1 Department of Biochemical Science, Kyushu Institute of Technology, 680-4
Kawatsu, lizuka, Fukuoka 820-8502, Japan
2 Department of Biochemistry, Institute of Medical Science, University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
* Author for correspondence (e-mail: miya{at}bse.kyutech.ac.jp)
Accepted 21 October 2002
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Summary |
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Key words: WAVE, WASP, NG108, Actin, Filopodia, Growth cone
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Introduction |
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To date, many aspects have been made clear about the extension of
lamellipodia (Borisy and Svitkina,
2000; Small et al.,
2002
), but almost nothing is known about the way filopodia are
assembled and disassembled in vivo. In this study, we aimed to clarify the
localization and roles of WAVE isoforms in the formation and extension of
lamellipodia and filopodia in the growth cones of cultured neuroblastoma
NG108-15 cells. To confirm the endogenous expression of WAVE isoforms, we
observed cells immunostained with an antibody reacting against all these
isoforms. Additionally, we characterized the localization and roles of the
WAVE isoforms in living cells transfected with fusion constructs of enhanced
green fluorescent protein (EGFP) and each WAVE isoform, and subsequently
observed a temporal change in their localization in the protruding
lamellipodia and filopodia. Furthermore, to clarify which domain and region of
WAVE isoforms control the localization, we observed the distribution of
various truncated fragments tagged with EGFP. To understand how actin bundles
are involved in lamellipodial extension and filopodial elongation at the
growth cone, we studied the actin bundles' localization and simultaneous
correlation with WAVE isoforms.
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Materials and Methods |
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Cell culture
NG108-15 cells (gift from Haruhiro Higashida, Kanazawa University Graduate
School of Medicine, Japan) were cultured in DMEM supplemented with 10% FCS,
and penicillin/streptomycin at 37.5°C in 5% CO2. In preparation
for immunofluorescence microscopy observation, cells were briefly trypsinized
(0.05% in 1 mM EDTA) and replated on a glass coverslip or glass bottom dish.
These coverslips were coated with poly-L-lysine. To induce the formation of
axons and growth cones in cells, the medium was supplemented with 1 mM
dibutyryl cyclic AMP (Bt2cAMP)
(Furuya and Furuya, 1983).
Expression vectors and transfection
WAVE1-, WAVE2- and WAVE3-EGFP were generated by polymerase chain reaction
(PCR) with the corresponding human cDNA as template. WAVE1 SHD (amino acid
residues 1-171) and SHD-basic region (SHD-BR) (residues 1-225) were amplified
by PCR with human WAVE1 cDNA. WAVE2 N-terminal (NT) region, NT54 (residues
1-54) and NT83 (residues 1-83), and proline-rich (Pro-rich) region (residues
265-400) were amplified by PCR with human WAVE2 cDNA
(Suetsugu et al., 1999). All
5' and 3' primers contained a XhoI restriction
endnuclease site. In the 5' primer for the WAVE2 Pro-rich region, the
ATG sequence for the first methionine was inserted between the XhoI
site and complementary sequence. Amplified fragments were subcloned using the
TOPO TA cloning kit and were sequenced. The subcloned fragments were inserted
into XhoI-digested pEGFP-N1. In all constructs, the sequence for EGFP
was fused to the 3' terminus of the insert. NG108-15 cells were
transfected at 1x105 cells in 35 mm dishes, using 6 µl of
TransIt-LT1 and 2 µg of DNA. After being transfected overnight, cells were
replated on dishes with fresh medium.
SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting were carried out as previously described by
Nakagawa et al. (Nakagawa et al.,
2000).
Immunofluorescence
Observation by immunofluorescence microscopy was performed as described by
Nakagawa et al. (Nakagawa et al.,
2000). Cells on glass coverslips were fixed with 3% formaldehyde
in PBS (137 mM NaCl, 2.7 mM Na2HPO4, 8.1 mM KCl and 1.5
mM KH2PO4), permeabilized with 0.1% Triton X-100 in PBS,
and reacted with primary and secondary antibodies. Rhodamine-phalloidin was
diluted in the secondary antibody solution. All observations were made with a
confocal laser-scanning microscope, LSM510 (Carl Zeiss, Germany).
Living cell observation
A few hours before the observation, the medium of the cells attached to
glass bottom dishes was changed to nutrient mixture F-12 Ham with 1 mM
Bt2cAMP. Under the microscope, the culture dishes were placed in an
open heating chamber maintained at 37.5°C in air. Differential
interference and fluorescence images were acquired through the confocal
laser-scanning microscope, LSM510.
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Results |
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Temporal change in the localization of WAVE isoforms
To date three isoforms, WAVE1, WAVE2 and WAVE3, have been identified in
mammalian tissues (Suetsugu et al.,
1999). We prepared constructs in which the cDNA of these isoforms
was tagged with EGFP at the C-terminus, and transfected each into NG108 cells
by lipofection; we subsequently induced the formation of lamellipodia and
filopodia at growth cones by treatment with Bt2cAMP as shown in
Figs 4 and
5. The expression of fusion
proteins of WAVE tagged with EGFP in the C-terminus was very low, therefore
the fluorescence signal from GFP was substantially weak. So, to clarify
whether fluorescence for EGFP is really emitted only from the fusion protein
of EGFP and WAVE isoform expressed in the cell, we immunostained these cells
with antibody against EGFP to enhance the fluorescence signal, and
simultaneously labeled actin filaments fluorescently with
rhodamine-phalloidin. We confirmed that the localization of WAVE isoforms
detected by immunofluorescence with anti-GFP sufficiently coincided with that
from EGFP fluorophores in both lamellipodia and filopodia
(Fig. 4). We had already
obtained the preparatory result that the functional domain to control the
localization might exist in the N-terminus, SHD region, of each WAVE. Hence,
in this study EGFP was consistently tagged with the C-terminus. In ruffled
regions of lamellipodia, the continuous distribution of WAVE1 along the
leading edge indicated no significant changes during observation
(Fig. 5A,B). WAVE1-EGFPs became
more concentrated with time, especially in the extending parts of
lamellipodia. By contrast, in the retracting areas of lamellipodia, the
fluorescence intensity from WAVE1-EGFPs gradually weakened. At the microspikes
and filopodia, we failed to observe the localization of WAVE1 either at the
tips or in the shafts. These findings show that WAVE1 is involved in
lamellipodial extension but not filopodial formation and elongation. We paid
special attention to the growth cones in which the microspikes were emerging
and growing into filopodia. WAVE2-EGFPs concentrated as dot-like spots at the
sites where the distal ends of actin bundles encountered the leading edge
(Fig. 5C,D). With time, the
microspikes appeared with ends at sites corresponding to
WAVE2-EGFP-concentrated points. In addition, these WAVE2 spots moved along the
leading edge with the lateral motion of microspikes throughout growth-cone
lamellipodia. These results show that WAVE2 on the leading edge controls the
microspike formation and movement. By contrast, WAVE2 disappeared at the tips
of retracting filopodia. WAVE3 also localized at the tips of microspikes and
filopodia similarly to WAVE2 (Fig.
5E,F). These findings imply that WAVE2 and/or WAVE3 might regulate
actin polymerization at the tips of filopodia.
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Functional domain to control the localization of WAVE isoforms
We previously reported that WAVE should be recruited to the lamellipodial
leading edge through the SHD region in the fibroblast cells
(Nakagawa et al., 2001). To
clarify the functional domain that regulates the concentration of WAVE
isoforms at the filopodial tips, we made various truncated fragments of each
WAVE and investigated their localization at the neuronal growth cones. WAVE1
SHD-EGFP concentrated at the filopodial tips
(Fig. 6B). An N-terminal
fragment of WAVE1 SHD plus basic region (SHD-BR-EGFP) similarly localized at
the filopodial tips (Fig. 6C).
Full-length WAVE1, however, failed to localize at the filopodial tips (Figs
4 and
5). This suggests that Pro-rich
+ VCA region of WAVE1 might influence an inhibitory effect to the
localization. For another possibility, perhaps full length of WAVE1 might not
localize at the filopodial tips because it usually exists in heterotetrameric
complex, as reported recently (Eden et al.,
2002
). To clarify the detailed functions of SHD of WAVE2 that
contributed mainly to the filopodia formation, we studied the localization of
various N-terminal fragments. Among them, an N-terminal 54 amino-acid fragment
of SHD tagged with EGFP (WAVE2 NT54-EGFP) migrated neither to the leading edge
nor to filopodial tips (Fig.
6D), but an 83 amino-acid fragment (WAVE2 NT83-EGFP) localized to
the filopodial tips (Fig. 6E).
A proline-rich region of WAVE2 (Pro-rich-EGFP) distributed diffusely
throughout the growth cone (Fig.
6F). These results indicate that 29-amino-acid residues from 54 to
83 of WAVE2 SHD are essential to the localization at the filopodial tips. Miki
et al. reported that this region of SHD contains the putative
leucine-zipper-like motif (Miki et al.,
1998
). The above results show that this motif might play a crucial
role in the localization at the filopodial tips.
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Discussion |
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Signal pathways from small G proteins to WAVE isoforms and their
localization at the growth cone
The signal pathways that lead to the Rho family of small G-proteins, Cdc42
and Rac, via various intermediators such as tyrosine kinase and
phosphatidylinositol 4,5-bisphosphate after accepting guidance cues have
almost been elucidated (Nobes and Hall,
1995; Hall, 1998
).
However, these molecules cannot directly reorganize the actin cytoskeleton.
Recently, WASP family proteins have received attention as mediators that
intervene between these two steps (Pollard
et al., 2000
; Takenawa and
Miki, 2001
), whereas the details as to the localization and the
roles of these proteins at neuronal growth cones remain vague. In our study,
we found that WAVE1 distributed continuously along the leading edge alone, and
WAVE2 and WAVE3 showed a discrete and dynamic localization at the initiation
sites of microspikes on the leading edge. They also continued to remain at the
tips of filopodia during elongation. These findings indicate that WAVE2 and
WAVE3 are involved in the emerging site induction of the filopodia and
subsequently promote their elongation. Additionally, the recent reports that
filopodia can be formed even in fibroblasts isolated from N-WASP-deficient
mice (Snapper et al., 2001
;
Lommel et al., 2001
) indicate
that N-WASP does not always participate in the formation and elongation of
filopodia. However, WAVE isoforms have no GBD/CRIB (GTPase-binding
domain/Cdc42 and Rac interactive binding) region different from WASP and
N-WASP, and do not directly bind small G-proteins, Rac and Cdc42 as previously
reported (Miki et al., 1998
).
WAVE isoforms commonly have a wave homology domain (WHD)/SHD region at the
N-terminus. Previously, we confirmed that the localization of SHD-EGFP
coincides with that of WAVE at fibroblast lamellipodia
(Nakagawa et al., 2001
). In
the present study we showed that WAVE isoforms localized at the filopodial
tips through the SHD region, exactly by its leucine-zipper-like motif. To
clarify the more detailed function of WAVE isoforms we will continue to search
for the protein interacting with this leucine-zipper motif at the growth cone.
Recently, a scaffold protein IRSp53, which activates WAVE2 by binding Rac-GTP,
was isolated (Yeh et al.,
1996
; Miki et al.,
2000
). IRSp53 transforms WAVE2 into an active form that
subsequently mobilizes the Arp2/3 complex and supplies monomeric actins via
its VCA region (Takenawa and Miki,
2001
; Suetsugu et al.,
2002
). These results show that WAVE isoforms convert the signals
to the reorganization of the actin cytoskeleton in the peripheral region of
growth cones. However, Ena/VASP (Enabled/vasodilator-stimulated
phosphoprotein) family proteins have been reported to localize at the leading
edge of lamellipodia and also at the filopodia tips
(Nakagawa et al., 2001
). VASP,
localized to the leading edge of lamellipodia, correlates with actin filaments
(Rottner et al., 1999
).
Additionally, Mena (mammalian Ena) has been reported to localize at filopodia
tips (Lanier et al., 1999
).
However, more recently, Bear et al. have reported that Ena/VASP proteins are
not always necessary for filopodia to protrude at the neuronal growth cone
(Bear et al., 2002
).
Consequently, we think it is more reasonable that WAVE isoforms, whose
localization at the growth cone was confirmed in this study, regulate the
formation and extension of lamellipodia and filopodia. The evidence that WAVE2
is a ubiquitous protein and especially well expressed in neuronal cells
(Suetsugu et al., 1999
) also
supports the hypothesis that WAVE2 is a major regulator of growth cone
morphogenesis.
Formation and extension of filopodia by actin bundle
reorganization
In the periphery of the growth cone, the distal ends of actin bundles
(barbed ends) fuse with others at the site where WAVE2 is concentrated at the
leading edge (Fig. 7A).
Therefore, the actin bundles with their tips at these sites are organized to
constitute the core of microspikes. This is consistent with findings reported
previously (Katoh et al.,
1999b; Oldenbourg et al.,
2000
). Additionally, the WAVE-concentrated point becomes an
emergent site of microspikes and protrudes outward nub-like
(Fig. 7B). In the vicinity of
the WAVE molecule, the actin-barbed ends interact loosely with the VCA region,
and monomeric actins bind to the ends stochastically with Brownian motion of
filaments. The filopodial portions of actin bundles are fastened by fascin
(Cohan et al., 2001
) and the
proximal ends join the actin network and microtubule in the filopodial base in
the lamellipodia cytoplasm (Schaefer et
al., 2002
). Therefore, they suppress the backward Brownian motion
of the proximal portion of actin bundles and cause a ratchet effect on the
whole bundle. Consequently, actin polymerization at the tips produces a
mechanical force to drive extension in the forward direction as theoretically
described (Mogilner and Oster,
1996
; Theriot,
2000
). G-actin molecules once polymerized in the bundle move
towards the base by a retrograde flow
(Kirschner, 1980
; Watanabe and
Mitchson, 2002). If the polymerization rate at the tip is faster than the
velocity of retrograde flow, filopodia continue to elongate (Mallavarapu and
Mitchson, 1999). Our observations show that clusters of WAVE isoforms remained
at the filopodial tips during elongation. By contrast, when filopodia and
lamellipodia retracted, the WAVE isoforms disappeared at the leading edge and
tips (Fig. 7C). Recently,
Steketee and Tosney proposed another working model of how filopodia emerge and
elongate after the formation of a focal ring at the periphery of the veil
(Steketee and Tosney, 2001
;
Steketee and Tosney, 2002
).
However, their model explains the emergence and extension of new filopodia
only if mature filopodia already exist in the growth cone. By contrast, our
model describes the case in which new filopodia simultaneously emerge and
elongate from the growth cone of lamellipodia in response to guidance cues. In
the latter case, our model is more reasonable. In the near future we will,
hopefully, shed light on the detailed roles of profilin, Arp2/3 and WAVE
isoforms at the filopodia tips.
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Acknowledgments |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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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.[Medline]
Bentley, D. and O'Connor, T. P. (1994). Cytoskeletal events in growth cone steering. Curr. Opin. Neurobiol. 4,43 -48.[Medline]
Borisy, G. G. and Svitkina, T. M. (2000). Actin machinery: pushing the envelope. Curr. Opin. Cell Biol. 12,104 -112.[CrossRef][Medline]
Cohan, C. S., Welnhofer, E. A., Zhao, L., Matsumura, F. and Yamashiro, S. (2001). Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia. Cell Motil. Cytoskeleton 48,109 -120.[CrossRef][Medline]
Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. and Kirschner, M. W. (2002). Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418,790 -793.[CrossRef][Medline]
Furuya, S. and Furuya, K. (1983). Ultrastructural changes in differentiating neuroblastoma x glioma hybrid cells. Tissue Cell 15,903 -919.[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]
Hall, A. (1998). Rho GTPases and the actin
cytoskeleton. Science
279,509
-514.
Heath, J. P. and Holifield, B. F. (1993). On the mechanisms of cortical actin flow and its role in cytoskeletal organisation of fibroblasts. Symp. Soc. Exp. Biol. 47, 35-56.[Medline]
Katoh, K., Hammar, K., Smith, P. J. and Oldenbourg, R.
(1999a). Birefringence imaging directly reveals architectural
dynamics of filamentous actin in living growth cones. Mol. Biol.
Cell 10,197
-210.
Katoh, K., Hammar, K., Smith, P. J. and Oldenbourg, R.
(1999b). Arrangement of radial actin bundles in the growth cone
of Aplysia bag cell neurons shows the immediate past history of filopodial
behavior. Proc. Natl. Acad. Sci. USA
96,7928
-7931.
Kim, M. D., Kolodziej, P. and Chiba, A. (2002).
Growth cone pathfinding and filopodial dynamics are mediated separately by
Cdc42 activation. J. Neurosci.
22,1794
-1806.
Kirschner, M. W. (1980). Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vivo. J. Cell Biol. 86,330 -334.[Abstract]
Lanier, L. M. and Gertler, F. B. (2000). From Abl to actin: Abl tyrosine kinase and associated proteins in growth cone motility. Curr. Opin. Neurobiol. 10, 80-87.[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]
Lewis, A. K. and Bridgman, P. C. (1992). Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity. J. Cell Biol. 119,1219 -1243.[Abstract]
Li, Z., Aizenman, C. D. and Cline, H. T. (2002). Regulation of rho GTPases by crosstalk and neuronal activity in vivo. Neuron 33,741 -750.[Medline]
Lommel, S., Benesch, S., Rottner, K., Franz, T., Wehland, J. and
Kuhn, 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
-870.
Mallavarapu, A. and Mitchison, T. (1999).
Regulated actin cytoskeleton assembly at filopodium tips controls their
extension and retraction. J. Cell Biol.
146,1097
-1106.
Miki, H., Suetsugu, S. and Takenawa, T. (1998).
WAVE, a novel WASP-family protein involved in actin reorganization induced by
Rac. EMBO J. 17,6932
-6941.
Miki, H., Fukuda, M., Nishida, E. and Takenawa, T.
(1999). Phosphorylation of WAVE downstream of mitogen-activated
protein kinase signaling. J. Biol. Chem.
274,27605
-27609.
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]
Mogilner, A. and Oster, G. (1996). Cell motility driven by actin polymerization. Biophys. J. 71,3030 -3045.[Abstract]
Nakagawa, H., Yoshida, M. and Miyamoto, S. (2000). Nitric oxide underlies the differentiation of PC12 cells induced by depolarization with high KCl. J. Biochem. 127,113 -119.[Abstract]
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.
Nikolic, M. (2002). The role of Rho GTPases and associated kinases in regulating neurite outgrowth. Int. J. Biochem. Cell Biol. 34,731 -745.[CrossRef][Medline]
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]
Oldenbourg, R., Katoh, K. and Danuser, G.
(2000). Mechanism of lateral movement of filopodia and radial
actin bundles across neuronal growth cones. Biophys.
J. 78,1176
-1182.
Pollard, T. D., Blanchoin, L. and Mullins, R. D. (2000). Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29,545 -576.[CrossRef][Medline]
Rottner, K., Behrendt, B., Small, J. V. and Wehland, J. (1999). VASP dynamics during lamellipodia protrusion. Nat. Cell Biol. 1,321 -322.[CrossRef][Medline]
Schaefer, A. W., Kabir, N. and Forscher, P.
(2002). Filopodia and actin arcs guide the assembly and transport
of two populations of microtubules with unique dynamic parameters in neuronal
growth cones. J. Cell Biol.
158,139
-152.
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]
Steketee, M. and Tosney, K. W. (2001).
Filopodial initiation and a novel filament-organizing center, the focal ring.
Mol. Biol. Cell 12,2378
-2395.
Steketee, M. B. and Tosney, K. W. (2002). Three
functionally distinct adhesions in filopodia: shaft adhesions control lamellar
extension. J. Neurosci.
22,8071
-8083.
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]
Suetsugu, S., Miki, H. and Takenawa, T. (2002). Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins. Cell Motil. Cytoskeleton 51,113 -122.[CrossRef][Medline]
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.
Theriot, J. A. (2000). The polymerization motor. Traffic 1,19 -28.[CrossRef][Medline]
Theriot, J. A. and Mitchison, T. J. (1991). Actin microfilament dynamics in locomoting cells. Nature 352,126 -131.[CrossRef][Medline]
Watanabe, N. and Mitchison, T. J. (2002).
Single-molecule speckle analysis of actin filament turnover in lamellipodia.
Science 295,1083
-1086.
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.
271,2921
-2928.