Regulation of actin cytoskeleton architecture by Eps8 and Abi1
© Roffers-Agarwal et al; licensee BioMed Central Ltd. 2005
Received: 24 May 2005
Accepted: 14 October 2005
Published: 14 October 2005
The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive.
Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects.
Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.
Remodeling of the actin cytoskeleton is critical for mediating changes in cell shape, migration, and adhesion. Actin filament architecture is regulated by a large group of actin binding proteins that modulate actin assembly, disassembly, branching, and bundling . Actin organization is also regulated by growth factor signals that stimulate the activity of Rho family GTPases, which mediate actin remodeling and formation of stress fibers, filopodia, and membrane ruffles . Although much has been learned about the general properties of actin binding proteins, the mechanisms by which these proteins control actin architecture in vivo are poorly understood.
Eps8 (EGF receptor pathway substrate 8) was originally identified as a substrate of the EGF receptor  and is the founding member of a multigene family of Eps8-like proteins named Eps8L1, Eps8L2, and Eps8L3 [4, 5]. Eps8 is thought to transduce growth factor signals by acting as a scaffold protein to support the formation of multi-protein signaling complexes that promote the activation of Rho family GTPases. Consistent with this model, studies in Eps8 null fibroblasts showed that Eps8 is required for growth factor-induced Rac activation as well as Rac-dependent actin remodeling and membrane ruffling . Eps8 is a critical component of a complex that contains the p85 regulatory subunit of phosphoinositide 3-kinase, Abi1, and Sos1, which acts as a guanine nucleotide exchange factor (GEF) for Rac [6, 7]. Eps8 interacts directly with Abi1 through its SH3 domain, which possesses a novel peptide binding specificity , and this binding is thought to relieve auto-inhibition of Eps8 .
Eps8 also directly binds actin, suggesting that it may function by localizing Rac to sites of actin remodeling . Eps8 binds actin through its C-terminal effector domain and expression of the effector region in serum-starved cells elicits Rac-dependent actin remodeling and membrane ruffling . Studies using deletion mutants of Eps8 show that the C-terminal effector domain is required for localizing Eps8 to membrane ruffles and the transduction of signals to Rac . A recent study revealed that C-terminal fragments of Eps8 also possess actin barbed-end capping activity in vitro and can substitute for capping protein in actin-based motility assays, suggesting a mechanism by which Eps8 might regulate actin filament dynamics in vivo . Interestingly, full-length Eps8 on its own lacks capping activity in vitro, but can block actin polymerization in the presence of Abi1 . The capping activity of Eps8 does not require Rac indicating that Eps8 can modulate actin dynamics through Rac-dependent and -independent mechanisms. Together, these data implicate Eps8 as a key regulator of actin filament dynamics and suggest that its activity is modulated through association with distinct sets of interacting regulatory proteins.
Eps8 has also been shown to bind Dishevelled (Dsh) , a key regulator of canonical and non-canonical Wnt signaling [12, 13]. Dsh is required for the establishment of cell polarity and directed migration during gastrulation in vertebrates [14–16]. The mechanism by which Dsh controls cell polarity and migration is unclear, but is hypothesized to involve the modulation of actin dynamics through activation of RhoA and Rac [17, 18]. The ability of Eps8 to bind both Dsh and actin and stimulate Rac activation suggests that Eps8 may play an important role in regulating Dsh function during gastrulation, but this possibility has not been investigated.
In this study, we utilized cultured mammalian cells and Xenopus embryos as model systems to investigate the mechanism by which Eps8 regulates actin filament architecture in vivo. Our results provide evidence that Eps8 can stimulate the assembly of distinct types of actin-based structures in cells and that the morphology of the actin structures induced by Eps8 is dependent on its interactions with Abi1. In addition, we show that Eps8 can recruit actin regulatory proteins, such as N-WASP and Dsh, to actin filaments and that mis-expression of Eps8 impairs cell movements during gastrulation in Xenopus embryos. Together, these data suggest that the role of Eps8 in modulating actin organization is multifaceted and is dependent on its participation in several potentially distinct multi-protein actin regulatory complexes.
Enhanced formation of filopodia-like structures in cells expressing Eps8
Abi1 modulates Eps8-dependent actin remodeling
Eps8 induces actin remodeling in Xenopus embryos
Abi1 modulates the activity of Eps8 in Xenopus embryos
Recruitment of Actin Regulatory Proteins to Eps8/Abi1-induced actin structures in Xenopus
WASP/Scar proteins play an important role in stimulating actin filament nucleation by the Arp2/3 complex [25–27]. To test whether the formation of actin stars involves recruitment of WASP proteins we analyzed the distribution of N-WASP-GFP in animal cap cells expressing Eps8 and Abi1. N-WASP co-localized with Eps8 and actin (Figure 5E–H), indicating that WASP proteins are recruited to Eps8/Abi1-induced actin structures. We also tested whether N-WASP activity is required for Eps8/Abi1-induced actin star formation by co-expressing Eps8, Abi1 and a dominant negative form of N-WASP (N-WASP-CA). We found that N-WASP-CA expression did not significantly alter the actin structures induced by Eps8 and Abi1 (data not shown). These data suggest that Eps8 and Abi1 can recruit actin nucleators to specific sites in the cell, although N-WASP function may not be strictly required for Eps8/Abi1-induced actin remodeling.
Members of the Ena/VASP family are critical regulators of actin filament dynamics and are thought to antagonize actin filament capping at the leading edge of migrating cells . Given this central role, we tested whether increased or decreased Ena/VASP activity would affect Eps8/Abi1-induced actin star formation. Expression of a dominant negative protein (FP4-mito-GFP, [28, 29]) that specifically neutralizes the function of all Ena/VASP proteins was used to knockdown Ena/VASP activity whereas expression GFP-tagged Xenopus VASP (Xvasp) was used to increase Ena/VASP activity. The ability of the FP4-mito dominant negative to mis-localize Ena/VASP proteins in Xenopus was confirmed by showing that it caused the redistribution of endogenous Ena from the cell periphery to the mitochondria surface (data not shown). We found that neither FP4-mito-GFP (Figure 5I–L) nor Xvasp-GFP (Figure 5M–P) had an effect on the presence of Eps8/Abi1-induced actin stars. In addition, Xvasp-GFP did not co-localize with the actin stars, indicating that Ena/VASP proteins are not recruited to these actin structures (Figure 5M–P).
Eps8 recruits Dsh to the membrane and actin filaments
Identification and developmental expression of Xenopus Eps8
To test the requirement for XEps8 during development, we utilized a morpholino (MO) antisense oligonucleotide targeted to the 5'-untranslated region to specifically knockdown levels of XEps8 protein during development. We found that the XEps8 MO could specifically block the expression of a myc-tagged version of XEps8, but injection of the XEps8 MO into 4-cell stage embryos resulted in embryos with no apparent phenotype (data not shown). The lack of a knockdown phenotype is not surprising since Eps8-/- mice also displayed no obvious phenotype . Since Eps8 is a member of a multi-gene family, we searched TIGR and NCBI databases for additional Xenopus Eps8 genes and found evidence for a second XEps8 gene as well as three XEps8-like genes. Therefore, the lack of a phenotype in XEps8 knockdown embryos is likely due to the expression of multiple XEps8 family members, including XEps8L1, XEps8L2, and XEps8L3, during early development (Roffers-Agarwal and Miller, unpublished results). Thus, assessing the role of Eps8 proteins in Xenopus will require novel knockdown techniques capable of simultaneously and specifically inhibiting the activity of multiple gene products during early development.
Expression of Eps8 disrupts cell movements during gastrulation
Since knockdown experiments produced negative results, we performed mis-expression experiments to test whether altering Eps8 activity would affect cell movements during gastrulation. Synthetic mRNA encoding mouse Eps8-myc or GFP as a control was injected into the equatorial region of both dorsal blastomeres at the 4-cell stage and resulting embryos were then examined for developmental abnormalities. Defects in Eps8-injected embryos were first apparent at stage 10.5 (early gastrula). At this stage, control embryos formed a well-defined dorsal lip indicative of the onset of gastrulation movements and involution of dorsal mesoderm. In contrast, Eps8-injected embryos showed a delay in the formation of the dorsal lip and when observed, the lip was disorganized (data not shown). By stage 12, Eps8-injected embryos displayed a severe delay in blastopore closure and buckling of tissue above the blastopore (Figure 7D). Eps8-injected embryos eventually complete gastrulation and tadpoles displayed a phenotype including a shortened and arched anterior-posterior axis and head defects (Figure 7F). The defects caused by Eps8 are dose dependent; low doses (50 pg) of Eps8 result in cyclopia and a shortened A-P axis, moderate doses (200 pg) show varying degrees of cyclopia, microcephaly, and shortening and arching of the A-P axis, and high doses (1 ng) result in varying degrees of anencephaly, shortening and arching of the A-P axis, and spina bifida. Control, GFP-injected embryos appeared normal at all stages examined (Figure 7C,E). These data are consistent with the idea that Eps8-induced actin re-organization leads to defects in cell movements during gastrulation in Xenopus.
The gross morphological defects caused by dorsal expression of Eps8 could be the result of defects in convergent extension or inhibition of mesoderm development, both of which would give superficially similar phenotypes. In order to distinguish between these two possibilities we performed histological analysis on injected embryos (Figure 7G,H). Histological sections of Eps8-injected embryos demonstrated that notochord, somites, and neural tissue are all present, showing that expression of Eps8 does not globally perturb specification of mesodermal or neural cell fates. Instead, expression of Eps8 resulted in broadening of the notochord along the mediolateral axis and morphological defects in the neural tube and somites. The widening of the notochord is consistent with the idea that expression of Eps8 impairs convergent extension movements of the axial mesoderm.
Here, we have investigated how Eps8 regulates actin filament architecture and how this activity impacts cell movements during gastrulation. Our results, together with previous studies, provide evidence that Eps8 plays multiple roles in regulating the actin cytoskeleton and that these functions are influenced by the participation of Eps8 in multi-protein actin regulatory complexes.
Based on in vitro studies, Eps8 is hypothesized to promote capping of actin barbed-ends in an Abi1-dependent manner . Our findings suggest that in addition to its proposed role as a barbed end capping protein, Eps8 might play additional roles in regulating actin organization in vivo. This idea is supported by the observation that Eps8 expression resulted in enhanced formation of actin-rich filopodia-like structures in cultured cells and enhanced formation of actin bundles and accumulation of actin at cell-cell junctions in Xenopus embryos. The presence of the filopodia-like structures on the dorsal surface of cells suggests that they are protrusive in nature and do not represent retraction structures, which are typically associated with sites of cell adhesion. Additional studies examining the dynamics of these Eps8-induced structures will help clarify the origin and nature of these structures. In addition, we found that Abi1 modulated Eps8 activity, promoting the formation of actin cables in cultured cells and actin stars in Xenopus, suggesting that Eps8 can regulate actin dynamics through Abi1-dependent and -independent mechanisms. Consistent with this idea, Abi1 did not co-localize with Eps8 at the tips of the filopodia-like structures in cultured cells suggesting that additional regulators of Eps8 remain to be identified.
The correlation between Eps8 expression and enhanced formation of filopodia-like structures and actin cables is consistent with the idea that Eps8 may regulate actin filament elongation in vivo. Regulation of barbed-end elongation and filopodia formation is thought to involve a balance between barbed-end capping and anti-capping activities. Proteins such as CP are hypothesized to block elongation and favor formation of a dendritic network , whereas proteins including Ena/VASP proteins, which antagonize capping, are hypothesized to promote actin filament elongation and filopodia formation [28, 36, 37]. Our work examining the regulation of Eps8 activity by CP, N-WASP, and Ena/VASP in Xenopus yielded largely negative results, however, making it difficult to discern the relative contribution of Eps8 capping activity versus other potential modes of activity in the regulation of actin architecture. Further biochemical analyses will help elucidate the molecular mechanism(s) by which Eps8 regulates actin dynamics in vivo.
Previous work [6, 7, 9, 38] and our results show that the ability of Eps8 to modulate actin organization is regulated by its interaction with distinct binding partners such as Abi1. We found that Abi1 can modulate Eps8 activity in cultured cells and Xenopus embryos. Abi1 binds to the SH3 domain of Eps8 [38, 39] and it has been proposed that this binding may alter the conformation or activity of the adjacent actin-binding domain of Eps8 . The mechanism by which Abi1 might regulate Eps8 activity remains unclear, but may involve recruitment of additional regulatory factors such as Dsh, Sos1, and Rac to the Eps8/Abi1 complex [7, 38]. In addition, our work shows that N-WASP is recruited to Eps8/Abi1-induced actin stars suggesting that the Eps8/Abi1 complex interacts either directly or indirectly with actin nucleating factors. This idea is supported by the observation that Eps8 can facilitate actin-based motility of N-WASP-coated beads in vitro in the presence of Arp2/3, ADF/cofilin, and profilin . Further studies will be required to examine how Abi1 modulates Eps8 activity and how Eps8 works with Abi1 and other regulatory factors to control actin organization in vivo.
Eps8 has been shown to bind Dsh , a component of the Wnt signaling pathway that is required for transduction of canonical Wnt/β-catenin and non-canonical signals [12, 13]. Here, we have shown that Eps8 expression recruits Dsh to actin filaments and the cell membrane in Xenopus. These data are significant because the role of Dsh in non-canonical Wnt/Polarity signaling is thought to be dependent on its localization to the membrane and its ability to affect cell polarity and migration through regulation of the actin cytoskeleton [14–18]. Dsh activity during gastrulation is dependent on both RhoA and Rac, and the formin homology protein DAAM1 is required for Dsh-mediated activation of RhoA [17, 18]. However, a link between Dsh and Rac has not been identified. The Eps8/Abi1/Sos1 complex is required for growth factor stimulated activation of Rac , suggesting that Eps8 might provide an important link between Dsh, Rac, and the actin cytoskeleton during development. Consistent with this idea, expression of Eps8 impaired cell movements during gastrulation and Eps8, Abi1, and Dsh co-localize in Xenopus suggesting that these proteins can form a tri-complex in vivo. Interestingly, we did not observe an effect of Eps8 on Dsh-mediated induction of Wnt/β-catenin target genes (siamois and Xnr3, JRA and JRM unpublished results) indicating that Eps8 does not participate in canonical Wnt/β-catenin signaling. Unfortunately, our attempts to analyze the requirement for Eps8 in Xenopus were unsuccessful due to the expression of multiple Eps8 family members during early development. Thus, additional studies are necessary to determine the potential role of Eps8 in the transduction of non-canonical Wnt signals and the potential role of Eps8 family members during gastrulation in vertebrates.
How might Eps8 regulate the actin cytoskeleton in vivo? Our findings together with data from previous studies support the idea that Eps8 might regulate actin architecture in multiple ways. Eps8 can bind to both barbed ends and the sides of actin filaments [9, 10] and it is possible that these different modes of actin binding mediate distinct effects on actin architecture in cells. Barbed-end capping activity might regulate actin filament dynamics and stabilize existing filaments whereas an alternative activity might promote the formation and maintenance of actin arrays required for protrusive force generation and cellular structures such as microvilli and filopodia. This idea is consistent with our observation that Eps8 is enriched at the tips of filopodia-like structures and localizes along the length of the filopodia-like structures and actin cables. This model is also in agreement with the observation that Eps8 localizes to microvilli in the intestinal epithelium of C. elegans and knockdown of Eps8 is associated with defects in microvilli formation . The formation of actin cables in cells expressing Eps8 and Abi1 and actin clusters in Xenopus embryos suggests that Abi1 is a critical modulator of Eps8's activity as an actin regulatory protein. The finding that Eps8 expression impairs cell movements during gastrulation provides further support for this view and underscores the idea that the proper balance of actin assembly, disassembly, and organization is essential for controlling morphogenetic movements during development. Thus, Eps8 has emerged as a critical regulator of actin filament dynamics and further analysis of Eps8 and its binding partners will help shed light on the mechanisms that mediate actin-based motility in vivo.
Expression constructs, antibodies, and cells
Constructs used were: mouse Eps8-myc pCS2+ (Eps8 cDNA provided by Dr. P.P. DiFiore, European Institute of Oncology, Milan ), human Abi1-GFP pCS2+ (Abi1 cDNA provided by Dr. Ann Marie Pendergast, Duke University; ), human capping protein α-GFP and β-GFP pCS2+ (capping protein cDNAs provided by Dr. Dorothy Schafer, University of Virginia ), FP4-mito-GFP pCS2+ (FP4-mito cDNA provided by Dr. Frank Gertler, MIT; ), RacN17 pCS2+ (RacN17 cDNA provided by Dr. Jennifer Westendorf, University of Minnesota), Xenopus Dsh-GFP pCS2+ , and Xenopus Dsh-flag pCS2+ . Xenopus N-WASP-mRFP pCS2+, Xenopus N-WASP-CA-mRFP pCS2+ and Xenopus VASP-GFP pCS2+ were constructed by PCR using full length IMAGE cDNAs obtained from ATCC. Details of vector construction are available upon request. Primary antibodies used were: mouse anti-c-myc 9e10 , mouse anti-flag (Sigma), rabbit anti-Eps8 (Santa Cruz Biotechnology), and anti-Rac (Transduction Labs). Anti-XEps8 rabbit polyclonal antibodies were raised against a peptide corresponding to the carboxyl terminal (NH2-SDSGVESFDEGNSH-COOH) conjugated to KLH (Sigma Genosys). A cysteine residue was added to the amino terminus of the peptide to facilitate conjugation to KLH. Secondary antibodies used were: Alexa568 goat anti-mouse, Alexa647 goat anti-mouse, and goat anti-rabbit Alexa568 and Alexa647 (all secondary antibodies were from Molecular Probes). Alexa568 phalloidin (Molecular Probes) was used to visualize F-actin. Cells used were B16F1 mouse melanoma cells (ATCC), MDA-MB231, and MDA-MB231BO (provided by Dr. Douglas Yee, University of Minnesota).
Cell culture, transfections, and imaging
B16F1, MDA-MB231, and MDA-MB231BO cells were grown in DMEM (CellGro) supplemented with 10% FBS (HyClone) at 5% CO2. For transfections, cells were plated on acid washed coverslips and transfected with Lipofectamine (Invitrogen). For imaging, cells were washed once with PBS and fixed in 4% formaldehyde in CSK buffer (10 mM Hepes pH 7.5, 150 mM sucrose, mM EGTA, 0.1% Triton X-100) for 15 min. at room temperature. Alternatively, cells were permeabilized with 0.1% Triton X-100 in PEM buffer (10 mM Pipes pH 7.4, 1 mM EDTA, 1 mM MgCl2) for 30 seconds and fixed with pre-warmed 4% paraformaldehyde in PEM buffer for 30 min. at 37°C. Fixed cells were then washed three times in PBS + 0.1% Triton X-100 (PBST), and incubated in PBST, 2% BSA, 10% normal goat serum (NGS) to prevent non-specific binding of antibodies. Staining with primary and secondary antibodies was performed in PBST, 2% BSA, 10% NGS for 2 hours at room temperature. Images were collected using a Zeiss spinning disc confocal microscope and digital images were processed using Adobe Photoshop.
Embryos, microinjections, imaging, RT-PCR, and Western blotting
Xenopus laevis eggs were fertilized in vitro and subsequently de-jellied in 2% cysteine (Sigma Chemical). Embryos were reared in 1/3× Marc's Modified Ringer's (MMR). Embryos were staged according to Nieuwkoop and Faber . Animal cap explants were prepared using a Gastromaster microsurgery instrument (Xenotek Engineering) and cultured in 1× Steinberg's in the presence of 10 ng/ml Activin (R&D Systems). For microinjections, embryos were placed in a solution of 4% Ficoll in 1/3× MMR and injected using a Harvard Apparatus microinjector and a Narashige micromanipulator. Injected embryos were reared in 4% Ficoll in 1/3× MMR supplemented with 10 μg/ml Gentamicin (Invitrogen) to stage 9 then washed and reared in 1/3× MMR + Gentamicin. Capped mRNA for injections was synthesized using mMessage Machine (Ambion) and purified using NucAway columns (Ambion).
For imaging, embryos and explants were fixed in 4% formaldehyde in CSK buffer at room temperature for 30 min., washed three times in PBST, and incubated in PBST, 2% BSA, 10% NGS to prevent non-specific binding of antibodies. Staining with primary and secondary antibodies was performed in PBST, 2% BSA for 2 hours at room temperature. Actin was visualized with Alexa568 phalloidin (Molecular Probes). Images were captured with a Zeiss spinning disk confocal microscope and digital images were processed with Adobe Photoshop.
RT-PCR analysis was performed using total RNA isolated from Xenopus explants. cDNA was prepared using SuperScript II reverse transcriptase (Invitrogen) and PCR was performed using GoTaq polymerase (Promega). Primers used for RT-PCR: XEps8: forward: 5'-attccctgagatgttgctccg-3', reverse: 5'-tagcagcagcgatttgccc-3'; XmyoD: forward: 5'-agctccaactgctccgacggcatgaa-3', reverse: 5'-aggagagaatccagttgatggaaca-3'; Xbra: forward: 5'-ggatcgttatcacctctg-3', reverse: 5'-gtgtagtctgtagcagca-3'; and ODC: forward: 5'-gccattgtgaagactctctccattc-3', reverse: 5'-ttcgggtgattccttgccac-3'.
Protein lysates for Western blots were prepared by homogenizing embryos in ice-cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100) supplemented with protease inhibitors (1 mM PMSF, 1 mM pepstatin, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Homogenates were cleared by centrifugation at 14,000 rpm for 10 min. at 4°C. SDS sample buffer was added to the cleared lysate and boiled for 4 min. prior to separation by SDS-PAGE. Approximately one embryo equivalent was loaded per lane on 10% gels (BioRad). Proteins were blotted to PVDF membrane (BioRad), blots were blocked in 5% milk in TBS + 0.1% Tween, and probed with anti-XEps8 antibodies (1:2000) for two hours at room temperature. Visualization was performed using a horseradish peroxidase conjugated anti-rabbit secondary antibody (Jackson ImmunoLabs) and enhanced chemiluminescence (Pierce).
List of abbreviations used
Epidermal Growth Factor substrate 8
Abl Interacting Protein 1
Reverse Transcriptase Polymerase Chain Reaction
The authors wish to thank members of the Miller lab for critical reading of the manuscript. We are grateful to Drs. Pier Paolo Di Fiore, Anne Marie Pendergast, Dorothy Schafer, Jennifer Westendorf, and Lorene Lanier for cDNA constructs. We thank our colleague Dr. Lorene Lanier for helpful insights and comments on the manuscript. This work was supported by a grant from the National Science Foundation to J.R.M. (0315767).
- Revenu C, Athman R, Robine S, Louvard D: The co-workers of actin filaments: from cell structures to signals. Nat Rev Mol Cell Biol. 2004, 5 (8): 635-646. 10.1038/nrm1437.View ArticlePubMedGoogle Scholar
- Raftopoulou M, Hall A: Cell migration: Rho GTPases lead the way. Dev Biol. 2004, 265 (1): 23-32. 10.1016/j.ydbio.2003.06.003.View ArticlePubMedGoogle Scholar
- Fazioli F, Minichiello L, Matoska V, Castagnino P, Miki T, Wong WT, Di Fiore PP: Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. Embo J. 1993, 12 (10): 3799-3808.PubMed CentralPubMedGoogle Scholar
- Tocchetti A, Confalonieri S, Scita G, Di Fiore PP, Betsholtz C: In silico analysis of the EPS8 gene family: genomic organization, expression profile, and protein structure. Genomics. 2003, 81 (2): 234-244. 10.1016/S0888-7543(03)00002-8.View ArticlePubMedGoogle Scholar
- Offenhauser N, Borgonovo A, Disanza A, Romano P, Ponzanelli I, Iannolo G, Di Fiore PP, Scita G: The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway. Mol Biol Cell. 2004, 15 (1): 91-98. 10.1091/mbc.E03-06-0427.PubMed CentralView ArticlePubMedGoogle Scholar
- Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S, Bjarnegard M, Betsholtz C, Di Fiore PP: EPS8 and E3B1 transduce signals from Ras to Rac. Nature. 1999, 401 (6750): 290-293. 10.1038/45822.View ArticlePubMedGoogle Scholar
- Innocenti M, Frittoli E, Ponzanelli I, Falck JR, Brachmann SM, Di Fiore PP, Scita G: Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J Cell Biol. 2003, 160 (1): 17-23. 10.1083/jcb.200206079.PubMed CentralView ArticlePubMedGoogle Scholar
- Kishan KV, Scita G, Wong WT, Di Fiore PP, Newcomer ME: The SH3 domain of Eps8 exists as a novel intertwined dimer. Nat Struct Biol. 1997, 4 (9): 739-743. 10.1038/nsb0997-739.View ArticlePubMedGoogle Scholar
- Disanza A, Carlier MF, Stradal TE, Didry D, Frittoli E, Confalonieri S, Croce A, Wehland J, Di Fiore PP, Scita G: Eps8 controls actin-based motility by capping the barbed ends of actin filaments. Nat Cell Biol. 2004, 6 (12): 1180-1188. 10.1038/ncb1199.View ArticlePubMedGoogle Scholar
- Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP: An effector region in Eps8 is responsible for the activation of the Rac- specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J Cell Biol. 2001, 154 (5): 1031-1044. 10.1083/jcb.200103146.PubMed CentralView ArticlePubMedGoogle Scholar
- Inobe M, Katsube K, Miyagoe Y, Nabeshima Y, Takeda S: Identification of EPS8 as a Dvl1-associated molecule. Biochem Biophys Res Commun. 1999, 266 (1): 216-221. 10.1006/bbrc.1999.1782.View ArticlePubMedGoogle Scholar
- Veeman MT, Axelrod JD, Moon RT: A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell. 2003, 5 (3): 367-377. 10.1016/S1534-5807(03)00266-1.View ArticlePubMedGoogle Scholar
- Wharton KA: Runnin' with the Dvl: proteins that associate with Dsh/Dvl and their significance to Wnt signal transduction. Dev Biol. 2003, 253 (1): 1-17. 10.1006/dbio.2002.0869.View ArticlePubMedGoogle Scholar
- Tada M, Smith JC: Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development. 2000, 127 (10): 2227-2238.PubMedGoogle Scholar
- Wallingford JB, Harland RM: Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis. Development. 2001, 128 (13): 2581-2592.PubMedGoogle Scholar
- Wallingford JB, Rowning BA, Vogeli KM, Rothbacher U, Fraser SE, Harland RM: Dishevelled controls cell polarity during Xenopus gastrulation. Nature. 2000, 405 (6782): 81-85. 10.1038/35011077.View ArticlePubMedGoogle Scholar
- Habas R, Dawid IB, He X: Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev. 2003, 17 (2): 295-309. 10.1101/gad.1022203.PubMed CentralView ArticlePubMedGoogle Scholar
- Habas R, Kato Y, He X: Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1. Cell. 2001, 107 (7): 843-854. 10.1016/S0092-8674(01)00614-6.View ArticlePubMedGoogle Scholar
- Fidler IJ: Biological behavior of malignant melanoma cells correlated to their survival in vivo. Cancer Res. 1975, 35 (1): 218-224.PubMedGoogle Scholar
- Cailleau R, Young R, Olive M, Reeves WJ: Breast tumor cell lines from pleural effusions. J Natl Cancer Inst. 1974, 53 (3): 661-674.PubMedGoogle Scholar
- Yoneda T, Williams PJ, Hiraga T, Niewolna M, Nishimura R: A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro. J Bone Miner Res. 2001, 16 (8): 1486-1495.View ArticlePubMedGoogle Scholar
- Mongiovi AM, Romano PR, Panni S, Mendoza M, Wong WT, Musacchio A, Cesareni G, Di Fiore PP: A novel peptide-SH3 interaction. Embo J. 1999, 18 (19): 5300-5309. 10.1093/emboj/18.19.5300.PubMed CentralView ArticlePubMedGoogle Scholar
- Cooper JA, Schafer DA: Control of actin assembly and disassembly at filament ends. Curr Opin Cell Biol. 2000, 12 (1): 97-103. 10.1016/S0955-0674(99)00062-9.View ArticlePubMedGoogle Scholar
- Wear MA, Cooper JA: Capping protein: new insights into mechanism and regulation. Trends Biochem Sci. 2004, 29 (8): 418-428. 10.1016/j.tibs.2004.06.003.View ArticlePubMedGoogle Scholar
- Bompard G, Caron E: Regulation of WASP/WAVE proteins: making a long story short. J Cell Biol. 2004, 166 (7): 957-962. 10.1083/jcb.200403127.PubMed CentralView ArticlePubMedGoogle Scholar
- Higgs HN, Pollard TD: Regulation of actin filament network formation through ARP2/3 complex: activation by a diverse array of proteins. Annu Rev Biochem. 2001, 70: 649-676. 10.1146/annurev.biochem.70.1.649.View ArticlePubMedGoogle Scholar
- Weaver AM, Young ME, Lee WL, Cooper JA: Integration of signals to the Arp2/3 complex. Curr Opin Cell Biol. 2003, 15 (1): 23-30. 10.1016/S0955-0674(02)00015-7.View ArticlePubMedGoogle Scholar
- Bear JE, Svitkina TM, Krause M, Schafer DA, Loureiro JJ, Strasser GA, Maly IV, Chaga OY, Cooper JA, Borisy GG, Gertler FB: Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell. 2002, 109 (4): 509-521. 10.1016/S0092-8674(02)00731-6.View ArticlePubMedGoogle Scholar
- Bear JE, Loureiro JJ, Libova I, Fassler R, Wehland J, Gertler FB: Negative regulation of fibroblast motility by Ena/VASP proteins. Cell. 2000, 101 (7): 717-728. 10.1016/S0092-8674(00)80884-3.View ArticlePubMedGoogle Scholar
- Sokol SY: Analysis of Dishevelled signalling pathways during Xenopus development. Curr Biol. 1996, 6 (11): 1456-1467. 10.1016/S0960-9822(96)00750-6.View ArticlePubMedGoogle Scholar
- Wallingford JB, Harland RM: Neural tube closure requires Dishevelled-dependent convergent extension of the midline. Development. 2002, 129 (24): 5815-5825. 10.1242/dev.00123.View ArticlePubMedGoogle Scholar
- Yang-Snyder J, Miller JR, Brown JD, Lai CJ, Moon RT: A frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr Biol. 1996, 6 (10): 1302-1306. 10.1016/S0960-9822(02)70716-1.View ArticlePubMedGoogle Scholar
- Howard JE, Smith JC: Analysis of gastrulation: different types of gastrulation movement are induced by different mesoderm-inducing factors in Xenopus laevis. Mech Dev. 1993, 43 (1): 37-48. 10.1016/0925-4773(93)90021-O.View ArticlePubMedGoogle Scholar
- Kwan KM, Kirschner MW: Xbra functions as a switch between cell migration and convergent extension in the Xenopus gastrula. Development. 2003, 130 (9): 1961-1972. 10.1242/dev.00412.View ArticlePubMedGoogle Scholar
- Mejillano MR, Kojima S, Applewhite DA, Gertler FB, Svitkina TM, Borisy GG: Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end. Cell. 2004, 118 (3): 363-373. 10.1016/j.cell.2004.07.019.View ArticlePubMedGoogle Scholar
- Han YH, Chung CY, Wessels D, Stephens S, Titus MA, Soll DR, Firtel RA: Requirement of a vasodilator-stimulated phosphoprotein family member for cell adhesion, the formation of filopodia, and chemotaxis in dictyostelium. J Biol Chem. 2002, 277 (51): 49877-49887. 10.1074/jbc.M209107200.View ArticlePubMedGoogle Scholar
- Lebrand C, Dent EW, Strasser GA, Lanier LM, Krause M, Svitkina TM, Borisy GG, Gertler FB: Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron. 2004, 42 (1): 37-49. 10.1016/S0896-6273(04)00108-4.View ArticlePubMedGoogle Scholar
- Innocenti M, Tenca P, Frittoli E, Faretta M, Tocchetti A, Di Fiore PP, Scita G: Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J Cell Biol. 2002, 156 (1): 125-136. 10.1083/jcb.200108035.PubMed CentralView ArticlePubMedGoogle Scholar
- Biesova Z, Piccoli C, Wong WT: Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene. 1997, 14 (2): 233-241. 10.1038/sj.onc.1200822.View ArticlePubMedGoogle Scholar
- Croce A, Cassata G, Disanza A, Gagliani MC, Tacchetti C, Malabarba MG, Carlier MF, Scita G, Baumeister R, Di Fiore PP: A novel actin barbed-end-capping activity in EPS-8 regulates apical morphogenesis in intestinal cells of Caenorhabditis elegans. Nat Cell Biol. 2004, 6 (12): 1173-1179. 10.1038/ncb1198.View ArticlePubMedGoogle Scholar
- Stradal T, Courtney KD, Rottner K, Hahne P, Small JV, Pendergast AM: The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr Biol. 2001, 11 (11): 891-895. 10.1016/S0960-9822(01)00239-1.View ArticlePubMedGoogle Scholar
- Schafer DA, Welch MD, Machesky LM, Bridgman PC, Meyer SM, Cooper JA: Visualization and molecular analysis of actin assembly in living cells. J Cell Biol. 1998, 143 (7): 1919-1930. 10.1083/jcb.143.7.1919.PubMed CentralView ArticlePubMedGoogle Scholar
- Cheyette BN, Waxman JS, Miller JR, Takemaru K, Sheldahl LC, Khlebtsova N, Fox EP, Earnest T, Moon RT: Dapper, a Dishevelled-associated antagonist of beta-catenin and JNK signaling, is required for notochord formation. Dev Cell. 2002, 2 (4): 449-461. 10.1016/S1534-5807(02)00140-5.View ArticlePubMedGoogle Scholar
- Evan GI, Lewis GK, Ramsay G, Bishop JM: Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985, 5 (12): 3610-3616.PubMed CentralView ArticlePubMedGoogle Scholar
- Nieuwkoop PD, Faber J: Normal Table of Xenopus laevis (Daudin). 1967, Amsterdam: North-HollandGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.