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A Drosophila gene regulated by rough and glass shows similarity to ena and VASP. rough (ro) encodes a homeobox transcription factor required for proper specification of photoreceptor cells R2 and R5 in Drosophila eye development. To identify the transcriptional targets through which ro acts to specify the R2/R5 neuronal sub-type, enhancer trap lines expressed in developing photoreceptors were screened for those whose expression patterns are altered when ro function is inactivated. In this way two potential ro targets were identified; these are also targets of the zinc finger transcription factor Glass. An enhancer trap line was identified that exhibits altered morphogenetic furrow expression in a ro mutant background. Finally, an enhancer trap line, AE33, was molecularly characterized that was identified in earlier screens as a target of both ro and gl. The transcript interrupted by AE33 (Sprouty-related protein with EVH-1 domain), subsequently shown to be a Sprouty-related suppressor of Ras signalling, shares similarity with the mammalian vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases that is associated with actin filaments, focal adhesions, and dynamic membrane regions. There is also similarity with Enabled, a substrate of the Drosophila Abl tyrosine kinase and with two human Expressed Sequence Tags (ESTs) (DeMille, 1996).
The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. This study shows that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. It is concluded that mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance (Chang, 2006).
Ena/VASP proteins are associated with cell-cell junctions in cultured mammalian cells and Drosophila epithelia, but they have only been extensively studied at the leading edges of migratory fibroblasts, where they modulate the protrusion of the leading edge. They act by regulating actin-filament geometry, antagonizing the effects of actin-capping protein. Embryos lacking the C. elegans Ena/VASP, UNC-34, display subtle defects in the leading edges of migrating epidermal cells but undergo normal epidermal morphogenesis. In contrast, embryos lacking both UNC-34 and the C. elegans N-WASP homolog have severe defects in epidermal morphogenesis, suggesting that they have parallel roles in coordinating cell behavior. GFP-tagged UNC-34 localizes to the leading edges of migrating epidermal cells, becoming redistributed to new junctions that form during epidermal-sheet sealing. Consistent with this, UNC-34 contributes to the formation of cadherin-based junctions. The junctional localization of UNC-34 is independent of proteins involved in Ena/VASP localization in other experimental systems; instead, junctional distribution depends upon the junctional protein AJM-1. Abelson tyrosine kinase, a major regulator of Enabled in Drosophila, is not required for UNC-34/Ena function in epithelia. Instead, the data suggest that Abelson kinase acts in parallel to UNC-34/Ena, antagonizing its function (Sheffield, 2007).
Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. This report shows that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, it is demonstrated that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus (Roffers-Agarwal, 2007).
Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates; evident in the adult is the failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton (Lanier, 1999).
Human and canine vasodilator-stimulated phosphoproteins (VASPs) share sequence similarity with ENA in three domains: the first 113 amino acids, the proline-rich central domain, and the 35 amino acids at the carboxyl terminus that are required for localization of VASP to focal adhesions (Gertler, 1995).
WASP, an ENA and VASP homolog, is a protein that is defective in the human Wiskott-Aldrich syndrome, characterized by thrombocytopenia, recurrent infections due to defects in T and B cell function, and eczema. The cellular defects are limited to hematopoietic lineages and include cytoskeletal abnormalities of T cells and platelets, failure of B cells to respond to polysaccharide antigens, and defective chemotaxis in neutrophils. This suggests that a defect in the organization of actin cytoskeleton may lie at the core of the syndrome. WASP contains an N-terminal sequence shared with ENA and VASP, an adjacent GTPase (Rac/CDC42Hs)-binding domain (GDP) common to WASP and PAK, a polyproline domain in common with ENA and VASP, and an C terminal proline rich domain, which is not present in ENA. Expression of WASP induces ectopic actin polymerization. It is suggested that the GTPase CDC42 integrates signals from G-protein coupled receptors to activate the JNK/SAPK (Drosophila homolog: JNK/Basket), and through its interaction with WASP, a subject of phosphorylation by non-receptor tyrosine kinases, regulates actin polymerization and cytoskeletal rearrangement (Symons, 1996).
A complex of N-WASP and WASP-interacting protein (WIP) plays an important role in actin-based motility of vaccinia virus and the formation of filopodia. WIP is also required to maintain the integrity of the actin cytoskeleton in T and B lymphocytes and is essential for T cell activation. However, in contrast to many other N-WASP binding proteins, WIP does not stimulate the ability of N-WASP to activate the Arp2/3 complex. Although the WASP homology 1 (WH1) domain of N-WASP interacts directly with WIP, the exact nature of its binding site has not been known. The N-WASP WH1 binding motif in WIP has now been identified and characterized in vitro and in vivo using Shigella and vaccinia systems. The WH1 domain, which is predicted to have a similar structural fold to the Ena/VASP homology 1 (EVH1) domain, binds to a sequence motif in WIP (ESRFYFHPISD) that is very different from the EVH1 proline-rich DL/FPPPP ligand. Interaction of the WH1 domain of N-WASP with WIP is dependent on the two highly conserved phenylalanine residues in the motif. The WH1 binding motif is conserved in WIP, CR16, WICH, and yeast verprolin (Zett, 2002).
VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. A 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrates that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data reveals that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with the identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, these results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation (Reinhard, 1995a).
Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. Vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases has a proline-rich domain that prompted an investigation of a possible interaction of VASP with profilins. VASP is a microfilament and focal adhesion associated protein that is also concentrated in highly dynamic regions of the cell cortex. VASP is demonstrated to be a natural proline-rich profilin ligand. Human platelet VASP binds directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. The subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. These data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation (Reinhard, 1995b).
VASP (vasodilator-stimulated phosphoprotein), a protein associated with microfilaments at cellular contact sites, has been identified as a ligand for profilin and zyxin, two proteins also involved in microfilament dynamics and organization at these regions. VASP also directly binds to vinculin, another component of adherens junctions. Competition experiments with a vinculin-derived peptide shows that a proline-rich motif, located in the hinge region that connects vinculin's head and tail domains, is involved in VASP binding. The same motif is present in zyxin but the interactions of VASP with vinculin and zyxin differ in detail. Hence, this motif may be recognized by VASP in different ways when presented in distinct cellular sites (Reinhard, 1996).
Focal adhesion sites are cell-matrix contacts that are regulated by phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent pathways. Vinculin is a major structural component of these sites and is thought to be engaged in multiple ligand interactions at the cytoplasmic face of these contacts. Cytoplasmic vinculin is considered to be inactive due to its closed conformation involving intramolecular head-tail interactions. The vasodilator-stimulated phosphoprotein (VASP), a substrate of either cyclic AMP-dependent kinases or cyclic GMP-dependent kinases, and a component of focal adhesion sites, has been shown to bind to vinculin. VASP-vinculin complexes can be immunoprecipitated from cell lysates and, using immunofluorescence, both proteins are found to colocalize in nascent focal adhesions. Consistent with the view that vinculin must be activated at these sites, it has been found that PIP2 (PIP2 levels are elevated during the early stages of adhesion) binds to two discrete regions in the vinculin tail, disrupting the intramolecular head-tail interaction and inducing vinculin oligomerization. Vinculin-VASP complex formation is greatly enhanced by PIP2 and both the EVH1 and EVH2 domains of VASP participates in vinculin binding. It is concluded that focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP2-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin-VASP complexes might bundle the distal ends of actin filaments in focal contacts. It is proposed that PIP2-dependent signaling modulates microfilament organization at cellular adhesion sites by regulating vinculin-VASP complexes (Huttelmaier, 1998).
The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. A motif repeated 4-fold within the primary sequence of ActA, E/DFPPPPXD/E, is identified as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, that are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling (Niebuhr, 1997).
Intracellular propulsion of Listeria monocytogenes is the best understood form of motility that is dependent on actin polymerization. In vitro motility assays of Listeria in platelet and brain extracts were used to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP functions in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity [0.5 x 10(8) M-1]; dephospho-VASP binds 40-fold less tightly. A molecular ratchet model is proposed for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament (Laurent, 1999).
Bovine profilin isoforms (See Drosophila Chickadee) bind both the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) (the target of Phospholipase C - see Protein kinase C for more information) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Compared with profilin II, profilin I has a higher affinity for PIP2. However, proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes on binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, profilin II was identified as the preferred ligand for VASP. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways (Lambrechts, 1997).
The Ena/VASP family of proteins is characterized by a common overall structural domain organization consisting of conserved N- and C-terminal domains separated by a less-conserved central proline-rich region. This N-terminal 113-amino-acid domain, or the Ena/VASP homology domain 1 (EVH1), is 58% identical between Drosophila Ena and human VASP. The EVH1 domain mediates VASP and Mena binding to the focal adhesion-associated protein zyxin as well as Listeria Act A. The EVH1 domain is also similar to the WP1 domain found in Wiskott-Aldrich syndrome protein. Wiskott-Aldrich syndrome is characterized by cytoskeletal abnormalities in T cells and platelets. The central proline-rich regions of the Ena, Mena, and VASP proteins, which vary greatly in length, are important for binding to both Src homology 3 (SH3) domains and the actin-binding protein profilin. The C-terminal 35 amino acids of these proteins, or the EVH2 domain, is 31% identical between Drosophila Ena and human VASP and consists of a series of conserved charged repeats with a spacing predicted to form an extended highly charged alpha helix. Homology to this region is also seen in a human expressed sequence tag and the mouse cDNA NDPP-1. Several functions have been proposed for the EVH2 domain, including subcellular localization and mediation of multimer formation, although conclusive evidence for these proposed functions has not been reported (Ahern-Djamali, 1998 and references).
Genetic, biochemical, and cell biological approaches were used to demonstrate the functional relationship between Ena and human VASP. When expressed in mammalian cells, both Drosophila Ena and VASP are detected at actin filaments and focal adhesion contacts. Thus both proteins have domains sufficient to permit binding in vivo to proteins in the actin cytoskeleton and focal adhesions The roles of Ena domains, identified as essential for Ena's activity in vivo, have been defined. VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila. To define sequences that are central to Ena function, a characterization was carried out of molecular lesions present in two lethal ena mutant alleles that affect the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that results in an amino acid substitution in the EVH1 domain eliminates in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. Both proteins bind to the proline-rich region of zyxin but not to the LIM domains consistent with results obtained for VASP and MENA. A nonsense mutation that results in a C-terminally truncated Ena protein lacking the EVH2 domain fails to form multimeric complexes and exhibits reduced binding to zyxin and the Abelson Src homology 3 domain. This analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena (Ahern-Djamali, 1998).
Zyxin binding is thought to be a mechanism for localizing Ena/VASP proteins to focal adhesions. Thus, multimerization of Ena/VASP proteins may be necessary for proper subcellular localization of these proteins to focal adhesions and the actin cytoskeleton. This would be consistent with results demonstrating that removal of the C-terminal 100 amino acids of VASP, which includes the EVH2 domain, results in an absence of VASP in the focal adhesions. Both EnaA97V and the EnaK636Stop mutants show a rather diffuse intracellular localization and are clearly absent from focal contacts when expressed in mammalian cells. Localization of members of the Ena/VASP family of proteins at focal adhesions places them at cellular structures where bidirectional signal transduction takes place. Thus, it is of particular interest that in addition to being cytoskeletal proteins, this family of proteins represents docking sites and substrates for signal transduction molecules. Several kinases phosphorylate Ena, Mena, and VASP, indicating that multiple signaling pathways act on these proteins to regulate cytoskeleton assembly (Ahern-Djamali, 1998).
The development and functional analysis of a monoclonal antibody (16C2) are reported; the antibody recognizes vasodilator-stimulated phosphoprotein (VASP; an established substrate of both cAMP- and cGMP-dependent protein kinase) only when serine 239 is phosphorylated. VASP serine 239 represents one of the best characterized cGMP-dependent protein kinase phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP constructs with mutated phosphorylation sites (S157A, S239A, T278A) and experiments with intact cells (human/rat platelets and other cells) treated with cyclic nucleotide-elevating agents demonstrate the specificity of the monoclonal antibody 16C2. Quantitative analysis of the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2 monoclonal antibody (VASP serine 239 phosphorylation) in human platelets stimulated by selective protein kinase activators confirms that serine 239 is the VASP phosphorylation site preferred by cGMP-dependent protein kinase in intact cells. Immunofluorescence experiments with human platelets treated with cGMP analogs show that the 16C2 monoclonal antibody also detects VASP serine 239 phosphorylation in situ at established intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method presently available to measure cGMP-dependent protein kinase activation in intact cells. Also, the 16C2 antibody promises to be an excellent tool for the evaluation of VASP function in intact cells (Smolenski, 1998).
The neural protein FE65 contains two types of protein-protein interaction modules or binding domains: a single binding domain for WW, and two different binding domains for phosphotyrosine. The WW domain contains four well conserved aromatic amino acids among which are two tryptophan residues, hence the name WW domain. The carboxyl-terminal phosphotyrosine binding domain of FE65 interacts in vivo with the beta-amyloid precursor protein, which is implicated in Alzheimer's disease. To understand the function of this adapter protein, binding partners were identified for the FE65 WW domain. Proline-rich sequences sharing a proline-proline-leucine-proline core motif were recovered by screening expression libraries for ligands of the FE65 WW domain. Five proteins from mouse brain lysates of molecular masses 60, 75, 80, 140, and 200 kDa were purified by affinity to the FE65 WW domain. Two of these five proteins were identified as the 80- and 140-kDa isoforms encoded by Mena, the mammalian homolog of the Drosophila Enabled gene. Using the SPOTs technique of peptide synthesis, the sequences in Mena were identified that interact with the FE65 WW domain; they were found to contain the signature proline-proline-leucine-proline motif. Mena binds to FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. The specificity of the Mena-FE65 WW domain association was confirmed by competition assays. Further characterization of the FE65-Mena complex may identify a physiological role for these proteins in beta-amyloid precursor protein biogenesis and may help in understanding the mechanism of molecular change that underlies Alzheimer's disease. FE65 may serve as an adapter protein that brings other partner molecules into a complex with beta-amyloid precursor protein, which would affect beta APP secretion, internalization, and/or trafficking. In light of this, Mena, being a cytoskeletal protein involved in microfilament assembly, is a good candidate to participate in cellular network of proteins interacting with beta APP in the cytoplasm (Ermekova, 1997).
The vasodilator-stimulated phosphoprotein (VASP) is associated with actin filaments and focal adhesions, which form the interface between the cytoskeleton and the extracellular matrix. VASP is phosphorylated by both the cAMP- and cGMP-dependent protein kinases in a variety of cells, including platelets and smooth muscle cells. Since both the cAMP and cGMP signalling cascades relax smooth muscle and inhibit platelet activation, it was speculated that VASP mediates these effects by modulating actin filament dynamics and integrin activation. To study the physiological relevance of VASP in these processes, the VASP gene was inactivated in mice. Adult VASP-deficient mice have normal agonist-induced contraction, and normal cAMP- and cGMP-dependent relaxation of intestinal and vascular smooth muscle. In contrast, cAMP- and cGMP-mediated inhibition of platelet aggregation is significantly reduced in the absence of VASP. Other cAMP- and cGMP-dependent effects in platelets, such as inhibition of agonist-induced increases in cytosolic calcium concentrations and granule secretion, are not dependent on the presence of VASP. These data show that two different cyclic, nucleotide-dependent mechanisms are operating during platelet activation: a VASP-independent mechanism for inhibition of calcium mobilization and granule release and a VASP-dependent mechanism for inhibition of platelet aggregation that may involve regulation of integrin function (Aszodi, 1999).
Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. The distribution of Mena in wild-type adult organs was compared to that of EVL (Ena-VASP-like) and VASP. The 140 kDa form of Mena is detected only in the brain, while the 80 kDa form of Mena is expressed predominantly in brain, testis, ovaries, and fat. In contrast, EVL and VASP are most highly expressed in thymus and spleen, and the relative intensities of the phospho and dephospho forms vary from tissue to tissue, suggesting that EVL and VASP may be differentially regulated in the brain and organs. Because Mena is expressed at high levels in the brain, while both VASP and EVL are expressed at low levels, the distribution of Mena in adult and developing brain was characterized in greater detail. The 80 and 140 kDa forms of Mena are detected in all regions of the adult brain, with highest levels in the hippocampus, cortex, and midbrain, and lowest levels in the striatum and cerebellum. The 140 kDa form of Mena is expressed at relatively high levels in serum-free cortical cultures (which are enriched for neurons) and is not detected in glial cultures, suggesting the 140 kDa form is indeed neuron-specific and that it may be the predominant form of Mena in neurons. In embryonic brains, all three forms of Mena are detected at embryonic day 11 (E11), the earliest time point examined. Expression of the 88 kDa form decreases steadily and becomes almost undetectable by E16, while expression of the 140 kDa form begins to increase at E13 and peaks between E16 and E18. In contrast to Mena, EVL expression in the brain is first detected at E15. VASP expression appears to be fairly constant throughout development of the brain, but then decreases to relatively low levels in the adult brain (Lanier, 1999).
At E8.5, Mena is particularly enriched in the neuroepithelium, the forebrain, and the somites. Mena is highly expressed in the edges of the neural folds. By E10.5, Mena expression is detected in the brain, dorsal root ganglia (DRG), somites, and limb buds. In addition, Mena is highly expressed in the branchial and pharyngeal arches, neural crest-derived structures that give rise to portions of the face and neck. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. High levels of Mena expression are detected in distinct bands of cells in the developing cortex at E16, a time when neurons are migrating from the ventricular zone to the cortical layers and axons are beginning to project across the corpus callosum. In the adult brain, Mena expression is detected in laminae 2/3 and 5 of the cortex and is particularly enriched in the hippocampus and the septum. In neurons, Mena is highly enriched in the lamellipodium and at the tips of the axonal growth cones. Similar Mena localization is seen in dendritic growth cones and at various stages of differentiation (Lanier, 1999)
Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and fail decussation of the corpus callosum. Fibers in the corpus callosum appear to reach a point just medial to the cingulum bundle as normal but then failed to project medially and cross the midline. Defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton. The neural tube closure defects seen in the Mena;profilin mutant animals reveal that Mena plays a critical role in neurulation in addition to its function in axon guidance. Mena function in neurulation involves Profilin and therefore might be linked to regulation of the actin cytoskeleton. Cephalic neural tube closure depends on actin-driven changes in the shape of cells within the dorsal-lateral hinge point of the neuroepithelium (Lanier, 1999).
Ena/VASP proteins have been implicated in cell motility through regulation of the actin cytoskeleton and are found at focal adhesions and the leading edge. Using overexpression, loss-of-function, and inhibitory approaches, it has been found that Ena/VASP proteins negatively regulate fibroblast motility. A dose-dependent decrease in movement is observed when Ena/VASP proteins are overexpressed in fibroblasts. Neutralization or deletion of all Ena/VASP proteins results in increased cell movement. Selective depletion of Ena/VASP proteins from focal adhesions, but not the leading edge, has no effect on motility. Constitutive membrane targeting of Ena/VASP proteins inhibits motility. These results are in marked contrast to current models for Ena/VASP function derived mainly from their role in the actin-driven movement of Listeria monocytogenes (Bear, 2000).
WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. The localization of the WASP family proteins, N-WASP and WAVE (Drosophila homolog (SCAR), and the Ena/VASP family protein, Mena, is observed in protruding lamellipodia. Rat fibroblast cell line 3Y1 protrudes lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 are concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena are observed to localize at the lamellipodium edge. Interestingly, Mena tends to concentrate at the microspike tips but WAVE does not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, is much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, is localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE is distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42 (Nakagawa, 2001).
In fibroblasts, Ena/VASP proteins are most highly concentrated in focal adhesions, the leading edge of lamellipodia, and at the tips of filopodia. Lamellipodial protrusion rate has been directly correlated with the accumulation of Ena/VASP proteins at leading edges, suggesting that Ena/VASP proteins promote leading edge protrusion. In addition, Ena/VASP proteins are required for full motility of the actin-dependent intracellular pathogen Listeria monocytogenes. Paradoxically, Ena/VASP negatively regulates cell translocation. To resolve this paradox, the function of Ena/VASP during lamellipodial protrusion was analyzed. Ena/VASP-deficient lamellipodia protrude slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contain shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contain longer, less branched filaments. In vitro, Ena/VASP promotes actin filament elongation by interacting with barbed ends, shielding them from capping protein. It is concluded that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia (Bear, 2002).
It is proposed that Ena/VASP proteins interact with actin filaments at or near their barbed ends and prevent or delay binding of capping protein to filaments while permitting continued elongation. Ena/VASP proteins associate with free barbed ends of actin filaments in vitro and, based on cytochalasin D displacement, free barbed ends are required for leading edge localization of ena/vasp proteins in vivo. the Ena/VASP-dependent changes in actin filament length in lamellipodia are consistent with VASP's antagonism of capping protein seen by in vitro polymerization assays. the results suggest that, in cells, cytochalasin D treatment largely counteracts the effects of Ena/VASP proteins on cell motility, lamellipodial dynamics, and the architecture of the actin network. Given the known effects of cytochalasin D on barbed end elongation and the reported effects on filament length in vivo, these observations are consistent with the proposed model for Ena/VASP proteins as 'anticapping' proteins. while ena/vasp proteins may represent the first known proteins with this activity, there may be other proteins with similar functions just as there are multiple types of filament-capping proteins. The inhibition of capping need not be particularly long lasting to produce the observed effects in cells. A transient inhibition of capping would still produce the long filaments observed in the lamellipodia of the FPPPP-CAAX cells (cells in which Ena/VASP is artificially recruited to the membrane). Since the dissociation rate of capping protein from filaments is slow (t1/2 = 28 min), capping is essentially a terminal event. Ena/VASP proteins may simply slow the association of capping protein with barbed ends (Bear, 2002).
Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. This paper reports on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (-/-) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, these results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton (Galler, 2006).
Focal adhesions are specialized regions of the cell surface where integrin receptors and associated proteins link the extracellular matrix to the actin cytoskeleton. To define the cellular role of the focal adhesion protein zyxin, the phenotype of fibroblasts was characterized in which the zyxin gene was deleted by homologous recombination. Zyxin-null fibroblasts display enhanced integrin-dependent adhesion and are more migratory than wild-type fibroblasts, displaying reduced dependence on extracellular matrix cues. Differences were identified in the profiles of 75- and 80-kD tyrosine-phosphorylated proteins in the zyxin-null cells. Tandem array mass spectrometry identified both modified proteins as isoforms of the actomyosin regulator caldesmon, a protein known to influence contractility, stress fiber formation, and motility. Zyxin-null fibroblasts also show deficits in actin stress fiber remodeling and exhibit changes in the molecular composition of focal adhesions, most notably by severely reduced accumulation of Ena/VASP proteins. It is postulated that zyxin cooperates with Ena/VASP proteins and caldesmon to influence integrin-dependent cell motility and actin stress fiber remodeling (Hoffman, 2006; full text of article).
Lamellipodial protrusion is regulated by Ena/VASP proteins. Lamellipodin (Lpd) has been identified as an Ena/VASP binding protein. The N terminus of Lpd (AA 1-50) is highly charged and is followed by a putative coiled-coil motif, Ras-association (RA), and PH domains. The C terminus is proline-rich, harboring eight potential SH3 binding sites, three potential Profilin binding sites, and four clusters containing a total of six putative EVH1 binding sites. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to Enteropathogenic E. coli (EPEC) and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP (Krause, 2004).
Based on these results, it is proposed that Lpd acts as a convergence point between upstream signaling pathways and cytoskeletal remodeling at the plasma membrane. This hypothesis is supported by the recruitment of Lpd to two pathogens, EPEC and Vaccinia, that exploit cellular signaling processes at the plasma membrane to reorganize the actin cytoskeleton for their own motility. Furthermore, Lpd is concentrated at the tip of growth cone filopodia, a structure that plays a key role in guidance signal detection and growth cone motility. Lpd is likely to receive signals directly from both phosphoinositides through its PH domain and Ras superfamily proteins through its RA domain. The Lpd PH domain selectively binds PI(3,4)P2, a phosphoinositide associated with polarization signals in a variety of cell types (Krause, 2004).
One way that Lpd is linked to cytoskeletal dynamics is through Ena/VASP proteins. Lpd associates with Ena/VASP by direct binding and can be detected in protein complexes from primary neurons. Ena/VASP and Lpd colocalize in filopodia and lamellipodial tips, and both types of proteins play critical roles in regulating lamellipodial dynamics. Lpd overexpression induces an increase in lamellipodial ruffling, similar to phenotypes that are observed when Ena/VASP activity is elevated in mammalian cells, and the Lpd overexpression phenotype requires Ena/VASP function. Interestingly, knockdown of Lpd led to impairment of lamellipodia formation, a phenotype not observed in cells devoid of all Ena/VASP proteins. This suggests that Lpd signals to other regulators of the actin cytoskeleton in addition to Ena/VASP proteins (Krause, 2004).
Tight regulation of cell motility is essential for many physiological processes, such as formation of a functional nervous system and wound healing. Drosophila Abl negatively regulates the actin cytoskeleton effector protein Ena during neuronal development in flies, and it has been postulated that this may occur through an unknown intermediary. Lamellipodin (Lpd) regulates cell motility and recruits Ena/VASP proteins (Ena, Mena, VASP, EVL) to the leading edge of cells. However, the regulation of this recruitment has remained unsolved. This study shows that Lpd is a substrate of Abl kinases and binds to the Abl SH2 domain. Phosphorylation of Lpd positively regulates the interaction between Lpd and Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd at the leading edge requires Abl kinases. Furthermore, transient Lpd phosphorylation by Abl kinases upon netrin-1 stimulation of primary cortical neurons positively correlates with an increase in Lpd-Mena coprecipitation. Lpd is also transiently phosphorylated by Abl kinases upon platelet-derived growth factor (PDGF) stimulation, regulates PDGF-induced dorsal ruffling of fibroblasts and axonal morphogenesis, and cooperates with c-Abl in an Ena/VASP-dependent manner. These findings suggest that Abl kinases positively regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to Lpd at the leading edge, and Lpd-Ena/VASP function in axonal morphogenesis and in PDGF-induced dorsal ruffling. These data do not support the suggested negative regulatory role of Abl for Ena. Instead, it is proposed that Lpd is the hitherto unknown intermediary between Abl and Ena/VASP proteins (Michael, 2010).
Drosophila ena was originally identified as a suppressor of lethality induced by mutations in Drosophlia abl, and it was postulated that abl and ena negatively regulate each other. Both the Abl tyrosine kinase family (Drosophila Abl, vertebrate c-Abl and Arg) and the Ena/VASP family (Ena, vertebrate Mena, VASP, and EVL) act downstream of the netrin-1 axon guidance receptor DCC and regulate cell motility. Abl kinases regulate platelet-derived growth factor (PDGF)-induced dorsal ruffling of fibroblasts, but it is not known whether Lamellipodin (Lpd) or Ena/VASP proteins function in this pathway (Michael, 2010).
Ena/VASP proteins play a crucial role in cell motility by antagonizing actin filament capping. This alters the geometry of the actin network toward longer, less-branched filaments, thereby changing the speed and persistence of lamellipodia. Ena/VASP proteins are recruited to the leading edge through interactions between their EVH1 domain and FP4 motifs within Lpd, a member of the MRL family of Ras effector proteins, which includes C. elegans MIG-10, vertebrate RIAM and Lpd, and Drosophila Pico (Lyulcheva, 2008). Lpd contains a proline-rich region harboring potential SH3-binding sites and a PH domain that mediates membrane targeting. It has been shown that Lpd is required for lamellipodia formation and that Lpd overexpression increases the speed of lamellipodial protrusion in an Ena/VASP-dependent manner (Michael, 2010).
Lpd-dependent recruitment of Ena/VASP proteins to the leading edge needs to be tightly regulated in order to precisely control lamellipodia formation, but it is not known how this is achieved. Studies in Drosophila have suggested that Abl regulates Ena localization. Yet how the localization of Ena/VASP proteins is controlled, and whether Abl plays a role in this process in vertebrates, remains unclear (Michael, 2010).
This study shows that phosphorylation of Lpd by c-Abl increases its interaction with Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd-positive lamellipodia requires Abl kinases. Evidence is provided that Lpd and Ena/VASP proteins regulate dorsal ruffling of fibroblasts upon PDGF treatment and that Lpd function in this process is controlled by Abl kinases and mediated by Ena/VASP proteins. Furthermore, it was demonstrated that both Lpd and c-Abl cooperate during axonal morphogenesis in an Ena/VASP-dependent manner. The data do not support the suggested antagonistic roles of Abl and Ena, and an alternative hypothesis is proposed that Abl kinases, via Lpd, positively regulate Ena/VASP proteins (Michael, 2010).
The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. RIAM was identified as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. The protein with highest homology to RIAM is Lamellipodin (Krause, 2004). Furthermore, RIAM is related to proteins CG11940 (AAF49029) in D. melanogaster and Mig-10 (P34400) in C. elegans. RIAM interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induces cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells have reduced content of polymerized actin. RIAM overexpression also induces integrin activation and cell adhesion. RIAM knockdown displaces Rap1-GTP from the plasma membrane and abrogates Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics (Lafuente, 2004).
Development of the multilayered cerebral cortex involves extensive regulated migration of neurons arising from the deeper germinative layers of the mammalian brain. The anatomy and formation of the cortical layers has been well characterized; however, the underlying molecular mechanisms that control the migration and the final positioning of neurons within the cortex remain poorly understood. Ena/VASP proteins, a protein family implicated in the spatial control of actin assembly and shown to negatively regulate fibroblast cell speeds, play a key role in cortical development. Ena/VASP proteins are highly expressed in the developing cortical plate in cells bordering Reelin-expressing Cajal-Retzius cells and in the intermediate zone through which newly born cells migrate. Inhibition of Ena/VASP function through retroviral injections in utero leads to aberrant placement of early-born pyramidal neurons in the superficial layers of both the embryonic and the postnatal cortex in a cell-autonomous fashion. The abnormally placed pyramidal neurons exhibit grossly normal morphology and polarity. These results are consistent with a model in which Ena/VASP proteins function in vivo to control the position of neurons in the mouse neocortex (Goh, 2002).
Netrins promote axon outgrowth and turning through DCC/UNC-40 receptors. To characterize Netrin signaling, a gain-of-function UNC-40 molecule, MYR::UNC-40 (an UNC-40 fusion protein in which the extracellular and transmembrane domains are deleted and replaced by sequences encoding a membrane-targeting myristoylation signal) is generated. MYR::UNC-40 causes axon guidance defects, excess axon branching, and excessive axon and cell body outgrowth. These defects are suppressed by loss-of-function mutations in ced-10 (a Rac GTPase), unc-34 (an Enabled homolog), and unc-115 (a putative actin binding protein: Drosophila homolog - unc-115). ced-10, unc-34, and unc-115 also function in endogenous unc-40 signaling. These results indicate that Enabled functions in axonal attraction as well as axon repulsion. UNC-40 has two conserved cytoplasmic motifs that mediate distinct downstream pathways: CED-10, UNC-115, and the UNC-40 P2 motif act in one pathway, and UNC-34 and the UNC-40 P1 motif act in the other. Thus, UNC-40 might act as a scaffold to deliver several independent signals to the actin cytoskeleton (Gitai, 2003).
WASP and Ena/VASP family proteins play overlapping roles in C. elegans morphogenesis and neuronal cell migration. Specifically, these studies demonstrate that UNC-34/Ena plays a role in morphogenesis that is revealed only in the absence of WSP-1 function and that WSP-1 has a role in neuronal cell migration that is revealed only in the absence of UNC-34/Ena activity. To identify additional genes that act in parallel to unc-34/ena during morphogenesis, a screen for synthetic lethals was performed in an unc-34 null mutant background utilizing an RNAi feeding approach. This is the first reported RNAi-based screen for genetic interactors. As a result of this screen, a second C. elegans WASP family protein, wve-1, was identified that is most homologous to SCAR/WAVE proteins. Animals with impaired wve-1 function display defects in gastrulation, fail to undergo proper morphogenesis, and exhibit defects in neuronal cell migrations and axon outgrowth. Reducing wve-1 levels in either unc-34/ena or wsp-1 mutant backgrounds also leads to a significant enhancement of the gastrulation and morphogenesis defects. Thus, unc-34/ena, wsp-1, and wve-1 play overlapping roles during embryogenesis and unc-34/ena and wsp-1 play overlapping roles in neuronal cell migration. These observations show that WASP and Ena/VASP proteins can compensate for each other in vivo and provide the first demonstration of a role for Ena/VASP proteins in gastrulation and morphogenesis. In addition, these results provide the first example of an in vivo role for WASP family proteins in neuronal cell migrations and cytokinesis in metazoans (Withee, 2004).
Netrins have been shown to promote outgrowth and guidance: vertebrate Netrin-1 was originally identified based on its ability to enhance axon outgrowth into a collagen matrix, and Netrin-1 knockout mice have defects in axon outgrowth in addition to axon guidance. Netrin can also orient axon outgrowth. Both of these effects of Netrin are dependent on the DCC receptor. The results of this study suggest that MYR::UNC-40 activates cytoplasmic signaling of the UNC-40 pathway in a constitutive, ligand-independent manner. The in vivo activation of signaling by the deletion of the extracellular and transmembrane domains suggests that these domains normally function to prevent UNC-40 activation but are disinhibited when UNC-6 binds to UNC-40. A similar disinhibition model has been proposed for the role of Netrin in activating the DCC-UNC-5 complex for axon repulsion (Gitai, 2003).
Double and triple mutant analysis indicates that all of the myr::unc-40 suppressors, unc-34, ced-10, and unc-115 are likely to participate in the endogenous unc-40 signaling pathway. These results suggest that myr::unc-40 activates the endogenous unc-40 signaling pathway, consistent with its acting as a constitutively active form of unc-40. unc-34, ced-10, and unc-115 were found to signal downstream of unc-40 in two parallel, partially redundant pathways. unc-34/Enabled also plays a partially redundant role in the sax-3/Robo pathway. The activation of parallel signaling modules with some functional overlap or redundancy may be a general feature of axon guidance signaling. It is worth noting that this apparent genetic redundancy could result from disrupting cell biological processes that are actually distinct. The activation of multiple pathways for cytoskeletal remodeling by guidance receptors may contribute to accurate guidance through various physical environments (Gitai, 2003).
MYR::UNC-40 is capable of inducing axon outgrowth, misguidance, branching, and cell body deformation. All of these phenotypes can be suppressed by unc-34, ced-10, and unc-115 or by deletions in the P1 and P2 motifs. These results suggest that distinct effects on cell morphology can be induced by the same signaling pathways, consistent with the observation that Netrin can signal through DCC to regulate cell migration, axon outgrowth, axon attraction, and axon repulsion (Gitai, 2003).
MYR::UNC-40 activity generates new outgrowths even in the adult stage, well past the normal period of neuronal development. It thus seems likely that downstream effectors of UNC-40 persist and remain functional into adulthood. Indeed, reporter gene fusions to unc-115 and ced-10 are expressed throughout the life of C. elegans. One possibility is that these genes function later in development to increase the size of the neuron as the size of the animal increases (Gitai, 2003).
Enabled was initially identified as a dosage-sensitive suppressor of Abl tyrosine kinase mutations in Drosophila. Enabled and its family members UNC-34, Mena, VASP, and EVL share a conserved domain structure that includes an N-terminal EVH1 domain and a C-terminal EVH2 domain. The EVH1 domain binds to proteins containing a FPPPP consensus sequence, found in actin-associated molecules such as zyxin and vinculin, whereas the EVH2 domain has been implicated in oligomerization as well as G and F actin binding (Gitai, 2003).
Enabled proteins can nucleate actin polymerization in vitro. In vivo, Ena proteins are important for a number of actin-based cellular processes including axon guidance, platelet shape change, and Jurkat T cell polarization. Ena proteins were initially thought to promote cellular outgrowth, since VASP enhances the actin-based motility of the intracellular pathogen Listeria monoctytogenes, and overexpression of Mena in fibroblasts produces actin-based outgrowths. However, this view was reversed when enrichment of Mena at the leading edge of fibroblasts was found to decrease motility, while depletion of Mena from the leading edge enhanced motility. These observations led to the idea that Ena proteins negatively affect outgrowth. This idea was reinforced when in Drosophila and C. elegans, UNC-34/Enabled was found to interact physically and genetically with the SAX-3/Robo guidance receptor to mediate axon repulsion. Furthermore, unc-34 mutants suppress the axon repulsion induced by ectopic expression of unc-5, suggesting a role for UNC-34 in mediating repulsion from UNC-6/Netrin. These results created a paradox between the observed role for Enabled in promoting actin-based activities generally associated with stimulation of outgrowth in vitro and its clear roles in axon repulsion and inhibition of cell motility in vivo (Gitai, 2003).
A recent paper provided a potential resolution for this paradox by examining the mechanism by which Mena inhibits fibroblast motility. Mena enrichment at the leading edge was actually found to enhance the dynamics of lamellipodial protrusion; the paradoxical decreased net motility results from the fact that these additional protrusions are not stabilized. These observations led to a proposal that Ena proteins function to stimulate the dynamics of protrusions at the leading edge. Whether the presence of additional protrusions promotes or inhibits cell migration or axon outgrowth may depend on whether the protrusions are stabilized or destabilized. It is thus possible that Mena-induced fibroblast protrusions are not stabilized because the actin filaments within them are isolated and unstable. It is proposed that actin filament bundling, observed in the filopodia of axonal growth cones, could provide a cellular context in which Mena-induced protrusions are stabilized. Thus, this new view of the mechanism of Enabled protein function is potentially consistent with a role for Enabled not just in axon repulsion and outgrowth inhibition, but also in axon attraction (Gitai, 2003).
The results provide direct evidence that UNC-34 can indeed function in an attractive axon guidance pathway: the endogenous UNC-6/UNC-40 pathway. These data establish the idea that Enabled proteins can promote outgrowth and attraction in vivo. In the AVM sensory neuron there is a remarkable example of Enabled's duality, since this single cell uses UNC-34/Ena downstream of both UNC-40 and SAX-3 to promote axon attraction and repulsion, respectively. The mild effect of unc-34 mutations on AVM axon guidance suggests that UNC-34 is not essential for either UNC-40 or SAX-3 function. This finding is consistent with the above model wherein Ena proteins promote outgrowth dynamics but are not dedicated factors required for a specific outgrowth response (Gitai, 2003).
These results identified two distinct pathways that mediate UNC-40 signaling: UNC-34/Enabled acts in one and CED-10/Rac and UNC-115/abLIM act in the other. Rac proteins have previously been shown to play roles in axon guidance, and Rac function is essential for repulsive axon guidance signaling by the Semaphorin receptor, Plexin. The involvement of a Rac protein in Netrin attraction is consistent with the observation that Rac promotes lamellipodial extension, since growth cones have a flattened area with some similarities to lamellipodia. Indeed, recent reports demonstrate that Netrin stimulation can activate Rac in vitro. It is interesting that ced-10 is important in the unc-40 pathway, but both mig-2, which encodes another C. elegans Rac-like protein, and unc-73, which encodes a Guanine Nucleotide Exchange Factor (GEF), are not. In preliminary studies, a mutation in rac-2(ok326), the third Rac-like gene in C. elegans, appears to partially suppress the excess outgrowth of MYR::UNC-40, suggesting that UNC-40 may signal to several, but not all, Rac proteins (Gitai, 2003).
The mechanisms by which Rac proteins cause changes in the actin cytoskeleton during axon guidance are largely unknown. The results suggest that UNC-115 acts as an element in the Rac signaling pathway. The UNC-115 protein contains three LIM domains and a villin headpiece domain. UNC-115 has been proposed to bind actin through its villin headpiece domain; thus, UNC-115 may provide a link between Rac and actin. A different LIM domain-containing protein, LIM-kinase, acts downstream of Rac through a PAK intermediate. The role of UNC-115 in axon guidance is not specific to C. elegans; a dominant-negative form of a vertebrate UNC-115 homolog, abLIM, can cause axon defects in retinal ganglion cells (Gitai, 2003).
Directed-turning toward an axonal attractant requires propagation of spatial information about the source of the attractant to downstream signaling events. Localized signaling might be achieved by localized nucleation of a signaling complex around the activated receptor. The activation of the UNC-34- and CED-10/UNC-115-dependent pathways by UNC-40 correspond to the specific conserved P1 and P2 motifs within the UNC-40 cytoplasmic domain. It is suggested that these actin-regulatory activities may remain closely associated with the activated receptor. UNC-40 may thus function as a scaffold for assembling several independent activities that regulate the cytoskeleton (Gitai, 2003).
Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems: microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Neuronal gephyrin has been shown to interact directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses (Giesemann, 2003).
Ena/VASP proteins play important roles in axon outgrowth and guidance. Ena/VASP activity regulates the assembly and geometry of actin networks within fibroblast lamellipodia. In growth cones, Ena/VASP proteins are concentrated at filopodia tips, yet their role in growth cone responses to guidance signals has not been established. This study finds that Ena/VASP proteins play a pivotal role in formation and elongation of filopodia along neurite shafts and growth cone. Netrin-1-induced filopodia formation is dependent upon Ena/VASP function and directly correlates with Ena/VASP phosphorylation at a regulatory PKA site. Accordingly, Ena/VASP function is required for filopodial formation from the growth cone in response to global PKA activation. It is proposed that Ena/VASP proteins control filopodial dynamics in neurons by remodeling the actin network in response to guidance cues (Lebrand, 2004).
The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. This issue was re-examined using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopus homologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells (RGCs). The specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly, despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations (Dwivedy, 2007).
Mammalian cortical development involves neuronal migration and neuritogenesis; this latter process forms the structural precursors to axons and dendrites. Elucidating the pathways that regulate the cytoskeleton to drive these processes is fundamental to understanding cortical development. Loss of all three murine Ena/VASP proteins, a family of actin regulatory proteins, causes neuronal ectopias, alters intralayer positioning in the cortical plate, and, surprisingly, blocks axon fiber tract formation during corticogenesis. Cortical fiber tract defects in the absence of Ena/VASP arise from a failure in neurite initiation, a prerequisite for axon formation. Neurite initiation defects in Ena/VASP-deficient neurons are preceded by a failure to form bundled actin filaments and filopodia. These findings provide insight into the regulation of neurite formation and the role of the actin cytoskeleton during cortical development (Kwiatkowski, 2007).
Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin Fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. This study identifies mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directs actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreases recruitment of endogenous VASP to the leading edge and results in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue (Moeller, 2004).
Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. This report explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts; E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, two distinct pools of Mena were identified within individual homophilic adhesions that cells make when they adhere to cadherin-coated substrata. These Mena pools localize with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity is necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. It is proposed that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts (Scott, 2006; full text of article).
The metameric organization of the vertebrate body plan is established during somitogenesis as somite pairs sequentially form along the anteroposterior axis. Coordinated regulation of cell shape, motility and adhesion are crucial for directing the morphological segmentation of somites. Members of the Ena/VASP family of actin regulatory proteins are required for somitogenesis in Xenopus. Xenopus Ena (Xena) localizes to the cell periphery in the presomitic mesoderm (PSM), and is enriched at intersomitic junctions and at myotendinous junctions in somites and the myotome, where it co-localizes with beta1-integrin, vinculin and FAK. Inhibition of Ena/VASP function with dominant-negative mutants results in abnormal somite formation that correlates with later defects in intermyotomal junctions. Neutralization of Ena/VASP activity disrupts cell rearrangements during somite rotation and leads to defects in the fibronectin (FN) matrix surrounding somites. Furthermore, inhibition of Ena/VASP function impairs FN matrix assembly, spreading of somitic cells on FN and autophosphorylation of FAK, suggesting a role for Ena/VASP proteins in the modulation of integrin-mediated processes. Inhibition of FAK results in defects in somite formation, blocks FN matrix deposition and alters Xena localization. Together, these results provide evidence that Ena/VASP proteins and FAK are required for somite formation in Xenopus and support the idea that Ena/VASP and FAK function in a common pathway to regulate integrin-dependent migration and adhesion during somitogenesis (Kragtorp, 2006; full text of article).
The inner ear is derived from a thickening in the embryonic ectoderm, called the otic placode. This structure undergoes extensive morphogenetic movements throughout its development and gives rise to all components of the inner ear. Ena/VASP-like (Evl) is an actin binding protein involved in the regulation of cytoskeletal dynamics and organization. The role of Evl was examined during the morphogenesis of the Xenopus inner ear. Evl (hereafter referred to as Xevl) is expressed throughout otic vesicle formation and is enriched in the neuroblasts that delaminate to form the vestibulocochlear ganglion and in hair cells that possess mechanosensory stereocilia. Knockdown of Xevl perturbs epithelial morphology and intercellular adhesion in the otic vesicle and disrupts formation of the vestibulocochlear ganglion, evidenced by reduction of ganglion size, disorganization of the ganglion, and defects in neurite outgrowth. Later in embryogenesis, Xevl is required for development of mechanosensory hair cells. In Xevl knockdown embryos, hair cells of the ventromedial sensory epithelium display multiple abnormalities including disruption of the cuticular plate at the base of stereocilia and disorganization of the normal staircase appearance of stereocilia. Based on these data, it is proposed that Xevl plays an integral role in regulating morphogenesis of the inner ear epithelium and the subsequent development of the vestibulocochlear ganglion and mechanosensory hair cells (Wanner, 2007)
The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, MenaINV. This study shows that Mena and MenaINV promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and MenaINV potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, MenaINV is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of MenaINV could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels (Philippar, 2008).
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