Blanchoin, L., May, R. C., Hall, M. E. and Pollard, T. D.
(1999). Scar1, a WASP-related protein, activates nucleation of
actin filaments by Arp2/3 complex. Proc. Natl. Acad. Sci.
USA 96,3739
-3744.
Miki, H. and Takenawa, T. (2002). WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac. Biochem. Biophys. Res. Commun. 293, 93-99.[CrossRef][Medline]
Miki, H., Sasaki, T., Takai, Y. and Takenawa, T. (1998a). Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 391, 93-96.[CrossRef][Medline]
Miki, H., Suetsugu, S. and Takenawa, T.
(1998b). WAVE, a novel WASP-family protein involved in actin
reorganization induced by Rac. EMBO J.
17,6932
-6941.
Miki, H., Yamaguchi, H., Suetsugu, S. and Takenawa, T. (2000). IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 408,732 -735.[CrossRef][Medline]
Miller, K. G. (2002). Extending the Arp2/3
complex and its regulation beyond the leading edge. J. Cell
Biol. 156,591
-593.
Nakagawa, H., Miki, H., Ito, M., Ohashi, K., Takenawa, T. and
Miyamoto, S. (2001). N-WASP, WAVE and Mena play different
roles in the organization of actin cytoskeleton in lamellipodia. J.
Cell Sci. 114,1555
-1565.
Nobes, C. D. and Hall, A. (1995). Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81,53 -62.[Medline]
Nozumi, M., Nakagawa, H., Miki, H., Takenawa, T. and Miyamoto,
S. (2003). Differential localization of WAVE isoforms in
filopodia and lamellipodia of the neuronal growth cone. J. Cell
Sci. 116,239
-246.
Pantaloni, D., le Clainche, C. and Carlier, M. F.
(2001). Mechanism of actin-based motility.
Science 292,1502
-1506.
Ridley, A. J. (2001). Rho family proteins: coordinating cell responses. Trends Cell Biol. 11,471 -477.[CrossRef][Medline]
Rottner, K., Behrendt, B., Small, J. V. and Wehland, J. (1999). VASP dynamics during lamelliodia protrusion. Nat. Cell Biol. 1,321 -322.[CrossRef][Medline]
Rottner, K., Krause, M., Gimona, M., Small, J. V. and Wehland,
J. (2001). Zyxin is not colocalized with
vasodilator-stimulated phosphoprotein (VASP) at lamellipodial tips and
exhibites different dynamics to vinculin, paxillin, and VASP in focal
adhesions. Mol. Biol. Cell
12,3103
-3113.
Small, J. V. Stradal, T. Vignal, E. and Rottner, K. (2002). The lamellipodium: where motility begins. Trends Cell Biol. 12,112 -120.[CrossRef][Medline]
Snapper, S. B., Takeshima, F., Anton, I., Liu, C. H., Thomas, S. M., Nguyen, D., Dudley, D., Fraser, H., Purich, D., Lopez-Ilasaca, M. et al. (2001). N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility. Nat. Cell Biol. 3,897 -904.[CrossRef][Medline]
Stradal, T., Courtney, K. D., Rottner, K., Hahne, P., Small, J. V. and Pendergast, A. M. (2001). The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr. Biol. 11,891 -895.[CrossRef][Medline]
Suetsugu, S., Miki, H. and Takenawa, T. (1998).
The essential role of profilin in the assembly of actin for microspike
formation. EMBO J. 17,6516
-6526.
Suetsugu, S., Miki, H. and Takenawa, T. (1999). Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. Biochem. Biophys. Res. Commun. 260,296 -302.[CrossRef][Medline]
Svitkina, T. M. and Borisy, G. G. (1999).
Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic
organization and treadmilling of actin Filament array in lamellipodia.
J. Cell Biol. 145,1009
-1026.
Takenawa, T. and Miki, H. (2001). WASP and WAVE
family proteins: key molecules for rapid rearrangement of cortical actin
filaments and cell movement. J. Cell Sci.
114,1801
-1809.
Westphal, R. S., Soderling, S. H., Alto, N. M., Langeberg, L. K.
and Scott, J. D. (2000). Scar/WAVE1, a Wiskott-Aldrich
syndrome protein, assembles an actin-associated multi-kinase scaffold.
EMBO J. 19,4589
-4600.
Yeh, T. C., Ogawa, W., Danielsen, A. G. and Roth, R. A. (1996). Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. J. Biol. Chem. 9,2921 -2928.[CrossRef]