See the embryonic expression pattern of ena at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Expression of ENA protein is first detected during germ-band extension at stage 10 and by stage 11 is detected in the endoderm and ectoderm of the extended germ band. During germ-band retraction [Images], ENA protein becomes restricted to the CNS and by stage 15 is observed primarily in the commissural and longitudinal axons of the CNS. During late embryogenesis (Stage 17), ENA protein is localized primarily to the longitudinal exons (Gertler, 1995).

Expression of ABL protein

ABL protein, present in all cells of the early embryo as the product of maternally contributed mRNA, transiently localizes to the region below the plasma membrane cleavage furrows as cellularization initiates. The function of this expression is not yet known. Zygotic expression of ABL is first detected in the post-mitotic cells of the developing muscles and nervous system midway through embryogenesis. In later larval and pupal stages, ABL protein levels are also highest in differentiating muscle and neural tissue including the photoreceptor cells of the eye. ABL protein is localized subcellularly to the axons of the central nervous system, the embryonic somatic muscle attachment sites and the apical cell junctions of the imaginal disk epithelium (Bennett, 1992).

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help in the understanding of animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. This study reports the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, these data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction (Fox, 2007).

Regulation of apical constriction during Drosophila VF formation is a paradigm for how signal transduction directs morphogenesis. This study identified Abl as a novel regulator of this process. The results suggest that Abl acts in parallel to the known signaling pathway that promotes apical myosin activation by helping to organize a continuous apical actin network. Furthermore, the results help to explain the greater severity of the RhoGEF2-mutant phenotype relative to other VF mutants by suggesting that RhoGEF2 plays crucial roles in both myosin and actin regulation (Fox, 2007).

Previous work established myosin as a key output of RhoGEF2 signaling during mesoderm internalization. However, ambiguities remained regarding the circuitry of this pathway, since the RhoGEF2 phenotype is much more severe than that of cta or fog mutants, suggesting that a simple linear pathway is unlikely. The data suggest that RhoGEF2 plays dual roles in actin and myosin regulation, and thus its inactivation has more severe effects (Fox, 2007).

From these data, a mechanistic model was developed for the regulation of apical constriction during VF formation. The regulation of actin localization by Abl and RhoGEF2 promotes organization of the apical actin network in constricting cells. It is suggested that Abl regulates actin by actively downregulating cortical Ena in mesoderm, thus leading to polarized actin accumulation, similar to the role that it was shown to play in follicle cells. RhoGEF2 plays a distinct, Cta-independent role in the effective assembly of organized apical actin. While RhoGEF2 and Abl are modulating actin assembly, the mesodermal transcription machinery activates Fog-Cta signaling, apically stabilizing RhoGEF2. This allows the efficient activation of apical myosin. Coupling of these two cues -- an organized apical actin ring at AJs and stable apical myosin activation -- cooperate to ensure highly coordinated actomyosin constriction throughout the sheet of mesodermal cells in a short timeframe (Fox, 2007).

This model helps explain the mutant phenotypes observed in this and previous studies. In abl mutants, Fog-Cta allow RhoGEF2 stabilization and myosin contraction, but the lack of organized mesodermal actin in these mutants, which results from inappropriate Ena regulation, prevents the uniform assembly of actin-based contractile rings. cta mutants lack a stabilizing signal for RhoGEF2, preventing uniform apical myosin activation and uniform constriction. However, some cells can constrict without Fog-Cta, accumulating apical myosin levels comparable to those in wild type. In RhoGEF2 mutants, the combined failure to stabilize/activate myosin and a lack of organized apical actin severely compromises apical constriction. The similarity between RhoGEF2 and cta;abl mutants supports this model, as both processes should be compromised (Fox, 2007).

The model suggests that organized apical actin is an essential prerequisite for cell constriction. Although both Abl and RhoGEF2 regulate actin localization, the data argue that each acts independently. First, actin defects arise during cellularization, when Abl and RhoGEF2 have non-overlapping localizations. Second, whereas Abl clearly acts through Ena, loss of RhoGEF2 disrupts actin without altering Ena localization. Finally, Abl is not a Rho effector in S2 cells (Fox, 2007).

Several unanswered questions remain. With respect to abl, a major question is why do some cells apically constrict while others fail? This phenotype resembles the cellularization defects of abl mutants, in which only some cells fail to reorganize actin into furrows. However, all cells exhibit excess apical Ena and thus form abnormally long, apical microvilli. Perhaps, in some cells, furrow actin assembly drops below a crucial threshold and furrows fail. In the VF, the absence of Abl may have similar effects. VF defects could result from both competition for cellular actin and recruitment of other regulators (e.g. the formin Diaphanous) to ectopic locations, preventing their action in VF formation. This may reduce actin assembly into contractile rings. When constriction initiates, stochastic variations in ring strength may lead some rings to fail, leading to unconstricted cells. Future work is needed to identify the full set of actin regulators involved, and to assess how they work. Interestingly, recent work implicates Abl in epithelial-mesenchymal transitions. Whereas Abl disrupts VF formation, Twist is normally localized in abl mutants, suggesting that this major regulator of such transitions is not an Abl target in flies (Fox, 2007).

The data also reveal the importance of mesodermal Ena downregulation. This may result from increased mesodermal Abl activity, suggested by elevated levels of mesodermal Abl relative to non-mesoderm; however, this remains to be tested. It is also necessary to identify the mechanism by which Abl regulates Ena. In some places, such as the syncytial blastoderm, Abl localizes to sites where Ena is normally absent and, in the absence of Abl, ectopic Ena is found at these sites. This suggests that Abl actively antagonizes Ena localization. At other times and regions, however, such as the leading-edge during dorsal closure, Abl co-localizes with Ena, and thus may hold it in an inactive state. In VFs, Abl localizes to the apical-lateral cortex, and Ena localizes to this site in its absence. Further studies of Abl action will be needed to clarify the mechanisms by which it downregulates Ena (Fox, 2007).

Interestingly, manipulating mammalian Ena/VASP can affect cell contractility Thus, Ena-downregulation may permit proper VF cell contractility. Testing this hypothesis will be important (Fox, 2007).

The results also raise questions regarding RhoGEF2. The model suggests that RhoGEF2 acts via two mechanisms, only one of which is Cta-dependent. Perhaps another upstream cue acts on RhoGEF2 to promote actin organization. Because RhoGEF2 mutants have actin-organization defects in all cells, this regulator may act in all cells prior to gastrulation. However, the data do not rule out a second mesoderm-specific RhoGEF2 regulator acting in parallel to Cta. Although Rho-Kinase is a potential Rho effector with respect to myosin, another effector may regulate actin organization. Attractive candidates are the Formins, which reorganize actin in many processes (Fox, 2007).

The data strengthen the idea that different cytoskeletal regulators direct distinct morphogenetic processes. Both Abl and Fog regulate mesodermal apical constriction but are dispensable for germband cell-cell intercalation. Thus, although both processes require dynamic myosin reorganization, distinct regulators act in each (Fox, 2007).

The picture becomes more complex when considering other roles of Fog, Cta and RhoGEF2. All are required for internalization of the posterior midgut and salivary glands, but these cells internalize in abl mutants. Thus, different types of apical constriction may be regulated differently. It will be interesting to explore the roles of Fog, Cta and RhoGEF2 during dorsal closure, which requires Abl (Fox, 2007).

This work supports mechanistic connections between VF formation and neural tube closure. Both involve actin-based apical constriction to internalize a sheet of cells into a tube. Mice lacking Abl and Arg kinases have neural tube defects, and actin organization in neuroepithelial cells appears altered; interestingly, these cells have ectopic actin that is less polarized than normal, similar to what was observed in abl-mutant VFs. Furthermore, double-mutant analysis suggests that mammalian Ena plays a role in neural tube closure in conjunction with Profilin. Thus, Abl-Ena signaling may represent a conserved mechanism of actin regulation during apical constriction. New mechanistic insights can now be pursued in mammals (Fox, 2007).

Rho also regulates neural tube closure. Mice lacking p190RhoGAP have neural tube defects. Interestingly, p190RhoGAP is an Arg substrate in the brain, suggesting possible direct links between Abl and Rho in apical constriction. The role of Drosophila p190RhoGAP in the VF has yet to be examined, but RhoGAP68F is implicated in VF formation. Future work in both flies and mice will provide further mechanistic insights into conserved mechanisms of apical cell constriction (Fox, 2007).

Although Abl> mutant flies die at the end of pupation or as young adults, flies that lack Abl function and have only one copy of ena survive to adulthood with no obvious defects. The fact that reduction in the level of ena compensates for the absence of Abl activity suggests that these two molecules normally function in opposition to one another in a precise balance. The most frequent CNS defect in enb mutants is a reduction in the integrity of axon bundles. Mutated axons have diffuse and loosely bundled longitudinal and commissural axon tracts. Some homozygous embryos exhibit less obvious phenotypes such as thinning of longitudinal connectives, increased number of axons exciting the CNS from the longitudinal axons or failure of commissural axons to separate into anterior and posterior axon bundles. The overall organization of the PNS is disrupted, spacing and organization of the neurons is irregular and some clusters of neurons are mislocalised. Some defects in axon guidance are apparent, with axons crossing between PNS elements in adjacent segments (Gertler, 1995).

During Drosophila embryogenesis, the Abelson tyrosine kinase (ABL) is localized in the axons of the central nervous system (CNS). Mutations in ABL have no detectable effect on the morphology of the embryonic CNS, and the mutant animals survive to the pupal and adult stages. In the absence of Abl function, however, heterozygous mutations or deletions of Disabled (Dab) exert dominant effects, disrupting axonal organization and shifting the lethal phase of the animals to embryonic and early larval stages. Embryos that are homozygous mutant for both Abl and Dab fail to develop any axon bundles in the CNS, although the peripheral nervous system and the larval cuticle appear normal. The genetic interaction between these two genes begins to define a process in which both the ABL tyrosine kinase and ENA participate in establishing axonal connections in the embryonic CNS of Drosophila (Gertler, 1989).

Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. Methods have been developed to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, several genes have been identified that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis (Gao, 1999).

The peripheral neurons in each hemisegment of the Drosophila embryo are grouped into dorsal, lateral, and ventral clusters. The neurons within each cluster can be further classified on the basis of their dendritic morphology; these categories are external sensory (es) neurons and chordotonal (ch) neurons, each containing a single dendrite; bipolar dendrite (bd) neurons, each with two simple unbranched dendritic projections; and multiple dendrite (md) neurons with extensive dendritic arborizations. The md neurons are thought to function as touch receptors or proprioceptors to sense body surface tension or deformation. The dendritic branching of md neurons does not begin until 16 hr after egg laying (AEL) and continues until and beyond hatching. Because impermeable cuticle already forms at 16 hr AEL, md neuron dendrites can not be visualized by standard antibody staining of whole mount embryos. It is possible to manually dissect individual embryos to allow antibody access; however, this technique is too laborious to be useful for a large-scale mutant screen. To circumvent these technical problems, an assay system was developed on the basis of expression of GFP in living embryos. First, a panel of Gal4 enhancer trap lines was screened to identify those that allow high levels of UAS-driven GFP expression in a subset of PNS neurons at the appropriate developmental stages. Of these, the Gal4 line 109(2)80 was chosen. Recombination was performed to create a second chromosome harboring both the Gal4 109(2) 80 transgene and a UAS-GFP transgene, but no background lethal mutations. A fly line homozygous for the Gal4 109(2) 80/GFP chromosome (denoted as Gal4 80/GFP) was then introduced. In the dorsal clusters of abdominal segments A1-A7, GFP expression labels both axons and dendrites of all six md neurons, one bd neuron, and one tracheal innervating neuron, but not the es neurons. In addition, high levels of GFP expression are detected in the lateral and ventral clusters, and in the antennomaxillary complex. Low levels of GFP fluorescence are also observed in a small subset of neurons in the central nervous system (CNS). The dendrites of dorsal cluster md neurons elaborate just underneath the epidermal layer. In larvae, these dendrites as revealed by Gal4 80/GFP are in tight association with a layer of epidermal cells labeled by Kruppel-Gal4/GFP. It is thus possible to visualize the md neuron dendrites in the dorsal cluster in living animals. A focus was placed on the development of these md neuron dendrites in wild-type as well as mutant embryos. To simplify this description, two types of easily detectable dendrites were defined: dorsal branches grow toward the dorsal midline and lateral branches grow along the approximate anterior-posterior axis toward segment boundaries (Gao, 1999).

The projection pattern of md neuron dendrites in a specific hemisegment is largely invariant from embryo to embryo, on the basis of observations on over thousands of embryos. The major characteristics of dendritic morphogenesis are summarized here. By 12-13 hr AEL, ch and es neurons have already sent out their initial dendritic projections. At this stage, bd neurons have also extended their dendrites. The primary dendrites of md neurons emerge at 13-14 hr AEL, 2 hr after the axons of PNS neurons have reached the CNS. The location of initial dendritic outgrowth and the orientation of this outgrowth are fairly invariant for md neurons. At 13 hr AEL, a dorsal dendrite first emerges from one md neuron in the anterior of the dorsal cluster; shortly after, a second dorsal dendrite emerges from a posterior md neuron of the same cluster. Both dorsal dendrites extend perpendicular to the anterior-posterior axis towards the dorsal midline. Each md neuron (in the dorsal cluster only) sends out one dorsally oriented primary dendrite; however, some md neurons have additional primary lateral dendrites. The dorsal extension essentially stops between 15 and 16 hr AEL, before the lateral branches start to develop. Between 15 and 17 hr AEL, numerous transient lateral branches extend and retract. These branches undergo constant remodeling. Only a subset is eventually stabilized between 18 and 20 hr AEL to become the final lateral branches. At this stage, dorsal and lateral branches are clearly distinguishable. The number of lateral branches in a particular segment is similar from embryo to embryo. In addition, the anterior and posterior dorsal branches within a hemisegment are clearly separated by an area devoid of dendrites. Before hatching (23-24 hr AEL), most lateral branches further elaborate tertiary branches before and after they reach the segment boundary, but only a small number of branches cross over into neighboring segments. At hatching, the dorsal branches have not yet reached the dorsal midline so there is a clear dendrite-free zone near the dorsal midline. After hatching, the dorsal branches resume elongation and reach the dorsal midline by the second instar stage. The length and the thickness of dendritic processes continue to increase with increasing larval body size (Gao, 1999).

Before hatching, the lateral branches are regularly spaced and project toward the segment boundaries. This pattern is relatively invariant from embryo to embryo for a specific hemisegment. To investigate how the dendritic patterning develops, dendrite formation was monitored in living embryos from 15 to 16 hr AEL and time-lapse analysis was carried out. Numerous lateral growth buds emerge anterior or posterior to the dorsal branches and then retract. Only a subset of these lateral branches elongates toward the segment boundaries and becomes stabilized. During this process, the length and orientation of dorsal branches remains largely unchanged. Numerous thin processes at the tips of the lateral branches undergo rapid extension and retraction. These thin processes are not labeled by a Tau-GFP fusion protein, indicating that they might not contain microtubules. This analysis reveals that dendritic development is a dynamic process (Gao, 1999).

Two approaches were used to identify genes involved in dendritic morphogenesis: (1) an investigation of the effects of previously isolated mutations, and (2) a systematic mutant screen. It was reasoned that dendrite development might share some common molecular mechanisms with axon and tracheal development, because all of these processes exhibit subcellular outgrowth and branching. Among the mutants that were examined, prospero mutants and embryos expressing a constitutively active form of Dcdc42 showed detectable dendrite phenotypes. A mutant shows a dendritic phenotype that is somewhat analogous to that in a kakapo mutant. The early budding and extension of lateral branches appear to be normal; however, at a late stage, some lateral branches turn dorsally instead of extending toward segment boundaries, resulting in an apparent reduction in the number of lateral branches. Approximately 60% of the mutant embryos exhibit the phenotype. The mutation that causes lethality and the dendritic phenotype is not complemented by a chromosomal deletion of the region 55F to 56C. Of the available lines carrying lethal P insertions in this region, l(2)02029 does not complement this mutant line. L(2)02029 carries a P-element insertion in the 5' region of the gene enabled. ena⁴⁶, isolated in this study, does not complement four different enabled alleles: ena²¹⁰, ena^GC5, ena^GC8, and ena²³. enabled encodes an Abl tyrosine kinase substrate. enabled is known to cooperate with Dlar to control motor axon guidance. These results indicate that enabled affects dendrite as well as axon development (Gao, 1999).

The ena gene was identified through its ability to suppress the Abl mutant phenotype. Since reductions in ena compensate for the absence of Abl, it has been hypothesized that a precise balance between Abl and Enabled activity is required for viability. Similar to the genetic interaction between Abl and ena, heterozygous mutations in ena can partially alleviate the trio mutant phenotype. One interpretation of this interaction is that a balance between trio and ena is required, and Trio may possess a biochemical function that is antagonistic to Enabled's. Since neural enriched isoforms of Mena, the murine homolog of Enabled, are believed to be involved in filopodia formation to extend the growth cone, a potential antagonistic role for Trio is the retraction of growth cones. Drosophila Trio's second DH domain stimulates the formation of stress fibers in REF-52 cells. In neurons, the formation of similar actin-myosin contractile filaments leads to neurite retraction. Therefore, a balance between the biochemical activities of Enabled and Drosophila Trio may be required for a proper balance between extension and retraction of the growth cone in response to attractive and repulsive pathfinding cues (Liebl, 2000).

Activation of the nonreceptor tyrosine kinase Abelson (Abl) contributes to the development of leukemia, but the complex roles of Abl in normal development are not fully understood. Drosophila Abl links neural axon guidance receptors to the cytoskeleton. This study reports a novel role for Drosophila Abl in epithelial cells, where it is critical for morphogenesis. Embryos completely lacking both maternal and zygotic Abl die with defects in several morphogenetic processes requiring cell shape changes and cell migration. The cellular defects are described that underlie these problems, focusing on dorsal closure as an example. Further, it is shown that the Abl target Enabled (Ena), a modulator of actin dynamics, is involved with Abl in morphogenesis. Ena localizes to adherens junctions of most epithelial cells, and it genetically interacts with the adherens junction protein Armadillo (Arm) during morphogenesis. The defects of abl mutants are strongly enhanced by heterozygosity for shotgun, which encodes DE-cadherin. Finally, loss of Abl reduces Arm and alpha-catenin accumulation in adherens junctions, while having little or no effect on other components of the cytoskeleton or cell polarity machinery. Possible models for Abl function during epithelial morphogenesis are discussed in light of these data (Grevengoed, 2001).

Several lines of evidence support the possibility that the morphogenetic defects of abl^MZ mutants result, at least in part, from Abl action at adherens junctions. (1) The effects on dorsal closure, germband retraction, and head involution are strongly enhanced by reducing the dose of DE-cadherin. (2) The defects in cell shape during dorsal closure resemble, in part, those of arm mutants. (3) The defects in morphogenesis are suppressed by mutations in ena, which is primarily found at adherens junctions. (4) A reduction in junctional Arm and alpha-catenin is seen in abl^MZ mutants. It is important to note, however, that any role for Abl at adherens junctions would be a modulatory one. It is not absolutely essential for adherens junction assembly or function. Of course, it remains possible that other tyrosine kinases may act redundantly with Abl. The relationship between the cadherin-catenin system, Abl, and Ena that may occur in epithelial cells could also exist in the CNS. Arm and DN-cadherin play roles in axon outgrowth in Drosophila, and in this role arm interacts genetically with abl (Grevengoed, 2001).

One target of Abl might be Ena, which could regulate actin dynamics in the actin belt underlying the adherens junction. Just as local modulation of actin dynamics likely regulates growth cone extension or stalling, the cell shape changes and cell migration characteristic of morphogenesis will require modulation of actin dynamics and junctional linkage. The idea that Ena may regulate cell-cell adhesion recently received strong support from work in cultured mammalian keratinocytes, where inhibiting Ena/VASP function prevented actin rearrangement upon cell-cell adhesion. This model is further supported by the demonstration that both Ab1 and Ena regulate actin polymerization at the adherens junctions of ovarian follicle cells in Drosophila (Grevengoed, 2001).

Actin and microtubules (MTs) are tightly coordinated during neuronal growth cone navigation and are dynamically regulated in response to guidance cues; however, little is known about the underlying molecular mechanisms. Drosophila Pod-1 can crosslink both actin and MTs. In cultured S2 cells, Pod-1 colocalizes with lamellar actin and MTs, and overexpression remodels the cytoskeleton to promote dynamic neurite-like actin-dependent projections. Consistent with these observations, Pod-1 localizes to the tips of growing axons, regions where actin and MTs interact, and is especially abundant at navigational choice points. In either the absence or overabundance of Pod-1, growth cone targeting but not outgrowth is disrupted. Taken together, these results reveal novel activities for pod-1 and show that proper levels of Pod-1, an actin/MT crosslinker, must be maintained in the growth cone for correct axon guidance (Rothenberg, 2003).

Preliminary genetic interaction data suggest that Pod-1 may function in part to transmit guidance signals to the cytoskeleton. For example, several observations were suggestive of a relationship between Pod-1 and Enabled. (1) Axon defects in embryos devoid of Pod-1 resemble defects in embryos mutant for enabled (ena). (2) Pod-1 overexpression recruits Ena to the ends of the neurite-like projections in S2 cells. (3) Extensive colocalization is observed between Pod-1 and Ena in S2 cells as well as in embryos. Therefore genetic interactions between pod-1, ena, and the robo receptor (one of several axon guidance receptors that directly binds to Ena) were tested to ask whether the genes might function together in midline repulsion, a specific axon guidance decision that involves Robo and Ena. Indeed, it was found that while pod-1 zygotic mutants, ena heterozygotes, or robo heterozygotes do not exhibit midline crossing errors, when gene dosages of ena or robo are reduced simultaneously with pod-1, frequent (i.e., in approximately 30% of abdominal segments) midline crossing errors are observed (Rothenberg, 2003).

Effect of mutations in Abl

The Drosophila Abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, abl/v-abl-chimeric Abelson murine leukemia viruses were constructed. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of Drosophila ABL protein. The chimeric Abelson murine leukemia viruses are able to mediate morphological and oncogenic transformation of NIH 3T3 cells and are able to abrogate the interleukin-3 dependence of a lymphoid cell line. A virus that contains both TPK and carboxy-terminal Drosophila ABL regions has no in vitro transforming activity for primary bone marrow cells and lacks the ability to induce tumors in susceptible mice. A virus that replaces only a portion of the v-abl TPK region with that of Drosophila ABL has low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of ABL TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity (Holland, 1990).

Mutations in the Drosophila Abelson tyrosine kinase have pleiotropic effects late in development that lead to pupal lethality or adults with a reduced life span, reduced fecundity and rough eyes. Evidence for ABL function was obtained by analysis of mutant phenotypes in the embryonic somatic muscles and the eye imaginal disk. The expression patterns and mutant phenotypes indicate a role for ABL in establishing and maintaining cell-cell interactions (Bennett, 1992).

The axonal localization of the Drosophila ABL protein and its genetic interactions with Dab and fasciclin I (Abl has a synergistic effect with fas I in axon guidance) implicates ABL in axonal pathfinding. Several changes at the amino terminus of ABL permit proper function and localization of the altered protein. In contrast, the presence of human c-Abl type 1a amino-terminal sequences or the murine c-Abl carboxy-terminal domain interfers with function and axonal localization. Rescue of phenotypes caused by mutations in Abl alone does not require tyrosine kinase activity, indicating a novel kinase-independent function for the properly localized Abl protein. However, ABL kinase activity is required to rescue the mutant phenotypes in genetic backgrounds also mutant for disabled (Henkemeyer, 1990).

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase (Hill, 1995).

The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Genetics and cell biology have been combined to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. Profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, Capulet (Capt), the Drosophila homologue of Adenylyl Cyclase Associated Proteins, functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. It is therefore proposed that Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila (Baum, 2001).

Initial analysis of Capt in the Drosophila germline shows that this protein is required for proper oocyte polarity. To determine whether Capt is also required for the establishment and/or maintenance of follicle-cell apical-basal polarity, the localization of microtubules, Crumbs, alpha-spectrin and ß-spectrin was examined in capt clones. With these markers, it is found that capt mutant follicle cells retain many aspects of their wild-type epithelial polarity despite the profound change in actin organization. Thus, it is possible that apical actin-filament formation in capt mutant clones is localized by the action of the cell's apical-basal targeting machinery. Alternatively, proximity to the germline could define the site of preferential apical actin-filament formation in a capt mutant cell. To distinguish between these two possibilities clones were generated within the columnar epithelium that lacked both capt and lethal(2)giant larvae [l(2)gl] gene functions (because l(2)gl is required cell-autonomously for the maintenance of epithelial polarity). In capt l(2)gl double-mutant cells, F-actin is frequently seen accumulating at a single, randomly positioned site within the cell. Thus, although components of the apical-basal targeting machinery are not required to limit actin-filament formation to a single site, they are necessary to target actin-filament formation to the apical surface of capt mutant cells. These data led to a postulate of the existence of a protein that is targeted to apical junctions and that promotes local actin-filament formation after the loss of Capt. Ena was considered a potential candidate (Baum, 2001).

Ena family members are thought to be key regulators of actin-filament dynamics. In mammalian cells they promote actin-filament formation and are localized at adherens junctions and focal contacts. To determine whether Drosophila Ena shares similar properties, the distribution and function of Ena within follicle cells was analysed. In early egg chambers, Ena protein is concentrated at the apical cell cortex of wild-type follicle cells. Subsequently, as the epithelium begins to migrate, the level of Ena increases, particularly within posterior follicle cells and in motile border cells. At this stage the protein remains localized together with F-actin at the apical surface of the epithelium. However, Ena is also observed in punctate cytoplasmic structures that lack coincident actin filaments. These structures are present in follicle cells throughout oogenesis but become more striking as the egg chamber develops. Finally, at late stages of oogenesis, Ena protein becomes concentrated at the basal surface of the follicular epithelium, flanking stress-fibre-like actin filaments. To locate Ena more precisely, the distribution of Ena was compared with that of Armadillo (Arm). Arm is the Drosophila ß-catenin homologue and is localized at apical adherens junctions interconnecting cells within the epithelium. It was found that Ena and Arm localize together apically in wild-type follicle cells. Thus, through much of oogenesis, Ena is concentrated together with F-actin and Arm at follicle-cell adherens junctions, the site of ectopic actin-filament formation in capt mutant cells (Baum, 2001).

As a test of Ena function, follicular ena clones were generated. Cells homozygous for hypomorphic ena alleles (ena²¹⁰, ena²³) lose cortical actin filaments from apical, basal and lateral sites. However, whereas chic clones preferentially lose basal actin filaments, ena mutant cells also exhibit a marked decrease in the amounts of apical F-actin. This might reflect the fact that Ena is concentrated at apical junctions in the wild type, whereas profilin has a broader cellular distribution. It is concluded that Ena facilitates actin-filament formation in Drosophila, much as it does in mammalian cells (Baum, 2001).

In mammalian cells, the overexpression of Ena homologues is able to induce ectopic actin-filament formation. To determine whether Ena can promote excessive actin-filament formation in Drosophila, Ena was overexpressed in follicle cells and, in a separate experiment, in the germline. In both cell types Ena is able to induce the formation of novel F-actin structures at sites where Ena aggregates accumulate. Thus, Ena is likely to be a critical determinant of the subcellular distribution of actin filaments within a cell (Baum, 2001).

Given Ena's important role in the control of epithelial actin organization, ena capt double-mutant clones were generated to test whether Ena is also required for the formation of apical actin aggregates in the capt mutant. Because ena and capt genes are located on opposite arms of chromosome II, this is an experimental challenge. However, the mutants were recombined onto a double FRT chromosome to generate double-mutant cells that were identified by the absence of green fluorescent protein (GFP) and the loss of Capt. F-actin aggregates do not form in the double mutant lacking both Ena and Capt functions, and ena capt clones frequently lose actin filaments. This result places Ena genetically downstream of Capt. Thus, like profilin, Ena is required for the synthesis or nucleation of actin filaments at adherens junctions in the capt mutant.

Given that the Abl tyrosine kinase binds mammalian Capt and antagonizes the function of Ena in Drosophila, whether Abl, like Capt and Ena, might have a role in the control of F-actin organization in the follicular epithelium was tested. Clonal analysis reveals that loss of Abl causes subtle defects in F-actin organization. In abl⁴ mutant cells, apical actin filaments are often mislocalized, appearing at elevated levels at lateral cell cortices. In addition, abl clones exhibit severe defects in epithelial architecture, with mutant tissue forming a multilayered epithelium close to the posterior pole of the egg chamber and, to a smaller extent, at the anterior pole. A similar phenotype has been described in dlg/l(2)gl/scrib mosaic egg chambers. Because these genes are required for proper epithelial cell polarity, Abl might also regulate the polarity of follicle cells. As a further perturbation of Abl function the heat-shock Gal4 driver was used to express the protein at high levels within the follicular epithelium. Like the abl loss of function, high-level overexpression of Abl also perturbs epithelial architecture, leading to the formation of multiple layers of cells at the posterior pole of the egg chamber. Thus Abl functions to modulate follicle-cell F-actin organization and cell polarity and must be tightly regulated in follicle cells to maintain proper epithelial character (Baum, 2001).

To test genetically for an interaction between Abl and Capt, Abl was expressed at more moderate levels (using the T155 Gal4 driver) in egg chambers containing capt mutant clones. Although a modest overexpression of Abl has little visible effect on wild-type follicle cells, an increase in Abl expression in capt mutant follicle cells has profound effects. Increased levels of Abl protein alter both the level and distribution of actin filaments in capt mutant cells. The formation of large localized F-actin aggregates seems to be suppressed and actin filaments often become more widely distributed around the cell cortex, as observed in abl mutant clones. In addition, in a minority of egg chambers, the combination of Abl overexpression and loss of Capt causes a profound disruption of epithelial morphology. This genetic interaction between capt and Abl implies that the two genes have related functions in the control of epithelial F-actin organization (Baum, 2001).

Finally, having found genetic evidence to suggest that Ena and Abl cooperate with Capt in the control of epithelial F-actin, the distribution was examined of Ena and Abl proteins in capt mutant follicle cells. In the capt mutant, Ena's distribution is altered so that the majority of the protein becomes localized with apical actin filaments. Thus Ena is found tightly associated with apical F-actin both in the wild type and in capt mutant cells. In contrast, significant amounts of Ena are not observed at the apical surface of capt chic double-mutant cells, in which apical F-actin aggregates are not formed. Therefore, both in the wild type and in various mutants, the amount of Ena present at adherens junctions closely parallels the level of apical F-actin. The localization of Abl was also examined in the wild type and in capt mutant tissue. Abl, like Capt, seems to have a diffuse staining pattern within wild-type follicle cells. However, in capt clones, Abl protein becomes concentrated at the apical cell surface, partly localizing with Ena. Thus, a loss of Capt leads to a change in localization of both Ena and Abl. Because these proteins act together with Capt to control the spatial organization of the follicular actin cytoskeleton, their altered distribution is likely to contribute to the generation of the marked capt mutant phenotype (Baum, 2001).

This study has used Drosophila genetics and the follicular epithelium to characterize how various actin-binding proteins act to regulate the spatial organization of F-actin. The results show that actin dynamics are regulated by distinct mechanisms within different domains of a polarized epithelial cell. Capt, Ena and Abl seem to modulate apical actin-filament formation, whereas cofilin and profilin seem to have a more global function, regulating cortical actin-filament dynamics throughout the cell. Moreover, the accumulation of F-actin at apical, basal and lateral sites in tsr mutant follicle cells and the loss of cortical actin filaments in chic mutant cells indicates that cortical actin filaments are turned over continuously throughout the cell. This being so, it is striking that F-actin becomes so highly polarized in the capt mutant (Baum, 2001).

In vitro, Capt has been shown to inhibit actin polymerization, which is consistent with its role in limiting epithelial actin-filament formation. In such assays, Capt seems to block actin polymerization by sequestering actin monomers. However, Capt protein and monomeric actin seem evenly distributed within the follicular epithelium. Because the loss of a uniformly distributed, actin-monomer sequestering protein would not be expected to result in the polarized accumulation of F-actin, it is considered possible that Capt might function by inhibiting the activity of a protein at apical adherens junctions that promote local actin-filament formation. Given Ena's ability to promote F-actin-filament assembly in mammalian cells, it is considered that Ena is a potential target of Capt activity. Cell-biological and genetic analysis of Ena within the Drosophila follicular epithelium supports this idea. Ena is concentrated together with F-actin at apical adherens junctions, and the level of Ena protein closely parallels the extent of local actin-filament formation. These results indicate that Ena has an important role in dictating the spatial organization of the actin cytoskeleton in Drosophila follicle cells. Moreover, Ena is required for the synthesis of apical actin aggregates in the capt mutant, which adds strength to the hypothesis that Capt might inhibit apical actin-filament formation catalysed by Ena. However, it is important to note that Ena is also present in cytoplasmic aggregates that lack concomitant F-actin and that in the wild type, Ena localizes with F-actin primarily at the apical cell cortex. The presence of Ena is therefore not sufficient to induce local actin polymerization, and its ability to catalyse actin-filament formation might be augmented at adherens junctions. Finally, homologues of Ena have been shown to localize to focal contacts and adherens junctions in mammalian epithelial cells in culture, suggesting that Ena might have an evolutionarily conserved function to control actin-filament formation at these sites (Baum, 2001).

Although profilin binds Ena and is required for F-actin formation within the wild type and in capt mutant follicle-cell clones, profilin seems to differ from Ena in two respects: (1) it is found that the overexpression of profilin has little effect on the level or distribution of F-actin; (2) profilin protein is not localized within follicle cells. Therefore profilin might have a general function within cells, facilitating actin-filament formation throughout, whereas Ena catalyses actin-filament formation at specific subcellular sites (Baum, 2001).

Like Capt and Ena, Abl is also required for proper F-actin organization within the follicle-cell epithelium. Interestingly, Abl also exhibits a cell-polarity phenotype reminiscent of that seen in l(2)gl/dlg/scrib mutants. Furthermore, the activity of Abl has a potent effect on the organization of F-actin in follicle cells lacking Capt. This is different from that observed in capt l(2)gl mutant cells because excess Abl generates more diffusely localized ectopic F-actin. The synergistic interaction between Capt and Abl suggests that these two proteins act in the same pathway. However, because Abl alters the organization of actin within capt mutant cells, Abl and Capt are unlikely to be components in a simple linear signalling cascade. One possibility is that Abl modulates the integrity of adherens junctions, where Ena and Capt seem to act. In support of the idea that Capt and Abl have related functions, mammalian homologues of Capt and Abl have been shown to interact physically, indicating that this relationship might be conserved in mammals. Moreover, actin aggregates reminiscent of those seen in the fly capt mutant cells are formed in the neurons of abl mutant mice. Thus Abl might modulate actin-filament formation in multiple organisms and cell types, perhaps by cooperating with Ena and Capt (Baum, 2001).

Bringing these data together, a model is envisaged in an effort to explain the aetiology of the pronounced capt mutant phenotype. Normally, in the columnar epithelium, Ena protein is concentrated at adherens junctions, where it promotes local F-actin synthesis. This activity of Ena is counterbalanced by Capt, which limits the amount of apical F-actin. Excess F-actin therefore forms at apical junctions in capt mutant cells. This newly formed apical F-actin is able to recruit additional molecules of Ena from the cytoplasm, because Ena binds microfilaments, which leads to further actin-filament formation. This explains why, in the absence of apical F-actin aggregates, Ena does not become concentrated at the apical cortex of cells in the capt chic mutant. Thus, the loss of Capt initiates an explosive cycle of local actin polymerization and Ena recruitment at adherens junctions, culminating in the striking polar actin aggregates observed in capt mutant cells. It is speculated that within wild-type epithelial cells, controlled autocatalytic cycles of actin-filament formation of this type might help to limit the accumulation of actin filaments to a single site within a cell. For instance, during the formation of a Drosophila wing hair, a similar process might be required to generate a single bundle of actin filaments at the apical cortex of the epithelium (Baum, 2001).

In Drosophila, Ena and Abl are thought to be part of a signalling pathway that changes local actin polymerization within the growth cones of neurons to guide axonal pathfinding. However, it has not been possible so far to analyse the cytoskeletal consequences of perturbations in Ena and Abl function directly within Drosophila neurons. The easily visible, asymmetric actin cytoskeleton in follicle cells has allowed the definition of cell-biological roles for these actin-regulatory genes. In these cells, Capt, Ena and Abl modulate actin-filament assembly at specific subcellular sites, probably by altering local actin dynamics. Thus, by analogy, these proteins might alter the site of actin-filament formation in response to signalling in neurons. If so, it will be interesting to determine whether similar signals impinge on this putative Capt/Ena/Abl pathway in neurons and in epithelia. Finally, given the fact that Capt, Ena and Abl control actin cytoskeletal organization in multiple tissues and in different organisms, these genes might have a conserved function, acting together to control the distribution of actin filaments in many other types of polarized animal cell (Baum, 2001).

Neurons exhibit diverse dendritic branching patterns that are important for their function. However, the signaling pathways that control the formation of different dendritic structures remain largely unknown. To address this issue in vivo, the peripheral nervous system (PNS) of Drosophila was used as a model system. Through both loss-of-function and gain-of-function analyses in vivo, it has been shown that the nonreceptor tyrosine kinase Abelson (Abl), an important regulator of cytoskeleton dynamics, inhibits dendritic branching of dendritic arborization (DA) sensory neurons in Drosophila. Enabled (Ena), a substrate for Abl, promotes the formation of both dendritic branches and actin-rich spine-like protrusions of DA neurons, an effect opposite that of Abl. In contrast, p120 catenin (p120 ctn) primarily enhances the development of spine-like protrusions. These results suggest that Ena is a key regulator of dendritic branching and that different regulators of the actin cytoskeleton exert distinct effects on dendritic morphogenesis (Li, 2005).

Previous studies implicated Ena in the actin-dependent process of axon guidance. Ena, one of the founding members of Ena/VASP family proteins, is present at the leading edge of lamellipodia and at the tips of filopodia and directly binds to the actin monomer-binding protein profilin (Krause, 2003). In cultured hippocampal neurons, Ena/VASP activity is required for the normal formation of filopodia on growth cones and neurite shafts. Using both loss-of-function and gain-of-function approaches, it has been demonstrated that Ena regulates the dendritic branching of different subtypes of DA sensory neurons in Drosophila. Dendritic filopodia may be involved in dendritic growth and branching. As shown by time-lapse analysis, numerous filopodia-like thin processes at the tips of lateral dendrites of DA neurons undergo rapid extension and retraction during Drosophila embryogenesis. Some are stabilized and eventually become lateral branches. These immunostaining studies showed that Ena was localized in dendrites of DA sensory neurons and that the number of dendritic filopodia is significantly reduced in ena mutant embryos. Therefore, it is highly likely that Ena plays analogous roles in dendrites to control the formation of filopodia, precursors of stable dendritic branches (Li, 2005).

Abl, a nonreceptor tyrosine kinase, plays a key role in several developmental processes including axon guidance and epithelial morphogenesis and has a cell-autonomous function in limiting dendritic branching, opposite that of Ena. Loss of Abl activity results in an increased number of dendritic branches and spine-like protrusions, while overexpression of Abl inhibits the formation of dendritic branches and spine-like protrusions. A mutant Abl construct that abolishes its kinase activity has no effect on dendritic development, suggesting that phosphorylation of its substrates is required for its activity in this process. Thus, the extent of dendritic branching appears to be controlled, in part, by the balance between the activities of Ena and Abl (Li, 2005).

Several lines of evidence indicate that Abl also interacts with cadherin complexes. Abl mutations in Drosophila significantly enhance the axonal defects found in armadillo (Arm, the ß-catenin homologue) mutants. Drosophila E-cadherin interacts with Abl in controlling epithelial morphogenesis. Furthermore, Abl forms a complex with the axon guidance receptor Robo and N-cadherin, and the kinase activity of Abl is essential for phosphorylation of ß-catenin. Abl also interacts with and phosphorylates ß-catenin, a member of the p120ctn subfamily in mammals. Several components in the N-cadherin complex regulate various aspects of neuronal morphogenesis (Li, 2005 and references therein).

The potential interaction between Abl and p120ctn prompted an examination of the role of p120ctn in dendritic branching. Drosophila p120ctn mutant embryos do not show defects in adherens junctions, and p120ctn is not required for DE-cadherin function in vivo. However, studies in mammalian systems indicate that p120ctn plays a key role in maintaining normal levels of cadherin. In this study, morphological and molecular differences between dendritic branches and spine-like protrusions on some DA neurons are described. Although those spine-like protrusions do not make synaptic connections with other neurons, they do share some similarities with mammalian spines. Most notably, these processes are more or less perpendicular to dendritic shafts and are highly enriched in actin. Evidence is provided that p120ctn has a function in neural development. It was found that loss of p120ctn activity reduced the number of spine-like protrusions on dendrites of some DA sensory neurons, while overexpression of p120ctn promotes the formation of these fine dendritic structures. Since overexpression of ß-catenin, another cadherin-associated protein, increases spine density in cultured mammalian neurons, it remains possible that p120ctn regulates actin cytoskeleton dynamics through modulating the cadherin complex. Interestingly, ena and p120ctn interacted genetically, revealing a supporting role for p120ctn in modulating dendritic branching of a subset of DA neurons, which was not obvious when p120ctn was mutated alone. Taken together, these genetic analyses suggest that different regulators of the actin cytoskeleton exert their specific effects on dendritic morphogenesis through interactive molecular pathways (Li, 2005).

Studies in cultured cells and in vitro have identified many actin regulators and begun to define their mechanisms of action. Among these are Enabled (Ena)/VASP proteins, anti-Capping proteins that influence fibroblast migration, growth cone motility, and keratinocyte cell adhesion in vitro. However, partially redundant family members in mammals and maternal Ena contribution in Drosophila previously prevented assessment of the roles of Ena/VASP proteins in embryonic morphogenesis in flies or mammals. This study used several approaches to remove maternal and zygotic Ena function, allowing this question to be addressed. Inactivating Ena does not disrupt cell adhesion or epithelial organization, suggesting its role in these processes is cell type-specific. However, Ena plays an important role in many morphogenetic events, including germband retraction, segmental groove retraction and head involution, whereas it is dispensable for other morphogenetic movements. This study focused on dorsal closure, analyzing mechanisms by which Ena acts. Ena modulates filopodial number and length, thus influencing the speed of epithelial zippering and the ability of cells to match with correct neighbors. Filopodial regulation was explored in cultured Drosophila cells and embryos. These data provide new insights into developmental and mechanistic roles of this important actin regulator (Gates, 2007).

Drosophila Ena localizes to AJs and regulates cortical actin assembly in follicle cells, and Ena/VASP proteins help establish cell adhesion in keratinocytes and mammary epithelial cells (Scott, 2006). The hypothesis was tested that Ena/VASP proteins play key roles in cell-cell adhesion. Surprisingly Ena inactivation or mutation does not disrupt AJs or epithelial integrity in embryos or imaginal discs, suggesting that they do not play general roles in these processes. Of course, Ena/VASP proteins may play cell type-specific roles; e.g., filopodial zippers initiating keratinocyte adhesion may require Ena, whereas other mechanisms of establishing epithelial architecture may not. These data also reveal morphogenetic events in which Ena is nonessential; e.g. there were no apparent defects in ventral furrows or germband extension. Consistent with this, Mena/VASP/Evl triple mutant mice complete many morphogenetic processes without major defects (Gates, 2007).

These data reveal key roles for Ena in many morphogenetic events: germband retraction, head involution, segmental groove retraction and dorsal closure. These build upon previously documented roles for Ena in axon guidance and actin regulation in follicle cells, providing a view of the spectrum of biological events requiring Ena. It is hypothesized that Ena acts in a mechanistically similar manner in each process, as an actin anti-Capping protein promoting filament elongation. Flies use this tool in many different ways, promoting distinct cell behaviors. Some events can be linked fairly directly to known Ena/VASP functions. The role of Ena in axon guidance fits its actin regulatory role well. During dorsal closure, Ena inactivation reduces filopodia, consistent with its biochemical function. The data reveal that this slows epithelial zippering and disrupts precise alignment between the two sheets. Leading-edge filopodia have been proposed to function both as sensors directing proper cell matching, and to facilitate adhesion of cells from opposing edges in Drosophila and C. elegans. Although the data support a sensory role for filopodia, disrupted epithelial adhesion/fusion was not observed. The data support and contrast with work on embryos expressing dominant-negative Cdc42 (Cdc42DN), which produce rudimentary protrusions. Like ena mutants, Cdc42DN embryos display mismatching of cells from opposing edges, but, unlike ena mutants, Cdc42DN embryos have gaps between cells along the midline. Although Cdc42DN-expressing leading-edge cells produce only rudimentary protrusions, FP4mito-expressing cells produce robust lamellipodia. Because AJ formation in cultured mammalian cells can be mediated either by filopodia or lamellipodia, lamellipodia may mediate adhesion in the absence of filopodia (Gates, 2007).

Ena is also essential for morphogenetic events in which its cell biological role is more speculative. Germband retraction requires integrin-mediated adhesion of amnioserosa and epidermis to couple cell-shape changes in the two tissues. Ena inactivation mimics integrin loss. Ena/VASP proteins localize to focal adhesions, and Drosophila Ena colocalizes with integrins at ends of planar-polarized actin bundles in follicle cells. Ena/VASP inactivation does not disrupt focal adhesions, but may modulate their size and stress fiber robustness under mechanical stress. Drosophila Ena may strengthen the cytoskeleton during germband retraction, promote amnioserosal lamellipodia or regulate extracellular matrix (ECM) adhesion more directly, as VASP does in platelets and as Ena/VASP proteins may do in Xenopus somitogenesis. Less is known about mechanisms by which segmental grooves form and retract. However, Ena is planar-polarized to dorsal-ventral cell boundaries in these cells; perhaps it stabilizes actin attachment at borders where it is enriched. Defects in head involution result from alterations in dorsal-fold cell shape change, which may share mechanistic similarities with segmental groove formation (Gates, 2007).

One key challenge is to identify machinery required to generate filopodia and lamellipodia. a striking correlation was found between Ena activity and filopodial length and number. Inactivating Ena significantly decreased filopodial length and number, whereas increasing Ena activity increased filopodial length. Interestingly, maximum filopodial length was not substantially altered, and thus is probably not limited by Ena levels. However, Ena can be rate-limiting in filopodial formation as Ena overexpression generated filopodia on lateral epithelial cells that normally do not produce them. Together, these data suggest that Ena promotes both initiation and elongation of leading-edge filopodia (Gates, 2007).

Ena is concentrated at the tips of elongating filopodia, consistent with its influence on filopodial length and its biochemical function. Interestingly, GFP-Ena particles move rearward prior to retraction, presumably by retrograde flow, and some GFP-Ena is retained at filopodial tips as they retract. This was unexpected, since the anti-capping function of Ena/VASP suggested that Ena at filopodial tips would promote extension. Although this could be an artifact of GFP-Ena, it may indicate complexity in the control of filopodial dynamics. For example, whether filopodia continue extending or retract may be determined not only by actin polymerization rates at the tip, but also by depolymerization and/or retrograde flow rates at its base. In addition, filopodial dynamics may be regulated at individual filaments within filopodia rather than the structure as a whole. GFP-Ena particles may be locally inactivated Ena on individual filaments moving away from the tip by retrograde flow (Gates, 2007).

One unanswered question is whether different filopodial regulators act additively or in series. Mammalian Ena/VASP can act downstream of Cdc42 together with IRSp53, but IRSp53 can promote Ena/VASP-independent filopodia (Nakagawa, 2003). Formins also promote filopodia, but whereas Dictyostelium dDia2 and VASP directly interact, Ena/VASP:formin relationships remain unclear. The reduced number of short filopodia formed when Ena is inactivated is consistent with multiple mechanisms acting additively/synergistically to produce the appropriate filopodial number/length (Gates, 2007).

These data also test in vivo one aspect of the convergent elongation model. This proposes that tip complex proteins bind filaments and protect them from capping, allowing continued elongation, and then interact laterally, bundling filaments and forming filopodia. Ena/VASP proteins may supply anti-capping activity, and could also help mediate lateral association via tetramerization. GFP-Ena overexpression promoted large lamellipodia containing numerous actin microspikes; these often converged at their distal ends to form filopodia, supporting the convergent elongation model (Gates, 2007).

Filopodial regulation was also examined. The data demonstrate that Abl is a key negative regulator of filopodial extension in cultured cells and in vivo, inhibiting Ena accumulation at nascent filopodial tips. This idea is further supported by a parallel analysis of embryos expressing activated Bcr-Abl or excess wild-type Abl; both reduce filopodia on leading-edge and amnioserosal cells. This provides a means for signal transduction pathways to regulate cell behavior (Gates, 2007).

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