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α-Actinin is an evolutionarily conserved actin filament crosslinking protein with functions in both muscle and non-muscle cells. In non-muscle cells, interactions between α-actinin and its many binding partners regulate cell adhesion and motility. In Drosophila, one non-muscle and two muscle-specific α-actinin isoforms are produced by alternative splicing of a single gene. In wild-type ovaries, α-actinin is ubiquitously expressed. The non-muscle α-actinin mutant ActnΔ233, which is viable and fertile, lacks α-actinin expression in ovarian germline cells, while somatic follicle cells express α-actinin at late oogenesis. This latter population of α-actinin, termed FC-α-actinin, is shown to be absent from the dorsoanterior follicle cells, and evidence is presented that this is the result of a negative regulation by combined Epidermal growth factor receptor (EGFR) and Decapentaplegic signalling. Furthermore, EGFR signalling increases the F-actin bundling activity of ectopically expressed muscle-specific α-actinin. A novel morphogenetic event in the follicle cells is described that occurs during egg elongation. This event involves a transient repolarisation of the basal actin fibres and the assembly of a posterior β-integrin-dependent adhesion site accumulating α-actinin and Enabled. Clonal analysis using Actn null alleles demonstrated that although α-actinin is not necessary for actin fibre formation or maintenance, the cytoskeletal remodelling is perturbed, and Enabled does not localise in the posterior adhesion site. Nevertheless, epithelial morphogenesis proceeded normally. This work provides the first evidence that α-actinin is involved in the organisation of the cytoskeleton in a non-muscle tissue in Drosophila (Wahlström, 2006).
To understand how α-actinin is involved in the function of the follicle cells at late oogenesis, α-actinin localisation was studied in wild-type follicle cells at stages 10-14. For detection, used the monoclonal antibody MAC276, which recognises all three α-actinin isoforms was used, along with a staining protocol that does not allow simultaneous labelling of F-actin with phalloidin. The follicle cells are polarised with the apical side facing the germline and the basal side facing the epithelial sheath surrounding each string of developing egg chambers. At the time of egg chamber assembly, the basal surface of the follicle cells acquires a layer of stress fibre-like actin bundles, which is maintained throughout oogenesis. At stage 10A, α-actinin was localised at the cell cortex (not shown) and was especially abundant in the basal actin fibres. At stage 10B/11, the evenly stained actin fibres began to reorganise, and by stage 11, a distinct patch of α-actinin accumulation was detected in the posterior part of the cell. This pattern was seen in all main body follicle cells, i.e. ventral follicle cells and dorsal cells posterior to the dorsal appendages. In the dorsoanterior follicle cells, α-actinin was expressed at lower levels and showed less distinct localisation patterns. α-Actinin was also detected at the roof cell apices of the elongating dorsal appendages. At the end of stage 12, the basal α-actinin pattern in the main body follicle cells was reorganised again. The accumulation at the posterior end of the cell gradually dispersed, and at stage 13, α-actinin was concentrated at the lateral cell margins. The central actin fibres were less strongly labelled. By stage 14, when the basal actin fibres have disappeared, α-actinin displayed a cortical localisation (Wahlström, 2006).
The lateral stripes of α-actinin in the follicle cells at stage 13 correspond to the previously described adhesion sites shown to contain β-integrin and Ena. Integrins are transmembrane receptors for ligands in the extracellular matrix (ECM), and they mediate adhesion between the cell and the ECM. Ena is the sole Drosophila member of the conserved family of Ena/VASP proteins, which act as positive regulators of actin filament assembly. Co-localisation studies of α-actinin and Ena revealed a complete overlap in the basal cytoskeleton, including the posterior patch, during stages 11 and 12. At stage 13, there was also extensive co-localisation, although Ena appeared to be located closer to the cell margin than α-actinin. Thus, both α-actinin and Ena accumulate in a transient adhesion site-like structure that forms at the onset of egg elongation (Wahlström, 2006).
The basal stress fibres are aligned perpendicular to the A/P axis of the oocyte between stages 7 and 10, but then a phase of slight disorganisation occurs before the perpendicular alignment is reassumed by stage 13. The disorganised phase correlates with the relocalisation of α-actinin and Ena observed in the basal cytoskeleton. The remodelling could also be recognised by phalloidin-staining of the actin fibres, although they indeed appeared quite irregular in most cells. In several cells, they are polarised in the A/P direction, and they often also converge in a denser patch of F-actin, which overlaps with the posterior patch containing Ena and α-actinin. Thus, egg elongation involves an organised repolarisation of the basal actin fibres (Wahlström, 2006).
Analysis of the α-actinin expression pattern in the non-muscle mutant ActnΔ233 revealed that at least two separate populations of α-actinin are present in the follicle cells. α-Actinin produced from an mRNA that is transcribed from the upstream promoter (NC-α-actinin) is ubiquitously expressed in the egg chamber. The second α-actinin population, FC-α-actinin, corresponds to α-actinin that is present in certain non-muscle cells of all examined non-muscle-specific α-actinin mutants. FC-α-actinin is most likely produced from an mRNA transcribed from the downstream promoter and may include both non-muscle α-actinin and adult muscle-specific α-actinin. However, an analysis using isoform-specific antibodies or a complete sequencing of the mRNAs expressed in the egg chamber will be required in order to clarify this issue. FC-α-actinin protein was expressed in the main body follicle cells starting from stage 10, but excluded from the dorsoanterior cells. The dorsoanterior cells are patterned by the EGFR and Dpp signalling pathways, and the results showed that these two pathways together downregulate FC-α-actinin expression, but not the expression of NC-α-actinin (Wahlström, 2006).
The dorsoanterior and main body follicle cells undergo very different morphogenetic changes. The dorsoanterior cells elongate in the apicobasal direction and migrate (Dorman, 2004), an event that did not seem to require α-actinin. In contrast, the main body follicle cells flatten and expand their surfaces. These events are expected to involve different sets of cytoskeletal regulators, of which very little is yet known. The formation of a dense layer of basal actin fibres in the main body follicle cells may include upregulation of proteins known to be involved in stress fibre formation, such as α-actinin. It has been shown that the dorsal midline cells upregulate basal E-cadherin and FasIII, indicating increased cell-cell adhesion. These cells also lose their basal actin fibres, which may explain why less α-actinin, i.e., only NC-α-actinin, is expressed in these cells (Wahlström, 2006).
Throughout oogenesis, the basal cytoskeleton is organised into actin fibres aligned in parallel. Variation has been noted in the actin fibre polarity at the late stages of oogenesis. These observations are extended by showing that the basal cytoskeleton undergoes an organised remodelling during the final stages of oogenesis. The rapid increase in oocyte volume during nurse cell dumping at stage 11 requires that the follicle cells expand their surfaces in order to maintain a coherent epithelium. This process involves a transient change in the polarity of the basal actin fibres, from a perpendicular to a parallel orientation relative to the A/P axis of the egg chamber, and the assembly of a transient structure that accumulates α-actinin and Ena. The association of this structure with an accumulation of β-integrin and its dependence on integrin adhesion demonstrate that the cytoskeletal reorganisation is linked to a remodelling of integrin-based adhesion sites. The fact that α-actinin and β-integrin did not show a strict co-localisation is in good agreement with studies on mammalian cells showing that integrin, but not α-actinin, is present in nascent adhesion sites termed focal complexes. α-Actinin accumulation in the adhesion site occurs later, as the focal complexes mature into focal adhesions (Zaidel-Bar, 2003). The signal that induces the remodelling of the basal cytoskeleton remains to be identified. An intriguing possibility is that the mechanical stress applied to the epithelium during nurse cell dumping is transduced into biochemical signals that result in the observed reorganisation. Two different mechanisms are known to mediate mechanotransduction: stretch-activated ion channels or conformational changes within cell-matrix adhesion sites (Wahlström, 2006).
The current view is that the parallel basal actin fibres shape the oocyte during egg elongation by preventing axial expansion. However, since the basal actin fibres repolarise during egg elongation, the current model does not adequately explain how the oocyte acquires its final shape. The fact that Ena accumulates in the posterior of the cell during egg elongation suggests that a mechanism involving localised actin polymerisation and directed cell growth may also contribute to shaping the oocyte. It has been reported that egg elongation is blocked by mutations in the genes encoding α-integrin, β-integrin, the adhesion site components talin or tensin, the receptor tyrosine phosphatase Dlar or the ECM component Laminin A. In the case of β-integrin and Dlar, it has been shown that the actin fibre polarity is disturbed (Bateman, 2001; Frydman, 2001), and this has been suggested to be the cause of the short egg phenotype. However, the data presented in this work give reason to speculate that defective adhesion between the follicle cells and the ECM might play a role as well (Wahlström, 2006).
To explore the function of α-actinin in the main body follicle cells, clones of cells lacking α-actinin were generated. This experiment unexpectedly revealed that α-actinin is not required for the formation or maintenance of the basal actin fibres. Previous studies, relying on the introduction of truncated α-actinin molecules into cultured mammalian cells, have suggested that α-actinin is important for stress fibre maintenance. Furthermore, examination of transformed cells expressing different levels of α-actinin showed that cells with low α-actinin levels had poorly developed stress fibres, an effect was not observe in this study. It is possible that the follicle cell basal actin fibres are not true contractile stress fibres and therefore do not depend on α-actinin. Alternatively, an alternative pathway for stress fibre assembly that is independent of α-actinin might be activated in the follicle cells following removal of α-actinin (Wahlström, 2006 and references therein).
The clonal analysis revealed that while α-actinin was not necessary for the lateral accumulation of Ena at stage 13, it was cell-autonomously required for the posterior localisation of Ena at stages 11 and 12. The reason for this could be that α-actinin is specifically required for recruiting Ena to the posterior adhesion site, perhaps by recruiting their common binding partner zyxin. Alternatively, the posterior adhesion site may not form at all. The latter possibility is supported by observations on mosaic stage 10B/11 egg chambers that are in the process of assembling the posterior adhesion site. While wild-type cells are in the process of translocating Ena towards the posterior, neighbouring cells lacking α-actinin still showed a lateral Ena pattern. At stage 12/13, the lateral adhesion sites are assembled earlier in the mutant cells than in the wild-type cells, perhaps because the mutant cells had not reorganised their cytoskeleton to the same extent as the wild-type cells had. Thus, these results clearly demonstrate that adhesion site remodelling is altered in the absence of α-actinin. However, in contrast to the cells lacking β-integrin, the Actn mutant cells appear to maintain their adhesion to the ECM, since they appear equally well spread as the wild-type cells at stage 13 (Wahlström, 2006).
The results are in agreement with the current view that vertebrate α-actinin is involved in adhesion site disassembly. This is a strictly regulated process that involves signalling by phosphoinositides, tyrosine phosphorylation and proteolytic cleavage of individual components. α-Actinin is one of the targets for these activities. Phosphorylation of α-actinin by focal adhesion kinase (FAK) reduces α-actinin’s affinity for F-actin and regulates the activity of FAK itself, PtdIns(3,4,5)-P3 binding to α-actinin disrupts α-actinin binding to β-integrin and F-actin, and cleavage of α-actinin by calpain has been associated with cell shape changes in certain cell types. It has also been shown that α-actinin is essential for maintaining the link between the adhesion site and the stress fibre. This conclusion was reached based on an experiment showing that laser-mediated inactivation of α-actinin located in an adhesion site resulted in stress fibre detachment from the adhesion site. In the Drosophila follicle cells, α-actinin is clearly not required for actin fibre attachment. However, by the laser-mediated inactivation, α-actinin was removed from an adhesion site, whereas in the Actn null mutant follicle cells, the adhesion sites never contained α-actinin. Considering the large number of proteins that interact with α-actinin, it is expected that a signal targeted at α-actinin indirectly affects many other proteins and processes as well. An adhesion site lacking α-actinin may well be functional, but it may respond differently to various signals that induce adhesion site remodelling (Wahlström, 2006).
Interestingly, even very large clones of Actn mutant cells had no negative effects on egg morphology. This indicates that proper cytoskeletal remodelling and posterior localisation of Ena is not necessary for egg elongation. Apparently, the expansion of the main body follicle cells in the Actn mutant cells occurs by an alternative mechanism that is not dependent on α-actinin. This raises the question of whether the wild-type remodelling mechanism would become important under some specific conditions not prevailing in the laboratory. The impact of the environment on the development of mutant phenotypes has been well documented in the slime mould Dictyostelium discoideum. Lack of α-actinin results in only minor alterations in cellular functions and did not reduce viability. However, when the cells were grown under conditions resembling their natural habitat, specific developmental defects appeared (Wahlström, 2006).
This study contributes new data to the field of cytoskeletal dynamics in Drosophila follicle cells. A surprisingly complex regulation was undercovered underlaying α-actinin expression in the follicle cells. The basal cytoskeleton of the main body follicle cells undergoes an organised remodelling during egg elongation, and α-actinin is required in this process. This observation provides the first identified phenotype in a Drosophila non-muscle tissue lacking α-actinin. The fact that both loss of α-actinin and overexpression of α-actinin results in very distinct cellular phenotypes suggests that the follicular epithelium could serve as a very useful in vivo system for further studies on mechanisms that regulate α-actinin function and activity. Furthermore, the cytoskeletal remodelling may provide an easily accessible and genetically tractable model for studies on adhesion dynamics in vivo (Wahlström, 2006).
MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. This study reports here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. It is proposed that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics (Lyulcheva, 2008).
The construction of properly sized and functional tissues and organs during animal development requires tight control of cell growth, proliferation, differentiation, and death. Networks of intracellular signal transduction pathways that respond to various secreted ligands and cell surface proteins coordinate these processes. Elucidating the nature of the intracellular signaling networks that connect extracellular stimuli to basic cellular machinery controlling proliferation, growth, and morphology is not only critical for the understanding of tissue size regulation during normal development, but is also important for the identification of aberrant events underlying numerous disease processes, including cancer (Lyulcheva, 2008).
A number of pathways regulating cellular development are initiated by ligation of transmembrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor (EGFR). One of the key mediators of RTK signaling is the Ras GTPase, capable of activating proteins harboring Ras association (RA) domains to initiate downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade, and ultimately resulting in changes in gene transcription. The Ras/MAPK and other canonical RTK signaling pathways have been well characterized, yet they cannot account for all of the observed effects of their respective extracellular signals (Lyulcheva, 2008).
The MIG-10/Rap1-GTP-interacting adaptor molecule (RIAM)/lamellipodin (Lpd) (MRL) proteins are a family of recently identified molecular adaptors, harboring an RA, pleckstrin homology (PH), and several proline-rich domains (Krause, 2004; Lafuente, 2004). Several lines of evidence indicate that MRL proteins act downstream of Ras-like GTPases and transduce extracellular signals to changes in the actin cytoskeleton, cell motility, and adhesion. In particular, Lpd interacts with active Ras and RIAM with active Rap1. Consistent with this, only RIAM is required for Rap1-induced cell adhesion. Lpd also binds to PI(3,4)P2 via its PH domain, which is sufficient for membrane targeting after platelet-derived growth factor stimulation. Both Lpd and RIAM utilize their proline-rich motifs to directly interact with the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) actin regulators, known to regulate lamellipodia formation and cell migration. In addition, Lpd knockdown impairs lamellipodia formation, whereas Lpd overexpression increases speed of lamellipodia protrusion in an Ena/VASP-dependent manner. Finally, both Lpd and RIAM have been shown to alter the cellular ratio between monomeric (G) and filamentous (F) actin, suggesting a wider role in regulating cell metabolism. Indeed, control of the G:F actin ratio is an essential way for cells to regulate gene transcription via the transcription factor serum response factor (SRF), and has been linked to changes in proliferation, migration, and differentiation (Lyulcheva, 2008).
This study reports the characterization of the Drosophila MRL ortholog, which has been named pico on the basis of the retarded growth phenotype resulting from pico knockdown or loss-of-function mutant. Reduction in pico levels results in reduced rates of cell growth and proliferation, whereas ectopic expression of pico promotes coordinated cell growth and proliferation, leading to tissue overgrowth. pico's effect on cell proliferation is conserved in its mammalian ortholog, Lpd. Evidence is presented that pico and Lpd link extracellular signaling to tissue growth via changes in actin dynamics and SRF activation. This is the first time that MRL proteins have been implicated in controlling cell proliferation and tissue growth (Lyulcheva, 2008).
Phylogenetic analysis has shown that pico (CG11940) encodes the only member of the MRL family of proteins in Drosophila. Two transcripts that were identified are generated from alternative transcription start sites of the pico transcription unit: pico and pico-L. pico-L encodes a 1159 amino acid protein that is identical to the protein encoded by pico, except for the presence of an additional 128 N-terminal residues. Both pico proteins contain RA and PH domains and proline-rich Ena/VASP binding sites characteristic of the MRL proteins (Lyulcheva, 2008).
This study shows that pico, which encodes the only Drosophila member of the MRL family of proteins, and its mammalian ortholog, Lpd, have a conserved role in the regulation of cellular proliferation. Reduced pico or Lpd levels result in reduced rates of cellular proliferation, but do not impair cell survival. Too much pico promotes coordinated growth and proliferation, leading to larger tissues with more normal-sized cells. In this respect, the effect of pico is distinct from that of many known Drosophila growth drivers. Growth regulators, such as Drosophila S6K, cause cells to accumulate mass faster than they can divide, primarily due to effects on translation, leading to cellular hypertrophy. Other regulators, such as E2F, can drive cell division without stimulating cell growth, leading to hyperplastic cellular hypotrophy and/or apoptosis (Lyulcheva, 2008).
Attenuating EGFR signaling abrogates the effect of ectopic pico on both F-actin accumulation and tissue growth. pico acts cell autonomously and is therefore unlikely to act upstream of Egfr by affecting the level of EGFR ligands. To rule out that pico regulates levels of EGFR, receptor levels and distribution was examined in wing imaginal discs overexpressing pico or picoIR. EGFR levels and distribution in these genetic backgrounds resembled wild-type. Another possibility is that pico regulates EGFR activity. Although suitable reagents were not available to directly monitor EGFR activity levels in wing discs, effects on extracellular signal-regulated kinase (ERK) activation, which provides a molecular readout for EGFR/Ras/Raf signaling, were measured. Diphosphorylated (dp) ERK levels were not affected by ectopic pico. These data suggest that, rather than being upstream of EGFR, Pico needs to be activated by EGFR or a downstream component of EGFR signaling, such as activated Ras. Consistently, both Lpd and Pico bind to activated, but not wild-type, Ras. Furthermore, pico knockdown partially suppresses the effects of ectopic Egfr and activated Ras; in addition, Lpd knockdown impairs the EGF-induced increase in proliferation. Taken together, these data suggest that pico and Lpd are downstream effectors of EGFR (Lyulcheva, 2008).
Ena/VASP has been reported to act downstream of MRL proteins. Correspondingly, it was found that pico-mediated wing overgrowth and F-actin accumulation are sensitive to the levels of ena. Importantly, ena is also sufficient to cause overgrowth and F-actin accumulation when overexpressed. Changes in actin dynamics induced by Ena/VASP proteins can activate SRF-dependent gene expression in mammalian cells. Similarly, it was found that Pico and Lpd can activate SRF activity. Like pico, ectopic mal or bs/SRF in flies are sufficient to cause wing overgrowth. Pico-mediated overgrowth is sensitive to the levels of bs/SRF, but mal-induced overgrowth could not be suppressed by pico knockdown, suggesting that Mal/SRF may act downstream of pico in flies. Collectively, these data suggest that MRL proteins may exert their mitogenic effects by specifically interacting with Ena/VASP proteins and inducing SRF-responsive transcription. Interactions between EGFR, MRL proteins, Ena/VASP, and Mal may provide a mechanism linking growth factor signaling and Mal-mediated SRF activation (Lyulcheva, 2008).
Are MRL proteins uniquely able to stimulate Mal/SRF-mediated tissue growth? Although other actin regulators are known to activate Mal/SRF (Posern, 2006), there is currently little data to indicate that they play a role in proliferation control. This might be explained if different transcriptional responses occur at different Mal-dependent SRF activation thresholds, leading to diverse cellular outcomes. Alternatively, other actin regulators might influence processes that limit net tissue growth. For instance, Rho activates Mal/SRF in mammalian cells, but increased Rho activity in flies is associated with loss of epithelial integrity and cell extrusion, which may negate any potential mal-mediated growth-promoting effects. These issues warrant further study in both flies and mammals. Future studies are also needed to characterize transcriptional targets of Drosophila SRF and resolve the contribution of SRF targets to MRL-mediated growth and proliferation (Lyulcheva, 2008).
Lpd expression appears to be differentially regulated in cancer compared to normal tissues. The data, showing a conserved role for MRL proteins in proliferation control, may provide a potential mechanistic explanation for these observations. In this regard, it is interesting that loss of pico or Lpd can abrogate the effects of EGFR/Erb signaling, deregulation of which has also been implicated in cancer progression. Collectively, these data suggest that MRL proteins might play a role in the pathogenesis of certain cancers and may therefore represent novel molecular targets for therapeutic intervention (Lyulcheva, 2008).
Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. This study presents evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and the Actin regulator Enabled was identified as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary (Becam, 2011).
Cell divisions lead to cell rearrangements that may challenge straight and sharp compartment boundaries. The DV boundary of mid- and late third instar wing primordia is characterized by a reduced rate of cell proliferation which defines the zone of non-proliferating cells (ZNC). The contribution of the ZNC to the maintenance of the DV affinity boundary was proposed many years ago but this notion was subsequently questioned. This study provides evidence that the ZNC does indeed play a role in boundary formation. bantam miRNA positively modulates the activity of the E2F transcription factor and drives G1-S transition in Drosophila tissues. Notch-mediated downregulation of bantam miRNA defines the ZNC and contributes to maintain a stable DV affinity boundary. Induction of proliferation in boundary cells by the ectopic expression of bantam, the cell cycle regulators Cyclin E and String, or the proto-oncogene dMyc, which is known to drive G1-S transition, compromises the formation of a smooth DV affinity boundary. A similar reduction in proliferation rates is observed at the rhombomere boundaries in the developing hindbrain, suggesting that reduced rate of cell proliferation might often be used in compartment boundary formation (Becam, 2011).
Notch-mediated downregulation of bantam activity is not only required to define the ZNC but also to establish the actomyosin cables observed at the interface between boundary and non-boundary cells. Ena, a regulator of Actin elongation, was identified as a direct target of bantam that is involved in DV boundary formation. The multiple roles of bantam in promoting G1-S transition and tissue growth, blocking apoptosis and regulating Actin dynamics unveil a new molecular connection between these three processes that might have relevance in growth control and tumorigenesis (Becam, 2011).
Intriguingly, bantam miRNA has no major role in the maintenance of the anterior-posterior compartment boundary of the developing wing and this boundary is not affected upon depletion of Ena protein levels. Thus, different regulators of actin elongation might be at work to regulate the actomyosin cytoskeleton and direct cell sorting in diverse developmental contexts. Whether reduced levels of bantam miRNA and increased levels of Ena protein are required to maintain differential cell sorting in the embryonic ectoderm or other imaginal tissues remains to be elucidated (Becam, 2011).
Cut is a late target of Notch that is expressed in boundary cells and is required to induce a stable Notch signaling center. This study demonstrate that Cut activity has also a specific function in reducing Ena mRNA and protein levels in boundary cells. Although depletion of Cut compromises the formation of the actomyosin cables at the interface of boundary and non-boundary cells and the maintenance of a stable DV affinity boundary, cell lineage and clonal analysis of wild-type and cut mutant cells reveal that Cut plays a major role in sorting boundary from non-boundary cells. The finding that the Notch signaling pathway defines, through Cut, a distinct population of boundary cells at the DV interface reinforces the mechanistic similarities in the maintenance of compartment boundaries within the vertebrate hindbrain and the Drosophila wing. In both developmental contexts, Notch defines a distinct population of boundary cells and contributes to segregating boundary from non-boundary cells. Although Cut mediates the role of Notch in the Drosophila wing, the molecular effectors mediating the role of vertebrate Notch in boundary formation remain uncharacterized. The data indicate that the later subdivision into boundary and non-boundary cells contributes to the maintenance of a stable DV affinity barrier in the mature wing primordium (Becam, 2011).
Compartment boundary formation plays an important role in development by separating adjacent developmental fields. Drosophila imaginal discs have proven valuable for studying the mechanisms of boundary formation. This study examined the boundary separating the proximal A1 segment and the distal segments, defined respectively by Lim1 and Dll expression in the eye-antenna disc. Sharp segregation of the Lim1 and Dll expression domains precedes activation of Notch at the Dll/Lim1 interface. By repressing bantam miRNA and elevating the actin regulator Enabled, Notch signaling then induces actomyosin-dependent apical constriction and epithelial fold. Disruption of Notch signaling or the actomyosin network reduces apical constriction and epithelial fold, so that Dll and Lim1 cells become intermingled. These results demonstrate a new mechanism of boundary formation by actomyosin-dependent tissue folding, which provides a physical barrier to prevent mixing of cells from adjacent developmental fields (Ku, 2017).
This study attempted to unravel the molecular and cellular mechanisms of boundary formation in the Drosophila head. Focus was placed on the antennal A1 fold that separates the A1 and A2-Ar segments. The results showed that the expression of the selector genes Lim1 and Dll, which are expressed in A1 and A2-Ar, respectively, was sharply segregated. This step was followed by differential expression of Dl, Ser and Fng, as well as activation of N signaling at the interface between A1 and A2. N signaling then induced apical constriction and epithelial fold, possibly through repression of bantam to allow levels of the bantam target Ena to become elevated, with this latter inducing the actomyosin network. The actomyosin-dependent epithelial fold then provided a mechanical force to prevent cell mixing. When N signaling or actomyosin was disrupted, or when bantam was overexpressed, the epithelial fold was disrupted and Dll and Lim1 cells become mixed. Thus this study describes a clear temporal and causal sequence of events leading from selector gene expression to the establishment of a lineage-restricting boundary (Ku, 2017).
Sharp segregation of Dll/Lim1 expressions began before formation of the A1 fold, suggesting that fold formation is not the driving force for segregation of Dll/Lim1 expression. Instead, the fold functions to safeguard the segregated lineages from mixing. Whether Dll/Lim1 segregated expression is due to direct or indirect antagonism between the two proteins is not known (Ku, 2017).
Actomyosin-dependent apical constriction is an important mechanism for tissue morphogenesis in diverse developmental processes, e.g., gastrulation in vertebrates, neural closure and Drosophila gastrulation, as well as dorsal closure and formation of the ventral furrow and segmental groove in embryos. This study describes a new function of actomyosin, i.e., the formation of lineage-restricting boundaries via apical constriction during development (Ku, 2017).
This actomyosin-dependent epithelial fold provides a mechanism distinctly different from other known types of boundary formation. The cells at the A1 fold still undergo mitosis, suggesting that mitotic quiescence is not involved. Perhaps epithelial fold as a lineage barrier is needed in situations in which mitotic quiescence does not happen. Mechanically and physically, epithelial folds could serve as stronger barriers than intercellular cables when mitotic activity is not suppressed. The drastic and sustained morphological changes, including reduced apical area and cell volume, may be accompanied by increased cortical tension of cells along the A1 fold, with such high interfacial tension then preventing cell intermingling and ensuring Dll and Lim1 cell segregation. Although similar to actomyosin boundaries, the epithelial fold in the A1 boundary is distinctly different from the supracellular actomyosin cable structure in fly parasegmental borders, the wing D/V border, and the interrhombomeric boundaries of vertebrates. The adherens junction protein Echinoid, which is known to promote the formation of supracellular actomyosin cables, is not involved in A1 fold formation. Although actomyosin is enriched in a ring of cells in the A1 fold, it does not exert a centripetal force to close the ring, unlike the circumferential cable described in dorsal closure and wound healing (see review. In the A1 fold, the constricting cells become smaller in both their apical and basolateral domains, thus differing from ventral furrow cells where cell volume remains constant (Ku, 2017).
A tissue fold probably provides a strong physical or mechanical barrier to prevent cell mixing. In addition, whereas in a flat tissue where the boundary involves only one to two rows of cells, the tissue fold involves more cells engaging in cell-cell communication. The close apposition of cells within the fold may allow efficient signaling within a small volume. This may be an evolutionarily conserved mechanism for boundary formation that corresponds to stable morphological constrictions such as the joints in the antennae and leg segments (Ku, 2017).
Although N signaling has been reported to be involved in many developmental processes, a role in inducing actomyosin-dependent apical constriction and epithelial fold is a novel described function for N. For the A1 boundary, N activity is possibly mediated through repression of bantam and consequent upregulation of Ena. In the wing D/V boundary, N signaling is also mediated through bantam and Ena, but the outcome is formation of actomyosin cables, i.e., without apical constriction and epithelial fold [19]. Thus, the N/bantam/Ena pathway for tissue morphological changes is apparently context-dependent (Ku, 2017).
Tissue constriction also occurs later in joint formation of the legs and antennae. N activation also occurs in the joints of the leg disc and is required for joint formation. This role is conserved from holometabolous insects like the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum to the hemimetabolous cricket Gryllus bimaculatus. It is possible that for segmented structures that telescope out in the P/D axis, like the antennae, legs, proboscis and genitalia, N signaling is used to demarcate the boundaries between segments, which are characterized by tissue constriction. N-dependent epithelial fold morphogenesis has also been reported in mice cilia body development without affecting cell fate, suggesting that such N-dependent regulation in morphogenesis is evolutionarily-conserved (Ku, 2017).
It is proposed that N signaling is important in all boundaries that involve stable tissue morphogenesis. For those boundaries corresponding to stable morphological constrictions, e.g., the joints in insect appendages, N acts via actomyosin-mediated epithelial fold. The wing D/V boundary represents a different type of stable tissue morphogenesis. It becomes bent into the wing margin and involves N signaling via actomyosin cables, rather than apical constriction. In contrast, actomyosin-dependent apical constrictions do not involved N signaling and are involved in transient tissue morphogenesis, such as gastrulation in vertebrates, neural closure, Drosophila gastrulation, dorsal closure, as well as formation of the ventral furrow, eye disc morphogenetic furrow, and segmental groove in embryos (Ku, 2017).
N signaling is also involved in the boundary between new bud and the parent body of Hydra, where it is required for sharpening of the gene expression boundary and tissue constriction at the base of the bud [78]. Whether the role of N in these tissue constrictions is due to actomyosin-dependent apical constriction and epithelial fold is not known (Ku, 2017).
Boundaries may be established early in development. As the tissue grows in size through cell divisions and growth, boundary maintenance become essential. This study found that N activity is maintained by actomyosin, suggesting feedback regulation to stably maintain the boundary. Mechanical tension generated by actomyosin networks has been suggested to enhance actomyosin assembly in a feedback manner. Interestingly, the N-mediated wing A/P and D/V boundaries, which form actomyosin cables rather than tissue folds, did not exhibit such positive feedback regulation. Instead, the stability of the Drosophila wing D/V boundary is maintained by a complex gene regulatory network involving N, Wg, N ligands and Cut. Perhaps this is necessary for a boundary not involving tissue morphogenesis (Ku, 2017).
The segmented appendages of arthropods (antennae, legs, mouth parts) are homologous structures of common evolutionary origin. It has been proposed that the generalized arthropod appendage is composed of a proximal segment called the coxopodite and a distal segment called the telopodite, either of which can further develop into more segments. The coxopodite is believed to be an extension of the body wall, whereas the telopodite represents the true limb, and thus represents an evolutionary addition. Dll mutants lack all distal segments except for the coxa in legs and the A1 segment in antennae. Lineage tracing studies have shown that Dll-expressing cells contributed to all parts of the legs except the coxa. These results indicate that the leg coxa and antenna A1 segment correspond to the Dll-independent coxopodite, and that Dll is the selector gene for the telopodite. Therefore, the antennal A1 fold is the boundary between the coxopodite and telopodite. It is postulated that the same N-mediated epithelial fold mechanism also operates in the coxopodite/telopodite boundary of legs and other appendages (Ku, 2017).
Drosophila Enabled (Ena) was first identified as a genetic suppressor of mutations in the Abelson tyrosine kinase and subsequently was shown to be a
member of the Ena/vasodilator-stimulated phosphoprotein family of proteins. All members of this family have a conserved domain organization, bind the
focal adhesion protein zyxin, and localize to focal adhesions and stress fibers. Members of this family are thought to be involved in the regulation of
cytoskeleton dynamics. The Ena protein sequence has multiple poly-(L-proline) residues with similarity to both profilin and src homology 3 binding sites.
Ena can bind directly to Chickadee, the Drosophila homolog of profilin. Furthermore, Ena and profilin are colocalized in spreading
cultured cells. The proline-rich region of Ena is responsible for this interaction as well as for mediating binding to the src homology 3 domain
of the Abelson tyrosine kinase. These data support the hypothesis that Ena provides a regulated link between signal transduction and cytoskeleton assembly
in the developing Drosophila embryo (Ahern-Djamali, 1999).
To identify target proteins for
the C-terminal 243 amino acids of Ena, a yeast two-hybrid screen was performed. The C-terminal 243 amino acids of Ena, which
include the consensus binding site for profilin and a proline-rich consensus site for binding the Abl SH3 domain, were fused to the DNA binding domain of the yeast transcription factor GAL4 and used to screen a Drosophila third-instar
larval library whose inserts were fused to the activation domain of GAL4. The separately expressed domains are unable to
activate transcription of the reporter genes HIS3 and LacZ unless a protein-protein interaction takes place. Of 20.5 million clones screened, 9 interacted with
Ena as assessed by expression of both the HIS and LacZ reporter genes. One of these clones carried a cDNA encoding full-length Chickadee. The interaction is specific, because a construct with the Ena N-terminal domain fused to the DNA binding domain of GAL4 does not interact
with the same isolated Chickadee clone. Of the seven remaining clones, two were partial Ena cDNAs and the other five are unique sequences that are
yet to be described (Ahern-Djamali, 1999).
The region of the Ena protein used as bait in the yeast two-hybrid screen contains several matches to a putative profilin binding site. To test whether
these sequences are important for mediating the interaction with Chickadee, DNA encoding Ena amino acids 440-490 that contain these putative binding sites,
and DNA encoding Ena amino acids 490-684 were fused to the DNA binding domain of the yeast transcription factor GAL4. Yeast were cotransformed with each
of these constructs, and the chickadee cDNA was fused to the activation domain of GAL4 and tested for activation of transcription of the reporter genes HIS3 and
LacZ. An interaction is detected when chickadee is cotransformed with Ena amino acids 440-490 and not Ena amino acids 490-684, suggesting that this
interaction is mediated by proline-rich sequences in Ena. Ena and Chickadee have also been shown to interact in vitro (Ahern-Djamali, 1999).
Profilin has been shown to be localized to cortical microfilament webs and leading lamellae of spreading or
locomoting cells. In Drosophila, profilin is expressed ubiquitously throughout development, as for example, the high levels of profilin in the ventral nerve cord of stage
16 embryos. The Ena protein is localized to actin stress fibers and focal adhesions in cultured cells and is localized to the axonal tracts of the developing
Drosophila embryonic central nervous system, although the small size of these cells makes higher-resolution localization difficult. Because Ena and Chickadee
interact in vitro and are expressed in the nervous system of Drosophila embryos, it was speculated that these two proteins might interact in vivo in regions of dynamic
actin remodeling. The subcellular distribution of transfected Drosophila Ena and endogenous profilin were compared in cultures of spreading Ptk2 cells. Ena
and profilin colocalize to the periphery of the spreading cells. The colocalization, together with the biochemical interactions, suggests that Ena and
profilin associate in vivo (Ahern-Djamali, 1999).
The proline-rich region of Ena contains multiple consensus binding sites for the SH3 domain in addition to the profilin
binding sequences. It has been shown with a filter binding assay that Ena binds the Abl and Src SH3 domains in vitro. The
SH3 binding specificity of Ena was examined further by using a solution binding assay. Ena was expressed in Drosophila S2 cells, and the transfected cell lysates were incubated
with a series of GST-SH3 fusions. Ena binds specifically to the Drosophila and murine Abl-SH3 domains and the murine src SH3 domain. Ena also bind to the
C-terminal but not to the N-terminal SH3 domain of Drk. The two Ena peptides most closely matching
the Abl consensus binding motif partially and specifically block Ena binding to the Abl SH3 domain, although they do not block Src SH3 domain binding. Peptides derived from Ena proline-rich sequences most closely matching the optimal
sequences for Src or Drk SH3 binding do not compete for Ena binding with any of the SH3 domains tested (Ahern-Djamali, 1999).
To determine whether the proline motifs identified in the peptide binding experiment as Abl SH3 binding sites are sufficient to mediate Abl SH3 binding,
site-directed mutagenesis was employed to change eight prolines to alanine, thereby eliminating many of the PXXP motifs present in the sites. Serial two-fold dilutions
of transfected cell lysates containing either the mutant Ena protein (Ena8 P to A) or wild-type Ena were tested for solution binding to the Abl SH3
domain. At higher concentrations of protein, it is difficult to detect an effect of the proline-to-alanine mutations on binding. However, at lower concentrations of the
Ena proteins, binding of the mutant protein is markedly reduced when compared with the wild-type Ena protein. To examine the in vivo effect of the proline-to-alanine mutations on Ena function, transgenes expressing wild-type Ena and the Ena8 P to A mutant
proteins were tested for their ability to rescue ena mutant lethality. The ena8 P to A transgene rescues the embryonic lethality associated with
loss-of-function mutations in ena as well as the wild-type ena transgene. The ena8 P to A-rescued flies are phenotypically normal and have
viability and fertility comparable to wild-type ena-rescued flies. Thus, the proline-to-alanine mutations present in Ena8 P to A are
not sufficient to disrupt an essential function of the Ena protein (Ahern-Djamali, 1999).
Interestingly, there is some overlap in the binding sites for the Abl SH3 domain and some of the putative binding sites for P to A profilin. Because mutation of the prolines in these overlapping regions reduces binding to the Abl SH3 domain, it was hypothesized that these
mutations might also disrupt binding to Chickadee. The mutant ena8 P to A cDNA was subcloned in the pGex expression vector, and the resulting
mutant Ena fusion protein, GST-Ena8 P to A, was compared with wild-type GST-Ena in solution binding assays for its ability to pull down
Chickadee from serial dilutions of lysates prepared from adult Drosophila. The GST-Ena8 P to A fusion protein and wild-type GST-Ena pull
down approximately equivalent amounts of Chickadee, suggesting that different amino acids may be important for Ena binding to profilin and the Abl-SH3
domain, despite the overlap observed in some of their putative binding sites. It is worth noting that no SH3 domain-bearing proteins were isolated in the yeast two-hybrid screen that identified
profilin as a binding partner for Ena. Perhaps the proline-rich sequences present in the Ena bait are not the most important for binding to SH3 domains. Alternatively,
the conditions in the yeast two-hybrid screen may not favor detection of an interaction between an SH3 domain and proline-rich sequences. Another possibility is
that Ena's interaction with the Abl SH3 domain may be less physiologically relevant than its interaction with Chickadee. Indeed, mutations that disrupt
binding to the Abl SH3 domain in vitro have no effect in vivo when they are expressed from a heterologous promoter. It will be important to identify critical amino
acids for the interaction between chickadee and Ena and to examine whether these mutations have any in vivo effects (Ahern-Djamali, 1999).
In the absence of the Drosophila ABL protein-tyrosine kinase (PTK), loss-of-function mutations in either
Disabled or prospero produce dominant phenotypic effects on embryonic development. Molecular and genetic
characterizations indicate that the products of these genes interact with the ABL PTK by different
mechanisms. The interaction between ABL and Prospero, which encodes a nuclear protein required for correct
axonal outgrowth, is likely to be indirect. In contrast, Disabled may be a substrate for the ABL PTK. The Disabled protein is colocalized with ABL in axons, its predicted amino acid sequence contains 10 motifs similar to the major autophosphorylation site of ABL, and the protein is recognized by antibodies to phosphotyrosine (Gertler, 1993).
An ENA fusion protein containing amino acids 53-503 of ENA can bind murine C-ABL and SRC SH3 domains. In contrast ENA protein is not bound by CRK, GAP, NCK or GRB2. ENA contains phosphotyrosine in vivo and acts as a substrate for ABL. Drosophila SRC1 also can phosphorylate ENA in transfected cells. An ABL variant lacking the SH3 domain can phosphorylate ENA, indicating that ABL can phosphorylate ENA in the absence of an SH3-dependent ABL-ENA interaction. Additionally, ENA tyrosine phosphorylation is reduced in Abl mutants, while the level of ENA phosphorylation is unchanged in Src1 mutant pupae (Gertler, 1995).
The cytoplasmic domain of Roundabout homologs varies considerably in length across species and has very little sequence similarity with the exception of three proline-rich motifs of about ten or more amino acids in length. These three short regions are highly conserved and might potentially function as binding sites for SH3 domains or other domains in linker proteins functioning in Robo-mediated signal
transduction. The first conserved cytoplasmic motif contains a tyrosine and is a potential site for phosphorylation. The second conserved motif contains the sequence LPPPP and is a potential site for Drosophila Enabled or its mammalian homolog Mena. Given the role of Drosophila Abl tyrosine kinase in midline guidance and the function of enabled as a suppressor of mutations in Abl, it will be of interest to determine whether Enabled binds the Robo cytoplasmic domain (Kidd, 1998).
Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms that function downstream of Robo to mediate repulsion. Genetic and biochemical evidence is presented that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor (Bashaw, 2000).
Abl and Ena are complementary components of the signaling machinery downstream of the Robo repulsive axon guidance receptor. Genetic interactions indicate that loss of ena function partially disrupts Slit- and Robo-mediated repulsion from the midline. Limiting or removing ena function enhances partial loss-of-function robo phenotypes and suppresses robo gain-of-function phenotypes. In contrast, reduction of abl has the opposite consequence, suppressing the effects of a partial loss of robo function, while panneural overexpression of Abl antagonizes Robo function, leading to a phenotype resembling that of robo mutants (Bashaw, 2000).
Both Abl and Ena bind directly to Robo's cytoplasmic domain in vitro and Robo can act as a substrate for Abl kinase activity in vitro and in cell culture. Robo and Ena also show in vivo physical interactions. Furthermore, cytoplasmic domain mutants that reduce Ena binding to Robo result in impaired ability to rescue robo loss of function, while a Y-F mutation in a conserved tyrosine that can be phosphorylated by Abl in vitro has the opposite consequence, generating a hyperactive Robo receptor. These genetic and biochemical data support a model in which Abl and Ena play direct and opposing roles in the transmission of Robo's repulsive signal (Bashaw, 2000).
The implication of Ena in repulsive axon guidance is somewhat surprising in light of the previous results from the pathogen Listeria monocytogenes indicating that Mena is required for Listeria's actin-polymerization dependent motility. The Listeria data, together with the in vitro effects on actin of the Ena/VASP proteins has frequently been interpreted to suggest that Ena/VASP proteins function to promote actin polymerization, thereby promoting motility. On the contrary, the results presented here indicate that Ena is partially required for axon repulsion from the midline. These data suggest that Ena may have the opposite function, namely, to inhibit forward growth cone motility at sites where Robo encounters Slit (Bashaw, 2000).
In a companion paper (Bear, 2000), an independent study in mammalian cell culture has reached a similar conclusion. By expressing a multimerized EVH1 domain binding site attached to specific subcellular localization sequences, Ena/VASP family members can be efficiently targeted to different areas of cultured fibroblasts. This system has allowed a direct examination of the role of Ena/VASP proteins in cell motility. Surprisingly, when Ena/VASP proteins are directed away from the cell membrane, using a mitochondrial targeting sequence, the cells actually migrate more quickly. Conversely, targeting Ena/VASP proteins to the membrane, or overexpressing Mena, leads to a dose-dependent decrease in the rate of cell migration. A major conclusion of this study is that Ena/VASP proteins function in part to decrease the rate of whole cell motility. Whether Ena/VASP proteins achieve the observed in vivo effects on whole cell and growth cone motility by stimulating or inhibiting actin polymerization awaits future investigation (Bashaw, 2000).
While the dosage-sensitive genetic interactions between ena and robo support a role for Ena in midline repulsion, Ena clearly can not explain all of Robo's repulsive output. Indeed, although mild midline crossing defects are observed in ena mutants, on the whole, Robo-mediated repulsion works fairly well in the absence of Ena. In this light, it is perhaps not surprising that the Robo DeltaCC2 mutant receptor (in which the Ena binding site is deleted) still provides some repulsive activity and can partially rescue robo loss-of-function mutants. These results indicate that there must be other proteins that function downstream of Robo to mediate repulsion. One would predict that simultaneously removing ena and the as yet unknown additional factors would reveal stronger disruptions of midline repulsion (Bashaw, 2000).
Thus, Ena is only part of what must be a more complex repulsive output from Robo. Ena helps strengthen the output (perhaps by locally putting the break on the actin-based motility machinery), but is only part of the output. In this light, it is interesting to note that Robo2 also binds Slit and mediates repulsion (albeit apparently more weakly than Robo), but Robo2 does not have the Ena binding site and does not bind Ena (J. Simpson, personal communication to Bashaw, 2000).
An important question for future studies concerns whether Ena is always docked on Robo, or alternatively, whether Slit binding to Robo leads to the recruitment of Ena to Robo's cytoplasmic domain. From what is known about other receptor systems, this second alternative seems more likely, but it remains an open question and needs to be directly tested (Bashaw, 2000).
Genetic analysis shows that Abl antagonizes Robo-mediated repulsion. The two most likely possibilities are that Abl functions to antagonize this pathway by phosphorylating Robo or by phosphorylating Ena. Three results argue in favor of a direct interaction with Robo. (1) Certain kinds of dose-dependent genetic interactions between abl and robo are observed that are not observed between abl and ena, suggesting that the Abl and Robo proteins might directly interact. (2) Biochemical experiments have shown that Abl can directly phosphorylate Robo's cytoplasmic domain at one or more tyrosine residues. (3) A Y-F mutation in a conserved tyrosine that can be phosphorylated by Abl in vitro generates a hyperactive Robo receptor. Taken together, these genetic and biochemical data suggest that it is the dephosphorylated form of Robo that is most active (Bashaw, 2000).
How might Abl normally regulate the output of Robo signaling? Abl-mediated phosphorylation might normally modulate the output of Robo signaling. Alternatively, this phosphorylation might participate more directly in the ligand-gated signal. It is interesting to speculate that it is the binding of Robo to its ligand Slit that triggers dephosphorylation, and that this in turn activates the repulsive response (Bashaw, 2000).
The CNS-specific receptor protein tyrosine phosphatases (RPTPs) RPTP10D and 69D are candidates to be additional factors that contribute to Robo repulsion. Simultaneous removal of these two RPTPs results in substantial ectopic midline crossing, and the double mutant shows dose-sensitive genetic interactions with slit. Whether these two phosphatases interact directly with Robo and whether their phosphatase activity is required for their observed roles in repulsion await future investigation (Bashaw, 2000 and references therein).
In the model presented above, it is attractive to speculate that these two RPTPs function in opposition to the Abl kinase activity by directly dephosphorylating Robo upon Robo's interaction with Slit. Interestingly, the other two Robo family members in Drosophila (Robo2 and Robo3; J. Simpson, personal communication to Bashaw, 2000) share the phosphorylation sites in Robo that are phosphorylated by Abl in vitro. In addition, genetic interactions are observed between the RPTPs and Robo2. Together these observations suggest that perhaps a common mechanism is employed to regulate the signaling output of the three Robo receptors. It will be of interest to determine the in vivo significance of the conserved tyrosine phosphorylation sites in the three Robo receptors. The future elucidation of the events set in motion by ligand binding will require the development of cell culture systems that will allow analysis of the phosphorylation state and cytoplasmic domain associations of the Robo receptors before and after Slit stimulation (Bashaw, 2000).
In addition to their function during Robo signaling shown here, it is clear that both Abl and Ena function in multiple guidance signaling pathways, and thus that they are not committed to repulsion downstream of Robo. In the nematode C. elegans, ena acts as a suppressor of the axon migration defects associated with ectopic expression of the UNC5 repulsive Netrin receptor. This raises the possibility that ena functions downstream of diverse repulsive guidance receptors. In Drosophila, during motor axon pathfinding, ena and abl play roles in ISNb choice point control. Overexpression of abl or loss of ena generates an ISNb 'bypass' phenotype, where the ISNb fails to defasciculate and branch off at the appropriate location to enter its muscle target region. This phenotype is also observed in mutations in Dlar, the gene encoding a receptor protein tyrosine phosphatase (RPTP). Mutations in all three of these genes (ena, abl, and Dlar) give rise to only partially penetrant ISNb guidance phenotypes and appear to modulate guidance decisions at this choice point. At the midline, mutations in the genes encoding the ligand (Slit) and a key receptor (Robo) have strong and highly penetrant midline guidance phenotypes. In contrast, mutations in the genes encoding Ena, Abl, and RPTP10D and RPTP69D on their own have weaker and less penetrant phenotypes. This is consistent with Abl and the RPTPs modulating Robo receptor output, and Ena mediating only part of Robo output (Bashaw, 2000 and references therein).
If this same logic is applied to the motor axon ISNb choice point, then it is likely that some of the key components are still missing. At present, the only gene with a nearly 100% penetrant bypass phenotype at this choice point is sidestep. Side is an Ig superfamily transmembrane protein that is expressed on muscle surfaces and appears to function as an attractive ligand for motor axons. The Side receptor is not known. Whether the key receptor is the Side receptor or not, it is likely that the major growth cone receptor for the ISNb choice point has not yet been identified (Bashaw, 2000 and references therein).
In this context, it is tempting to speculate, by analogy with the proposed model for Robo signaling, that at the ISNb motor axon choice point, DLAR and Abl play complementary roles in modulating the output activity of the hypothetical guidance receptor, while Ena functions as part of the receptor output. In this way, the two guidance decisions -- to cross or not to cross the midline, and to fasciculate or defasciculate from other motor axons -- use different signals on the outside of the growth cone, but similar signaling and regulatory mechanisms on the inside. It is suggested that once the signal crosses the membrane, in both cases the output is regulated in opposing directions by Abl vs. one or more RPTPs, and that the output is partially mediated by Ena. This model provides a unifying way of viewing signal transduction during these two different guidance decisions. It will be interesting in the future to see to what degree this model holds up in terms of both the role of phosphorylation in modulating receptor output, and the role of Ena in mediating part of repulsive signaling (Bashaw, 2000).
Disabled interaction with Drk and Sevenless
Drk, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the Sevenless receptor tyrosine kinase (Sev). One role of Drk is to provide a link between activated Sev and the Ras1 activator Sos. The ability of activated Ras1 to bypass the requirement for Sev function during R7 development has suggested that the primary function of Sev is to activate Ras. However, the model suggesting that the sole function of activated Sev is to bind Drk-Sos has been questioned by genetic studies that suggest the existence of multiple intracellular signaling pathways downstream of Sev. For example, although the association of Drk and Sos does not depend on the carboxy (C)-terminal SH3 domain of Drk, mutations that affect this domain partially compromise Sev signaling. Furthermore, a C-terminal SH3 domain-truncated Drk cannot rescue the lethality associated with homozygous drk mutations. These data suggest that Drk-binding proteins other than Sos may play important roles in signaling by Sev and other RTKs. Biochemical studies performed with mammalian systems have provided evidence that such Grb2-binding partners do exist. These include Cbl, a proto-oncogene product, and GAB1, a downstream component of the insulin and epidermal growth factor receptors. The possibility that Drk performs functions other than binding to Sos has been been investigated by identification of additional Drk-binding proteins. The phosphotyrosine-binding (PTB) domain-containing protein Disabled (Dab) binds to the Drk SH3 domains (Le, 1998).
To characterize the nature of the in vitro Dab-Drk interaction, it was necessary to determine which domains of Drk are required for binding to Dab. To answer this question, well-characterized mutations were used that had been shown to inactivate the function of either the SH2 or SH3 domain of Grb2. For example, changing the proline 49 residue to leucine (P49L) inactivates the N-terminal SH3 domain, while the arginine 86-to-lysine (R86K) mutation disrupts the SH2 domain and the glycine 203-to-arginine (G203R) mutation affects the C-terminal SH3 domain. The corresponding mutations were introduced, individually or in combination (P49L, R85K, G199R, P49L/G199R), into the [32P]GTK-DRK fusion protein and the ability of the mutant proteins to interact with the lambda gt11-encoded beta-galactosidase-DAB fusion protein was tested. Mutation of the SH2 domain does not affect binding, indicating that the in vitro Dab-Drk interaction does not require a functional Drk SH2 domain. However, the Dab-Drk interaction is dependent on the function of the SH3 domains because simultaneous mutations of both SH3 domains abolish binding. Moreover, while Dab binds to both SH3 domains, it appears to interact more strongly with the C-terminal domain. In addition, in vitro interaction between Drk and Dab requires the presence of the proline-rich region of Dav and suggests that the SH3 domains of Drk bind directly to sequences within the Dab proline-rich core (Le, 1998).
Dab is expressed in the ommatidial clusters, and loss of Dab function disrupts ommatidial development. Intense anti-Dab staining is observed both in the morphogenetic furrow and in developing ommatidial clusters posterior to the furrow. An apical-to-basal cross section revealed that Dab is localized to a small region just below the apical surface of the retinal epithelium. To determine which cells express Dab, the discs were costained with an antibody to ELAV, a neuronal marker present in the nuclei of developing and mature photoreceptors. The results from these experiments showed that Dab accumulates at the apical membrane of the developing photoreceptor cells. However, it was not possible to assign Dab expression to particular photoreceptors due to the apical constriction of these cells. The subcellular localization of Dab is similar to that of Drk, consistent with its role as a Drk-binding partner (Le, 1998).
Numerous abnormalities are observed in dab homozygous mutant clones. The most common defects are the absence of the R7 cell and the lack of one or more outer photoreceptors (R1 to R6) in mosaic ommatidia. In addition, large dab mutant clones show extensive ommatidial disorganization. including regions in which no photoreceptors are present. This phenotype is observed with three different alleles of dab and resembles those observed in clones of cells homozygous for weak alleles of either Sos or Ras1. These results indicate that Dab has an important function during photoreceptor and ommatidial development. Reduction of Dab function attenuates signaling by a constitutively activated Sev. Biochemical analysis suggests that Dab binds Sev directly via its PTB domain, becomes tyrosine phosphorylated upon Sev activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that Dab is a novel component of the Sev signaling pathway (Le, 1998).
Disabled has been implicated in other RTK signaling pathways. A murine DAB-related protein, mDAB1, has been identified as a tyrosine-phosphorylated protein that binds to the non-receptor protein tyrosine kinase Src. Recently, several reports have shown that mice lacking mDAB1 function have neuronal defects similar to those seen in reeler mice, including abnormal cortical lamination resulting from disruptions of neuronal migration processes. These results suggest that mDAB1 might participate in a signaling pathway triggered by REELIN, a secreted protein released near the targets of migrating neurons. The neuronal defects associated with Drosophila and mouse dab mutations and the identification of DAB as a putative adaptor protein acting downstream of the receptor tyrosine kinase Sev suggest that Dab may function downstream of many RTKs, including ones required for proper development of the Drosophila central nervous system (Le, 1998).
Semaphorins have been intensively studied for their role in dendritic and axonal pathfinding, but less is known about their potential role in synapse formation. In the adult giant fiber (GF) system of fruit flies, it has been shown that transmembrane Semaphorin 1a is involved in synapse formation in addition to its role in guidance during pathfinding. Cell-autonomous rescue experiments show that Sema-1a is involved in assembly of a central synapse and that it is required both pre- and post-synaptically. Pre- but not post-synaptic gain-of-function Sema-1a is able to disrupt the GF-motor neuron synapse and the phenotype depends on a proline-rich intracellular domain that contains a putative Enabled binding site. Thus Sema-1a may signal via Enabled. It is suggested that transmembrane Sema-1a is part of a bi-directional signaling system that leads to the formation of the GF synapse and possibly acts as both a ligand and a receptor (Godenschwege, 2002).
Thus, in addition to its better-known role in pathfinding, Sema-1a also functions during synapse formation. There were two main phenotypes in the sema-1a loss-of-function mutants. In half of the semaP1 specimens, the GF axons had pathfinding defects and did not exit the brain. Ectopic expression of three sema-1a constructs in the brain could rescue these pathfinding defects with similar probability, suggesting that Sema-1a acts as a ligand (its well-known role) during giant fiber pathfinding (Godenschwege, 2002).
The idea that Sema-1a may also function as a receptor is supported by findings in both the rescue and the gain-of-function experiments. (1) SemaDeltacyt (with a deleted cytoplasmic domain) is less potent than SemaWT in rescuing the synaptic defects in the target area. This difference may be attributed to the functional requirement of a cytoplasmic domain, suggesting that Sem-1a may work as a receptor during GF-TTMn synapse assembly ([Note: TTM (tergotrochanteral motor neuron is the jump motorneuron)}. (2) The overexpression data in a wild-type background further supports a role for the Sema-1a cytoplasmic domain. The disruptive, bendless-like, gain-of-function phenotype is cell-autonomous and is found only with full-length Sema-1a. (3) The cytoplasmic domain of Sema-1a contains a putative Enabled binding site (LPQP). Enabled family proteins influence growth cone behavior by regulating actin dynamics. A small deletion removing the putative Enabled binding site (LPQP) abolishes Sema-1a's ability to induce the gain-of-function phenotype, suggesting that the presynaptic repulsive effect of Sema-1a may be mediated by signaling via Enabled. A role for Enabled is further supported by the finding that the reduction of ena gene dosage suppresses the Sema-1a gain-of-function phenotype, suggesting that Sema-1a may function as a receptor and signal via Enabled in the giant fiber (Godenschwege, 2002).
Whereas the rescue experiments suggested a requirement of Sema-1a function pre- and postsynaptically, the gain-of-function experiments showed a repulsive effect of Sema-1a presynaptically but not postsynaptically. This shows an asymmetry in Sema-1a function on the two sides of the synapse, which could be due to the fact that Enabled signaling has different effects in the GF and the TTMn. An alternative possibility is that Sema-1a involves different signaling pathways pre- and postsynaptically. In vertebrates, Sema-4c and Sema-4f bind to PSD-95, and Sema-4c interacts with the neurite outgrowth-related protein Norbin as well. Both proteins are involved in the assembly of the post-synapse and synaptic plasticity. Notably, the C-terminal of Sema-1a contains a motif (VYL) that can be recognized by class II PDZ domains and would allow Sema-1a to bind to a variety of cytoplasmic molecules. Therefore, Sema-1a's postsynaptic function may be mediated through a PDZ binding motif, which would be distinct from signaling presynaptically via Enabled (Godenschwege, 2002).
In summary, it is proposed that during target recognition, Sema-1a is present presynaptically as well as postsynaptically. The bi-directional signaling of Sema-1a and unknown receptors on both sides of the synapse may trigger the switch from pathfinding to synaptogenesis. It is speculated that Sema-1a functions presynaptically as a repulsive receptor when it reaches its target, thereby stopping or slowing the GF growth cone. Subsequently, for synaptogenesis to proceed, Sema-1a on the GF needs to be removed or down-regulated from the surface, so as not to disrupt synapse formation with its repulsive effects. In contrast, there is no requirement for removal of Sema-1a from the postsynaptic side, and Sema-1a has a function in the GF-TTMn synapse assembly. Bi-directional signaling of Sema-1a may cause the GF to grow along the TTMn dendrite and thereby promote synapse formation. Thus it ia thought that Sema-1a has a bi-directional and bi-functional role in the assembly of the giant synapse (Godenschwege, 2002).
The proto-oncogenic kinase Abelson (Abl) regulates actin in response to cell signaling. Drosophila Abl is required in the nervous system, and also in epithelial cells, where it regulates adherens junction stability and actin organization. Abl acts at least in part via the actin regulator Enabled (Ena), but the mechanism by which Abl regulates Ena is unknown. A novel role is described for Abl in early Drosophila development, where it regulates the site and type of actin structures produced. In Abl's absence, excess actin is polymerized in apical microvilli, whereas too little actin is assembled into pseudocleavage and cellularization furrows. These effects involve Ena misregulation. In abl mutants, Ena accumulates ectopically at the apical cortex where excess actin is observed, suggesting that Abl regulates Ena's subcellular localization. Other actin regulators were also examined. Loss of Abl leads to changes in the localization of the Arp2/3 complex and the formin Diaphanous, and mutations in diaphanous or capping protein beta enhance abl phenotypes (Grevengoed, 2003).
Genetic analysis suggests that in the early Drosophila embryo, the primary means by which Abl influences the cytoskeleton is through Ena. Reducing the Ena dose by half profoundly suppresses ablM; it is as effective as adding back a wild-type abl transgene. Ena is also a critical target of Abl during embryonic morphogenesis. Although the data suggest that the primary effect of loss of Abl is Ena deregulation, they do not rule out Abl acting on the cytoskeleton by other mechanisms (Grevengoed, 2003).
The mechanism by which Abl regulates Ena has remained mysterious. This study demonstrates that Abl regulates Ena by regulating its intracellular localization. In the absence of Abl, Ena localizes to ectopic sites. Alterations in Ena and actin localization have been observed at the leading edge of migrating epidermal cells in abl mutants during dorsal closure. This suggests that regulation of Ena localization by Abl may be a more general mechanism. It is hypothesized that Abl targets Ena to places where it is needed to modulate actin dynamics, perhaps by excluding it from other sites where Ena activity would be detrimental (Grevengoed, 2003).
There are many ways in which Abl could restrict Ena localization. Abl's kinase activity is essential, and thus Abl phosphorylation of Ena may restrict its localization by preventing Ena binding to partners that localize to particular cortical sites, or by promoting Ena binding to partners that sequester it in the cytoplasm. Phosphorylation of Ena by Abl in vitro inhibits binding of Ena to SH3 domains, whereas Mena/VASP phosphorylation by PKA alters binding to SH3 domains and actin. However, if direct phosphorylation were the only mechanism by which Abl regulated Ena, mutating Ena's phosphorylation sites should create a protein that can no longer be regulated and thus would localize to ectopic sites. Instead, mutation of all of the Abl phosphorylation sites in Ena modestly reduced Ena function, rather than making it ectopically active as is seen in abl mutants (Grevengoed, 2003).
Thus, Abl may regulate Ena by additional mechanisms. Abl may modulate Ena localization and restrict Ena activity by direct binding (this could still require Abl kinase activity, since auto-phosphorylation or phosphorylation of other partners may regulate protein-protein interactions). Abl might sequester Ena in the cytoplasm in an inactive state, or it might recruit Ena to appropriate sites. Alternately, binding of Abl's SH3 domain to the Ena proline-rich region might prevent Ena from binding to other partners, such as profilin, which might in turn modulate both Ena localization and activity. In thinking about these different possible mechanisms, it is interesting to note that Abl localizes to the actin caps and apical pseudocleavage furrows during syncytial stages and the apical portion of the cellularization furrow, the precise places where ectopic actin accumulation occurs in its absence. Thus, it is poised to act at this location. Working out the details of the mechanism by which Abl regulates Ena localization will be one of the next challenges (Grevengoed, 2003).
This work provides an in vivo test of the current model for Ena function, and allows extension of this model. The excess growth of microvilli seen when Ena is ectopically localized in early embryos fits well with work on Ena/VASP function in mammalian fibroblasts, where forced localization of Ena/VASP proteins to the leading edge promotes the formation of long, unbranched filaments. Ena also localizes to the ends of filopodia and microspikes, suggesting that Ena's role in promoting long unbranched actin structures is broadly conserved. Earlier experiments in fibroblasts artificially altered Ena localization. This study demonstrates that Ena localization is a normal regulatory point in vivo, and that Abl is a critical player in this process. Finally, in vitro experiments have suggested that Ena promotes filament elongation by antagonizing capping protein. Mutations in cpb enhance the effects of mutations in abl in the CNS and probably during oogenesis. These data are consistent with Ena and capping protein playing antagonistic roles in vivo, with Abl potentially influencing the outcome of this antagonism. However, Abl and capping protein may also work together independently of Ena in the regulation of actin dynamics (Grevengoed, 2003).
Different actin regulators play fundamentally different biochemical roles. Models often picture all of these regulators modulating actin assembly and disassembly at a single site, but of course individual cells target different actin regulators to distinct sites, creating actin structures with diverse functions. Syncytial embryos provide an excellent example. During interphase, they assemble actin-based microvillar caps above each nucleus. As they enter prophase, caps are disassembled and actin polymerization is redirected to the pseudocleavage furrows. This is likely to require new machinery: both Arp2/3 and the formin Dia are required for pseudocleavage furrow formation, but not for actin caps. Cellularization also requires distinct machinery to polymerize/disassemble apical microvilli and to recruit and modulate actin at the cellularization front. For transitions to occur smoothly, two fundamental changes have to occur: the location at which actin polymerization occurs must change, and a different constellation of actin regulators must be deployed to produce the distinct actin structures observed (Grevengoed, 2003).
The data support a hypothesis in which the balance of activity of different actin regulators at distinct sites is tightly regulated, influencing the nature of the actin structures produced. One regulator is Abl. In its absence, Ena localizes ectopically to the cortical region, upsetting the temporal and spatial balance of actin regulators. This leads to a change in both the location and nature of actin polymerization during mitosis. Excess actin is polymerized into microvillar projections that extend from the apical region of the furrows, whereas insufficient actin is directed to the pseudocleavage furrows. Similarly, during cellularization in ablM mutants, actin polymerization continues to be directed to apical microvilli, whereas in a wild-type embryo this ceases early in cellularization (Grevengoed, 2003).
The data also suggest that there is cross-talk between different modulators of actin polymerization, and that the balance of their activities determines the outcome. Although many actin modulators are unaffected in ablM mutants, both the Arp2/3 complex and Dia are recruited to sites of ectopic actin polymerization. However, genetic analysis suggests that although Ena mislocalization plays a critical role in the actin alterations seen in ablM mutants, Dia and Arp2/3 mislocalization may not. In fact, reduction of the dose of Dia enhanced the ablM phenotype. Dia normally promotes actin polymerization lining the furrows. In ablM mutants, the balance of actin polymerization is already shifted to the apical microvilli because of ectopic Ena localization. Reduction in the dose of Dia might further reduce actin polymerization in pseudocleavage furrows, resulting in the observed enhancement of the ablM phenotype. The abnormal recruitment of Dia to the apical regions in ablM mutants may also reduce pseudocleavage furrow formation (Grevengoed, 2003).
It will now be important to investigate how the cell regulates the distinct types of actin polymerization required for distinct cellular and developmental processes. One mechanism of cross-talk may involve direct or indirect recruitment of one type of actin modulator by another. Abl's ability to interact with both Ena and the Arp2/3 regulator WAVE1 is interesting in this regard. However, the recruitment of Arp3 and Dia to ectopic actin structures observed in ablM mutants may have a more simple explanation. Both are thought to have a higher affinity for newly polymerized, ATP-bound actin, which is likely to be increased where ectopic actin polymerization appears to occur (Grevengoed, 2003).
Drosophila Abl also functions in other contexts. It has a role in embryonic morphogenesis, where it also acts, at least in part, via Ena. However, in this context Abl also affects AJ stability. Since Ena is normally highly enriched in AJs, it is hypothesized that Abl helps restrict Ena localization to AJs, and thus helps initiate the proper organization of actin underlying AJs. In Abl's absence, Ena may localize to sites other than AJs, leading to ectopic actin polymerization at those sites and reduction in actin polymerization at AJs (analogous to the divergent effects on apical actin and pseudocleavage/cellularization furrows). Since the cortical actin belt underlying the AJ plays an important role in its stability, this could explain the phenotype of abl mutants. A similar model may help explain the roles of Abl and Ena in axon outgrowth. The network of actin filaments in the growth cone is complex, with different types of actin in filopodia and in the body of the growth cone. By regulating Ena localization, Abl may influence the balance of the different types of actin, thus influencing growth cone motility. Likewise, in fibroblasts, where Ena/VASP proteins regulate motility, the Arp2/3 regulators N-WASP and WAVE localize to sites at the leading edge distinct from those where Mena is found. Whether Abl or Arg regulate the localization of Ena/VASP family proteins in mammals remains to be determined. Likewise, it is possible that deregulation of Ena/VASP proteins underlie some of the alterations in cell behavior in Bcr-Abl–transformed lymphocytes. Experiments to test whether Ena/VASP activity is important for either mammalian Abl's normal function or for the pathogenic effects of Bcr-Abl will help answer these questions (Grevengoed, 2003).
The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. Genetic and/or physical interactions between Fra and these effector molecules suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline (Forsthoefel, 2005).
The interactions of Abl with Fra are intriguing, since they suggest that in Drosophila, as in other organisms, this evolutionarily conserved guidance receptor is regulated by tyrosine phosphorylation, and also that Fra may regulate Abl substrates. Other studies have demonstrated Netrin-dependent tyrosine phosphorylation of DCC, Netrin/DCC-dependent activation of the tyrosine kinases FAK, Src and Fyn, and the requirement of DCC tyrosine phosphorylation for Netrin-dependent Rac1 activation and growth cone turning. Interestingly, the tyrosine residue in DCC identified as the principal target of Fyn/Src kinases is not conserved in Drosophila Fra or C. elegans UNC-40, suggesting that the precise mechanisms by which Fra/DCC/UNC-40 signaling is regulated by tyrosine kinases may differ between organisms. Tyrosine phosphorylation of UNC-40 has also been observed, and although the kinase(s) responsible has not been identified, genetic interactions suggest that UNC-40 signaling is regulated by the RPTP CLR-1, supporting the idea that regulation of tyrosine phosphorylation is a consequence of UNC-6/Netrin signaling in C. elegans as well. In this study, more robust tyrosine phosphorylation of Fra was observed in cells with pervanadate stimulation than with Abl overexpression alone, raising the possibility that additional kinase(s) may function during Fra signaling. Further investigation will be needed to address this issue and to determine how Abl-mediated phosphorylation of Fra modulates commissural growth cone guidance (Forsthoefel, 2005).
Abl is thought to control actin dynamics in part through its ability to regulate other proteins through tyrosine phosphorylation. Thus, in addition to potential regulation of Fra, Fra may recruit Abl to regulate other Abl substrates. Abl interacts genetically with trio, and in this study, Trio was found to physically interact with Abl in vitro, and Trio tyrosine phosphorylation increases dramatically with co-expression of Abl. Phosphorylation of Trio may affect its activity, as observed for other GEFs. For example, Abl regulates phosphorylation and Rac-GEF activity of Sos1, and Lck, Fyn, Hck and Syk kinases tyrosine phosphorylate Vav GEF and stimulate its activity (Forsthoefel, 2005).
Trio physically interacts with Fra in vitro and in S2 cells, suggesting that Fra can recruit Trio directly. In addition, heterozygosity for trio dominantly modifies the Robo-Fra chimeric receptor phenotype, consistent with a positive role for Trio as a downstream effector of Fra signaling in vivo. As a Rac/Rho GEF, Trio may link Netrin-Fra signaling to the regulation of Rho-family GTPases in commissural axons. Rho-family GTPases have been rigorously studied with regard to their role in the regulation of cytoskeletal dynamics and axon guidance, outgrowth and branching. Although positive roles for GTPases in commissure formation in the Drosophila embryo have not been directly demonstrated, trio and GEF64C, a Rho GEF, interact genetically with fra leading to the dramatic disruption of commissures. Additionally, expression of constitutively active or dominantly negative isoforms of both Rac and Rho, as well as constitutively active Cdc42, causes axons to cross the CNS midline inappropriately. Recent studies have implicated Cdc42 and Rac1/CED-10 as effectors of DCC and UNC-40 signaling, but reaching an understanding of the biochemical mechanisms by which GTPases are regulated has been elusive. Future experiments must determine whether Netrin-Fra signaling modulates the GEF activity of Trio, and how this occurs (Forsthoefel, 2005).
Reducing the genetic dose of ena causes either more or fewer axons to cross the CNS midline, depending on the genetic background, suggesting that the role of Ena in the growth cone is complex. Heterozygosity for ena in embryos expressing the Robo-Fra chimeric receptor reduces the number of axon bundles that inappropriately cross the CNS midline, consistent with a role for Ena as a positive effector of Fra signaling. Ena/UNC-34 has been identified genetically as an effector of DCC/UNC-40 in C. elegans. In cultured mouse neurons, Ena/VASP proteins are required for Netrin-DCC-dependent filopodia formation, and Mena is phosphorylated at a PKA regulatory site in response to Netrin stimulation. In migrating fibroblasts, increasing Ena/VASP proteins at the leading edge leads to unstable lamellae and decreased motility; by contrast, increasing Ena/VASP levels at the leading edge in growth cones causes filopodia formation, possibly due to differences in the distribution of actin bundling or branching proteins. Although the role of Ena in actin reorganization in Drosophila has not been rigorously studied, Ena localizes to filopodia tips in cultured Drosophila cells, suggesting that the role of Ena in filopodia formation may be conserved (Forsthoefel, 2005).
No direct biochemical interaction was observed between Fra and Ena. However, Abl binds and phosphorylates Ena, and heterozygosity for both Abl and ena further suppresses the Robo-Fra phenotype, suggesting that Fra may recruit Abl to regulate filopodial extension through Ena. Alternatively, Fra may regulate Ena through other molecule(s), and the synergistic suppression of the Robo-Fra phenotype by Abl and ena is a result of the compromise of parallel pathway(s) regulated by Fra. It is important to note that the functional consequences of biochemical interactions between Abl and Ena are not yet understood. Therefore it will be of particular interest to determine whether Ena is tyrosine phosphorylated in response to Netrin-Fra signaling, and if Ena phosphorylation regulates its activity during filopodial extension (Forsthoefel, 2005).
In addition to suppressing the Robo-Fra chimeric receptor phenotype, mutations in ena also suppress the loss-of-commissure phenotype in fra, Netrin, trio and Abl mutant combinations. In Drosophila (as well as in C. elegans), Ena interacts genetically and biochemically with the repulsive receptor Robo, indicating that Ena may restrict axon crossing at the midline. Thus, the fact that mutations in ena dominantly suppress fra, Netrin, trio and Abl CNS phenotypes could simply reflect the compromise of a parallel, opposing signaling pathway. Consistent with this idea, some axons that cross the midline in ena heterozygous, trio,Abl homozygous embryos are Fas2 positive, indicating a partial reduction in repulsive signaling. However, ena also dominantly suppresses fra and Netrin commissural pathfinding defects, without causing longitudinal Fas2-positive axons to cross the midline. Reductions in Robo signaling therefore may not fully explain the ability of ena to suppress defects in fra, Netrin, Abl and trio mutants (Forsthoefel, 2005).
Based on the fact that mutations in ena suppress a number of Abl mutant phenotypes, it has been proposed that Abl antagonizes Ena function. In Abl mutant embryos, Ena and actin mislocalize during dorsal closure and cellularization, and apical microvilli are abnormally elongated, indicating that Abl regulates the localization of Ena. In migrating fibroblasts, increasing Ena/VASP levels at the leading edge results in long, unbranched actin filaments, unstable lamellae, and decreased motility due to increased antagonism of capping protein. Interestingly, mutations in the gene encoding Drosophila capping protein ß enhance CNS axon pathfinding defects in Abl mutants, including commissure formation. Therefore, if Fra and/or Abl regulate Ena localization in commissural axons, then in fra, Netrin or Abl mutants, Ena may be mislocalized in the growth cone, leading to inappropriate inhibition of capping protein and excessive F-actin filament elongation. Additionally, reducing regulation of Ena by Fra or Abl may also allow greater Ena regulation by Slit-Robo signaling. In either case, reducing the gene dose of ena in fra, Netrin and trio,Abl mutant embryos would partially relieve these effects, allowing axons to respond more efficiently to other cues and cross the midline, as was observed. Consistent with this idea, it was found that either increasing or decreasing Ena/VASP proteins at the leading edge impairs the elaboration of growth cone filopodia in response to Netrin-DCC signaling, suggesting that Ena/VASP levels must be tightly regulated in order for the growth cone to respond optimally to extracellular signals (Forsthoefel, 2005).
The role of Abl in the growth cone is also likely to be complex. The observations implicate Abl as an effector of attractive Fra signaling. In addition, tyrosine phosphorylation of Robo by Abl is thought to negatively regulate repulsive signaling by Robo. Paradoxically though, loss-of-function mutations in Abl, robo and slit interact genetically, resulting in inappropriate axon crossing at the midline, and indicating that Abl may also promote repulsion in longitudinally migrating growth cones. Obviously, much remains to be understood about the molecular basis for genetic interactions of Abl, particularly how Abl and its various substrates cooperate with different growth cone receptors to yield specific cytoskeletal outputs (Forsthoefel, 2005).
In summary, genetic and biochemical interactions indicate that Abl, Trio and Ena are integrated into a complex signaling network with Fra and the Netrins during commissure formation. These observations identify another receptor that acts through these effectors, and provide a framework for further investigation of signaling by this key, evolutionarily conserved guidance receptor (Forsthoefel, 2005).
Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. A zyx mutant was generated in Drosophila using TALEN endonucleases, and this was used to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, this study shows that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). These results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity (Gaspar, 2015).
The control of tissue size represents a major unsolved question in developmental biology. The conserved Hippo (Hpo) signaling pathway is thought to sense mechanical and nutritional cues to restrict tissue growth. Activation of the Ste20-like kinase Hpo (MST1/2 in mammals) and subsequent phosphorylation of the downstream Ndr-like kinase Warts (Wts-LATS1/2 in mammals) inhibits the transcriptional co-activator Yorkie (Yki-YAP/TAZ in mammals), via phosphorylation at S168. This prevents the interaction of Yki with transcription factor partners, such as Scalloped (Sd-TEAD1-4 in mammals), thereby inhibiting expression of pro-growth and survival genes (Gaspar, 2015).
The known upstream stimuli for Hpo signaling involve a number of regulatory proteins, many of which are associated with the actin cytoskeleton. In particular, the Drosophila proteins Expanded (Ex) and Merlin (Mer), which belong to the FERM (Four point one, Ezrin, Radixin, Moesin) domain family, and the protocadherins Fat (Ft) and Dachsous (Ds), were identified as tumor suppressors that prevent expression of Yki target genes. Whether Ex/Mer and Ft/Ds signaling represent entirely distinct branches of Hpo signaling remains unclear. For instance, Ft depletion leads to a reduction in apical Ex localization. However, Ft and Ex have been implicated in distinct functions: Ft/Ds are involved in the control of planar cell polarity (PCP), while Ex has strong effects on eye differentiation. The proposed mechanisms of Ft and Ex function are also distinct. In particular, Ex promotes cytoplasmic sequestration of Yki through direct binding and by promoting Hpo-Wts kinase activity, while Ft antagonizes the growth-promoting function of the atypical myosin Dachs (D), which, in turn, destabilizes Wts (Gaspar, 2015).
Several reports have highlighted the contribution of the actin cytoskeleton to Hpo signaling. The actin Capping Protein αβ heterodimer (CP), which prevents addition of actin monomers to F-actin barbed ends, antagonizes Yki activity, and thereby restricts tissue growth. Accordingly, in mammals, CapZ and other factors that restrict F-actin levels, have growth-restrictive effects via the control of YAP/TAZ subcellular localization, particularly in response to mechanical cues. Interestingly, YAP and TAZ respond to mechanical cues dependent on actomyosin networks and formin-dependent actin polymerization. Recently, the actin-associated LIM (Lin11, Isl-1, and Mec-3) domain protein Zyxin (Zyx) has been shown to mediate the effects of Ft-Ds signaling on Yki target genes, by promoting Wts destabilization via its interaction with D (Rauskolb, 2011). Importantly, Zyx provides a link to the actin polymerization machinery, since it directly interacts with the actin-binding proteins Enabled (Ena)/VASP via conserved F/LPPPP motifs, and promotes Ena function in barbed-end F-actin polymerization (Gaspar, 2015 and references therein).
The analysis of Drosophila zyx has been limited by the absence of a mutant. This study generated a zyx mutation and describe its effects on growth and Hpo signaling. Zyx is shown to strongly antagonize Ex function in growth control, eye differentiation and F-actin accumulation, while being largely dispensable for Ft-mediated tissue growth. Finally, this work suggests that Zyx's growth-promoting function requires its ability to bind the actin polymerization factor Ena (Gaspar, 2015).
Zyx was previously shown to promote Wts degradation in a mechanism based on a Zyx/Dachs interaction (Rauskolb, 2011). However, this study reports that zyx and dachs (d) have additive effects on tissue growth. In addition, zyx loss has a modest effect on ft growth phenotypes, which, in contrast, are highly sensitive to d mutations, highlighting the possibility of additional functions for Zyx in tissue growth (Gaspar, 2015).
Characterization of the zyx mutant shows that Zyx acts in the Ex branch of the Hpo pathway to control tissue growth. This is in contrast to a previous study using RNAi knockdown of zyx and ex, which concluded that zyx expression had only minor effects on the Ex branch (Rauskolb, 2011). The current results indicate that zyx loss can significantly reverse the lethality and growth defects of ex mutant animals. This antagonistic function of Ex and Zyx is not confined to growth regulation but extends to tissue differentiation. This study shows that Zyx restricts eye differentiation antagonistically to Ex and in parallel to Dachs but independently of Ft. Consistent with these observations, simultaneous loss of ex and ft leads to additive, and therefore apparently independent effects on eye differentiation. Therefore, it is proposed that Zyx is a key modulator of Ex function (Gaspar, 2015).
In growth control, Zyx function may be partially independent of Hpo-Wts signaling, as zyx is partially required for the overgrowth of hpo and wts mutant eye and wing but has no major effect on wts overexpression in the wing or Yki phosphorylation by Wts. Ex has been reported to sequester Yki in the cytoplasm through a direct interaction. However, since ex mutant overgrowth is suppressed by zyx loss, it is unlikely that Zyx directly antagonizes Ex protein. Instead, it is suggested that the interplay between Zyx and Ex in growth control is mediated through their antagonistic effects on F-actin (Gaspar, 2015).
This work links F-actin barbed-end polymerization with Zyx/Ex in the control of Yki activity and tissue growth. The Zyx domain encompassing the conserved LPPPP motif, which binds Ena, is required for Zyx to promote growth and to antagonize Ex function. Moreover, Zyx and Ena synergize to promote tissue growth. This supports the idea that Zyx promotes tissue growth via its interaction with Ena. Conversely, CP antagonizes Zyx-induced tissue growth and functions together with Ex in preventing F-actin polymerization. Therefore, an attractive possibility is that antagonistic effects on Yki activity between the activators Zyx/Ena on one hand and the inhibitors Ex and CP on the other hand is played out indirectly through their effects on F-actin polymerization. Consistent with this hypothesis, Zyx antagonizes the effect of Ex on apical F-actin accumulation (Gaspar, 2015).
Recent data suggest that the actin cytoskeleton acts in parallel to the core kinase cascade to control YAP/TAZ activity, with CapZ being proposed as one of the 'gatekeepers' restricting its nuclear translocation. Yki/YAP/TAZ may respond to the relative activities of Ena and CP, either by being sensitive to the presence of polymerizing actin barbed ends, or because Ena produces a specialized set of cortical actin filaments necessary for Yki/YAP/TAZ activation. The study of the mechanism(s) coupling F-actin and Yki/YAP/TAZ should resolve these issues. This study has shown that Zyx cortical localization is relevant for its function in promoting tissue growth. Since Zyx has been shown to rapidly relocalize to strained or severed actin filaments in cultured mammalian cells and Drosophila follicular epithelial cells, it is possible that Zyx may also link mechanical forces to growth control (Gaspar, 2015).
Finally, it is also interesting to note the possible redundancy in growth control between Zyx and other Ena-interacting proteins. Like Zyx, Pico/Lamellipodin contains an EVH1-interacting L/FPPPP motif, and its interaction with Ena promotes tissue growth in Drosophila. Since Ena localization is not strictly dependent on Zyx, it is tempting to speculate that Ena recruitment by multiple membrane-associated proteins, such as Zyx and Pico, is a common denominator in the regulation of growth by the actin cytoskeleton (Gaspar, 2015).
The oncoprotein BCR-ABL1 triggers Chronic Myeloid Leukemia. It is clear that the disease relies on the constitutive BCR-ABL1 kinase activity, but not all the interactors and regulators of the oncoprotein are known. This study describes and validates a Drosophila leukemia model based on inducible human BCR-ABL1 expression controlled by tissue specific promoters and thought as a versatile tool to perform genetic screens. BCR-ABL1 expression in the developing eye interferes with ommatidia differentiation and expression in the hematopoietic precursors increases the number of circulating blood cells. BCR-ABL1 interferes with the pathway of endogenous dAbl with which it shares the target protein Ena. Loss of function of ena or Dab, an upstream regulator of dAbl, respectively suppresses or enhances both the BCR-ABL1 dependent phenotypes. Importantly, in patients with leukemia decrease of human Dab1 and Dab2 expression correlates with a more severe disease and Dab1 expression reduces the proliferation of leukemia cells. Globally, these observations validate the Drosophila model that promises to be an excellent system to perform unbiased genetic screens aimed at identifying new BCR-ABL1 interactors and regulators to better elucidate the mechanism of leukemia onset and progression (Bernardoni, 2018).
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