Abl tyrosine kinase



The expression of the Abl protein has been examined throughout embryonic and pupal development; mutant phenotypes in some of the tissues expressing abl have been analyzed. In contrast to the ABL mRNA, the Abl protein is not maternally supplied to the oocyte. As cellularization initiates, Abl protein, present in all cells of the early embryo as the product of maternally contributed mRNA, transiently localizes to the region below the plasma membrane cleavage furrows and apical cell junctions. The function of this expression is not yet known. Abl is localized in cytoplasmic islands that form around the nuclei in the anterior end of the embryo. Zygotic expression of abl is first detected in the post-mitotic cells of the developing muscles and central nervous system midway through embryogenesis. Zygotic Abl in the CNS is limited to neurons; immunostaining is not detected in neuroblasts or ganglion mother cells. The Abl protein is concentrated in axons as they extend from neurons. The highest level of Abl throughout embryogenesis is observed in the axon scaffold of the CNS. At stage 14, Abl protein is detected in the visceral mesoderm and at a lower level in the somatic mesoderm. In the somatic muscle, immunostaining is concentrated at the muscle attachment sites (Bennett, 1992).

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Parallel genetic and proteomic screens identify Msps as a CLASP-Abl pathway interactor in Drosophila

Regulation of cytoskeletal structure and dynamics is essential for multiple aspects of cellular behavior, yet there is much to learn about the molecular machinery underlying the coordination between the cytoskeleton and its effector systems. One group of proteins that regulate microtubule behavior and its interaction with other cellular components, such as actin-regulatory proteins and transport machinery, is the plus-end tracking proteins (MT+TIPs). In particular, evidence suggests that the MT+TIP, CLASP (Chromosome bows), may play a pivotal role in the coordination of microtubules with other cellular structures in multiple contexts, although the molecular mechanism by which it functions is still largely unknown. To gain deeper insight into the functional partners of CLASP, parallel genetic and proteome-wide screens for CLASP interactors were performed in Drosophila. 36 genetic modifiers and 179 candidate physical interactors were identified, including 13 that were identified in both data sets. Grouping interactors according to functional classifications revealed several categories, including cytoskeletal components, signaling proteins, and translation/RNA regulators. The initial investigation focused on the MT+TIP Minispindles (Msps), identified among the cytoskeletal effectors in both genetic and proteomic screens. This study reports that Msps is a strong modifier of CLASP and Abl in the retina. Moreover, Msps functions during axon guidance and antagonizes both CLASP and Abl activity. The data suggest a model in which CLASP and Msps converge in an antagonistic balance in the Abl signaling pathway (Lowery, 2010).

The in vivo functions of cytoskeletal effector and regulatory proteins have been studied very effectively in Drosophila, with particular success at the earliest stages of embryonic development prior to zygotic gene expression, when depletion of maternal stores of such proteins often results in disruption of mitosis, cellularization, or other aspects of cell biology. However, the functions of cytoskeletal effectors at late stages of development are often obscured by early embryonic functions. In this regard, the existence of maternal stores of some key effectors has been helpful for analysis of late events in nervous system development, such as axonal and dendritic patterning, because such maternal supplies of protein are sometimes exhausted only at late stages when axons and dendrites emerge. However, zygotic mutations in many key cytoskeletal components disrupt early stages, making screens based on neuroanatomical phenotypes problematic. For this reason, genetic interaction screens were used to explore the network of cytoskeletal regulators linked to key guidance signaling molecules as a means of identifying candidates for deeper analysis during axonal development. These screens for modifiers of Abl kinase phenotypes led to the identification of CLASP as an effector essential for accurate growth cone navigation. By using CLASP as a starting point for a new generation of screens, new functional categories and individual players of the CLASP interactome were identified, including cytoskeletal components, signaling proteins, and translation/RNA regulators. In addition, a microtubule regulatory protein (the MT+TIP Msps) not previously associated with axonal pathfinding decisions was identified (Lowery, 2010).

To build functional neural networks, axonal growth cones must accurately interpret and translate multiple guidance cues into directional movement by coordinating both microtubule and F-actin networks. There appears to be significant interplay between the two cytoskeletal components, but a sophisticated understanding of the signaling and effector mechanisms by which both systems are coordinated in response to guidance cues has not yet been obtained. The Abl tyrosine kinase is one of the few known signaling molecules shown to transduce guidance cue signals to both actin and MT networks, although far less is known regarding how it regulates MTs. This work, and that of others, suggests that CLASP may be an important player in the MT-actin crosstalk machinery (Lowery, 2010).

The largest thematic group of CLASP-interacting genes identified is the actin-binding proteins, including Shot, Zip, Capu, Pnut, Jar, Bif, Kst, and the uncharacterized CG13366 (ortholog of calponin-homology domain containing CYTSA/B). These are all known actin-associated factors that are also predicted to bind to MTs, and their presence in the screen points to a role for CLASP in mediating actin dynamics in coordination with the MT network. This supports previous observations that vertebrate CLASPs may function as actin-MT crosslinkers. CLASPs possess actin-binding activity (Tsvetkov, 2007), and CLASP-decorated MT tips track along actin filament bundles in the growth cone peripheral domain. Moreover, CLASP was recently shown to bind to the actin-binding protein, IQGAP1, and phosphorylation of CLASP controls linkage of MTs to actin through IQGAP1 for cell migration (Watanabe, 2009). From the current study, it appears that CLASP may have numerous other effector proteins that can modulate its interaction with the actin network. IQGAPs have not been found in Drosophila, and so, it is speculated that the novel interactors identified, as well as others, may allow CLASP to link MTs and actin in different contexts (Lowery, 2010).

The novel CLASP MT+TIP interactor that were identified, Msps, emerged from the screens as a high priority for future analysis. Msps interacts with CLASP in both the genetic and proteomic screens, and it antagonizes CLASP and Abl signaling. The antagonism seen between Msps and CLASP in the Drosophila retina is consistent with cell culture studies, which have shown that CLASP regulates MT dynamics by specifically promoting the pause state (Sousa, 2007) whereas Msps-family proteins function as MT antipause factors (Brittle, 2005). More specifically, CLASPs have MT-stabilizing effects, and depleting cells of CLASP protein results in highly dynamic, constantly growing or shrinking MTs (Sousa, 2007). Alternatively, Msps family members can have the opposite effect (Popov. 2003), catalyzing the addition and removal of multiple tubulin dimers at MT plus-ends (Brouhard, 2008), and depletion of Msps results in a dramatic increase in MT pausing with little or no growth (Brittle, 2005). These opposite effects on MT behavior in cell culture studies suggest reciprocal functions in the regulation of MT dynamics in vivo, and Msps could thus be a component of the Abl signaling pathway that provides an antagonistic counterbalance to CLASP in regulating the growth cone cytoskeletal output downstream of guidance cues (Lowery, 2010).

In the context of CNS, it seems likely that CLASP and Msps drive axon guidance decisions through reciprocal regulation of growth cone turning toward or away from the source of axon guidance factors at the midline. Accurate navigation of both ipsilateral and contralateral axon pathways requires a combination of cues, to regulate both midline crossing behavior and also the stereotyped positions of longitudinal axon tracts. The lateral specification model proposes that for longitudinal axons to find and maintain a correct trajectory at a specific distance from the midline, they must reach a balance of turning responses to the attractive Netrins and repellent Slit secreted by midline glia. Perhaps this balance requires antagonism of Msps and CLASP downstream of Abl, such that reduction of either protein would bias the growth cone or reduce the fidelity of the overall navigation process. Abl has been shown to mediate both Slit and Netrin activity, thus providing a potential point of integration (Lowery, 2010).

While it is anticipated that CLASP and Msps will influence the directionality of growth cone advance in response to guidance cues, the cellular mechanism by which these two effectors guide axons is not yet known. Reciprocal control over MT advance toward the growth cone peripheral domain could account for the effects of CLASP and Msps. However, there are alternatives. For example, reciprocal modulation of growth cone cell adhesion by Msps and CLASP might underlie the two phenotypes observed in the different mutants. Reduction of adhesion has already been shown to be key in the midline repulsive response to Robo, whereas an increase in adhesion has long been known to be vital for fasciculation. Interestingly, the Abl kinase that interacts with both Msps and CLASP was also implicated in Robo-dependent modulation of cell adhesion. If CLASP were to play a role in Robo-mediated suppression of adhesion, then the ectopic midline crossing that occurs in CLASP LOF mutants could be explained by an increase in growth cone adhesion toward the midline, which is exacerbated when Msps is overexpressed. Consistently, the fasciculation morphology defects that occur in msps LOF (and are exacerbated by CLASP GOF) could be explained if the role of Msps is to promote adhesion. Although the effects of CLASP-family proteins on cell adhesion have not been directly measured, studies in nonneuronal contexts suggest that CLASP helps to drive MT-cortical interactions, which would presumably promote, not suppress, adhesion (Lowery, 2010).

In conclusion, this is the first study demonstrating that Msps functions during axon guidance. Numerous studies have analyzed its role in the regulation of MT stability in several systems including the mitotic spindle and in centrosomes, but its potential role(s) in the nervous system has never been previously addressed. In fact, the growth cone functions of most MT+TIPs are unknown; however, previous discoveries that MT+TIPs CLASP and APC, and now Msps, are important for axon guidance demonstrates that the MT+TIPs are an exciting class of guidance effectors worthy of further exploration and understanding (Lowery, 2010).

How Notch establishes longitudinal axon connections between successive segments of the Drosophila CNS

Development of the segmented central nerve cords of vertebrates and invertebrates requires connecting successive neuromeres. This study shows both how a pathway is constructed to guide pioneer axons between segments of the Drosophila CNS, and how motility of the pioneers along that pathway is promoted. First, canonical Notch signaling in specialized glial cells causes nearby differentiating neurons to extrude a mesh of fine projections, and shapes that mesh into a continuous carpet that bridges from segment to segment, hugging the glial surface. This is the direct substratum that pioneer axons follow as they grow. Simultaneously, Notch uses an alternate, non-canonical signaling pathway in the pioneer growth cones themselves, promoting their motility by suppressing Abl signaling to stimulate filopodial growth while presumably reducing substratum adhesion. This propels the axons as they establish the connection between successive segments (Kuzina, 2011).

The axons of the longitudinal pioneer interneurons of the Drosophila ventral nerve cord establish the initial connection between successive segments of the animal. The receptor Notch is crucial for making those first connections, performing two parallel, partially redundant but completely separate functions. Canonical Notch signaling in the interface glia constructs an unbroken track for longitudinal pioneer axons to follow by shaping a continuous band of neuronal membrane that bridges from segment to segment. Simultaneously, non-canonical Notch/Abl signaling in the pioneer neurons themselves promotes the motility of their growth cones, suppressing the activity of the Abl tyrosine kinase signaling module to stimulate filopodial development. Either signaling mechanism provides substantial rescue of a Notch mutant, but both are required for full activity in formation of longitudinal connections of the CNS (Kuzina, 2011).

The mechanism that guides the very first axon to establish the path of a nascent nerve is one of the most fundamental problems in neural development. For longitudinal pioneers of the Drosophila CNS, it is now seen that constructing their path requires coordinated contributions from four interacting cell types. First, the axons of commissural interneurons bearing the Notch ligand Delta contact interface glia. The glia respond by activating canonical Notch signaling, enhancing expression of Notch target genes, including prospero. The genetic program stimulated by Notch directly or indirectly allows the glial cells to attract a 'cap' of fine filopodial processes from nearby differentiating neurons, and shapes that cap into a continuous longitudinal band that bridges between segments. The neuronal cap atop the glia bears the Netrin receptor Frazzled (DCC), which in turn recruits soluble Netrin, thus constructing a domain of accumulation of Netrin protein that hugs the surface of the associated glia. Finally, the pioneer growth cones advance along the edge of that domain of immobilized Netrin until they meet and fasciculate with their partners pioneering from the next segment. The consequence of this choreography is a nerve trajectory that follows, indirectly, the shape of the row of interface glia (Kuzina, 2011).

This view suggests plausible explanations for many aspects of longitudinal axon development that have, up to now, been confusing. Previous investigators have documented that the pioneer growth cones extend amidst a thicket of filopodia that cap the interface glia. The provenance and significance of those filopodia were unknown, though it was clear that they did not derive from other axons. It is now seen that these filopodia come from surrounding, differentiating neurons, and that their function is to hold Frazzled, and therefore Netrin, in a pattern dictated by the positions of the overlying glia, creating the Netrin domain along which the pioneers extend. This explains why the positions of the interface glia correlate so closely with the axon trajectory even though the glia are not the direct substratum. The data, along with other recent results, also suggest why previous experiments investigating the guidance function of interface glia might have given such confusing results. Transformation of the glia into neurons in a gcm mutant would be predicted to place a row of DCC-expressing neurons in precisely the position of the wild-type filopodial carpet. Moreover, genetic experiments ablating or displacing the glia have relied on reagents targeting the progeny of the longitudinal glioblast, but it is now known that only nine of the ten interface glia come from this precursor; the tenth, M-ISNG, is from a different lineage. M-ISNG is appropriately positioned to anchor the filopodial carpet in the absence of the other interface glia and preliminary experiments suggest that it is sufficient for this. Moreover, it has been argued that in those rare segments where pioneer axons stall owing solely to manipulations of the interface glia, all ten of them, including M-ISNG, tend to be absent or displaced. Finally, as in previous studies, this stud found that the Netrin zygotic mutant has a mild, and genetically enhanceable phenotype, showing that the null for the gene is not null for the genetic pathway. It might be that there is a maternal contribution to Netrin, as there is for frazzled. Alternatively, because the receptor on longitudinal pioneers presumably recognizes a Netrin-Frazzled complex, it might be that this receptor has some affinity for Frazzled even in the absence of Netrin. Identification of the missing Netrin receptor will be necessary to clarify this point. It also seems likely that other neuronal components cooperate with the Netrin-Frazzled complex on the meshwork to provide substratum function, as expression of Fra in interface glia is not sufficient to rescue the Notch axonal phenotype (Kuzina, 2011).

Once the pathway for an axon has been constructed, there remains the problem of driving the motility of the growth cone along that pathway. Somehow, the information encoded in a pattern of occupancy of cell surface receptors must be transformed into a pattern of cytoskeletal dynamics that drives growth cone motion. At the level of the axon, this is the bedrock problem in axon guidance, and it, too, has resisted analysis. The current data reveal how Notch modulates an elementary property of the actin cytoskeleton to promote motility of longitudinal pioneer growth cones. Through its antagonism of the Abl signaling network, Notch de-represses the Abl antagonist Enabled and suppresses the Rac GEF Trio. Enabled directly promotes filopodial growth; suppressing Rac indirectly promotes filopodia, probably by redirecting various factors away from lamellipodia. Stimulating filopodial development probably promotes longitudinal axon growth in at least two ways. First, converging pioneer growth cones from successive segments need to encounter one another and fasciculate to establish the connection between segments. Extension of filopodia increases the area searched by an advancing growth cone, increasing the probability that it will encounter its partners advancing from the adjacent segment. Second, filopodia promote neurite growth by promoting microtubule invasion of the leading edge (Kuzina, 2011).

In parallel with stimulating filopodia, suppression of Trio, and thus of Rac, is expected to reduce substratum adhesion. When the pioneers are growing towards the segment border, small gaps in the Frazzled-Netrin pattern are not uncommon, so release of the advancing growth cone from the substratum is likely to aid its forward motion. Moreover, initially there is more Frazzled-bound Netrin within the neuromeres than there is at the segment border, requiring advancing pioneers to go down a gradient of Netrin, towards a region with less Netrin. It is possible that both of these properties make it helpful to limit substratum adhesion of the growth cone via reduction of Rac signaling by Notch (Kuzina, 2011).

It might seem paradoxical that Notch promotes axon growth by suppressing Abl signaling when Abl has been the archetype of a motility-promoting signaling pathway. Indeed, genetic studies of Abl in axon guidance have often appeared to be confusing and contradictory. In part, this reflects pleiotropy. Abl appears to act in the glia, and in the cells providing the filopodial carpet, in addition to the pioneers. As the phenotype in a whole-animal mutant reflects the sum of unrelated functions in different cells, seemingly similar experiments can produce contradictory results if different cellular processes become limiting. For longitudinal axons, for example, if pathway establishment is limiting (in Abl- or fra- animals), reduction of Notch interferes with axon growth synergistically; if growth cone function is limiting (in Notch- animals), reduction of the Abl pathway restores axon growth. It was therefore essential in the current work to control gene activity, and analyze phenotypes, in single, identified cells (Kuzina, 2011).

Beyond pleiotropy, however, complexity arises because the effect of signaling molecules in motility is profoundly context dependent. Ena promotes actin polymerization but often restricts cell motility; cofilin severs actin filaments but can promote net actin polymerization and cell migration. As axon growth is achieved by throughput through a cycle of actomyosin dynamics, it requires a balance among the steps of that cycle. Excessive activity or inactivity of any single step in the process inhibits motility by impairing progression through the cycle. The data now reveal that, for Drosophila longitudinal pioneers, an essential aspect of growth cone movement is restraint of Abl activity to allow filopodial development, and perhaps also to limit substratum adhesion (Kuzina, 2011).

The data reported in this study reveal that Notch promotes CNS longitudinal axon growth in two very different ways, constructing a pathway using its canonical signaling mechanism and promoting motility via the Notch/Abl interaction. This dual role bears striking parallels to the dual role of Notch in radial migration of neurons in the mammalian cortex. There, as in the fly, canonical signaling by Notch is essential for the development of glial cells that define a migration pathway, whereas interaction with the Abl pathway protein Disabled controls neuronal motility and adhesion. Further study will be required to assess whether these parallels between Notch function in the fly and vertebrate nervous systems reflect a deeper mechanistic similarity. Similarly, it will be interesting to see whether formation of longitudinal nerve tracts in the spinal cord uses machinery homologous to that which this study has described in the fly (Kuzina, 2011).

Control of dendritic morphogenesis by Trio in Drosophila melanogaster

Abl tyrosine kinase and its effectors among the Rho family of GTPases each act to control dendritic morphogenesis in Drosophila. It has not been established, however, which of the many GTPase regulators in the cell link these signaling molecules in the dendrite. In axons, the bifunctional guanine exchange factor, Trio, is an essential link between the Abl tyrosine kinase signaling pathway and Rho GTPases, particularly Rac, allowing these systems to act coordinately to control actin organization. In dendritic morphogenesis, however, Abl and Rac have contrary rather than reinforcing effects, raising the question of whether Trio is involved, and if so, whether it acts through Rac, Rho or both. This study shows that Trio is expressed in sensory neurons of the Drosophila embryo and regulates their dendritic arborization. trio mutants display a reduction in dendritic branching and increase in average branch length, whereas over-expression of trio has the opposite effect. It is further shown that it is the Rac GEF domain of Trio, and not its Rho GEF domain that is primarily responsible for the dendritic function of Trio. Thus, Trio shapes the complexity of dendritic arbors and does so in a way that mimics the effects of its target, Rac (Shivalkar, 2012).

Trio has been associated with both Rho family GTPases and the Abl tyrosine kinase. Both these pathways control dendritic arborization in Drosophila, but they do so in different ways, with Rac, for example, promoting dendritic branching and Abl limiting it. This made it important to determine whether Trio plays a role in dendrogenesis, and if so, whether it was functioning in association with Rac or with Rho, and how its effects compared with those of Abl. This study shows that Trio also shapes dendritic structure in the fly. In both simple Class I sensory neurons and complex Class IV sensory neurons, Trio promotes formation of dendritic branches: over-expression of trio produces more elaborately branched dendritic trees whereas loss of trio reduces the number of dendritic branches. In both cases, the effect of Trio is concentrated on higher-order branches, which others have shown to be actin-dominated and more dynamic, and not in the primary branches, which tend to be microtubule-dominated and more stable (Shivalkar, 2012).

Trio not only affects dendritic branching but also dendritic length. In most assays, Trio limits the average length of some or all orders of dendritic branches to a degree that roughly offsets the increase in branch number, leading to a modest net change or no change in total dendritic length. The compensation is not exact, however. For example, in trio mutants, while average dendritic length is unchanged in Class I neurons, an increase in average branch length is seen in Class IV neurons but it is not enough to counteract the decrease in branch number, leading to an overall decrease in total length. Conversely, in trio over-expression, both Class I neurons and Class IV neurons show no net change in total length in spite of an increase in the average length of dendrites. This variability may suggest that total dendritic length is not strictly invariant for a given sensory neuron, with a fixed length parceled among a variable number of branches, but rather that Trio may have separate, and opposite, effects on branch length and number. Further experiments will be necessary, however, to test this idea (Shivalkar, 2012).

Expression of constructs bearing mutations in each of its GEF domains suggests that Trio acts primarily through its Rac GEF domain, and not its Rho GEF domain, to affect dendritic morphogenesis of the PNS sensory neurons. Thus, a Trio derivative lacking Rac GEF activity does not alter dendritic structure whereas a derivative lacking Rho GEF activity produces effects that are indistinguishable from those of the wild type protein. This is consistent with the similarity between the phenotype observed for gain and loss of trio function and that reported for gain and loss of Rac, and also with data from axonal development, both in embryonic motor neurons and adult photoreceptors showing that the Rac-specific GEF1 domain is the key effector domain of Trio in axons. It is in contrast, however, to results from the adult Drosophila mushroom body, in which trio mutant clones showed overextension of neurites similar to that in RhoA mutant clones in the dendritic portion of the structure (the calyx). Perhaps Trio pairs with different GTPases in different developmental settings, as has been observed for C. elegans Trio. The results also indicate that the dendritic phenotypes seen upon over-expression of trio are not due to changes in expression of the important neuronal class specific transcription factors, Abrupt and Knot, thus arguing against the idea that changes of cell fate are responsible for changes in dendritic morphology in these experiments (Shivalkar, 2012).

In contrast to the concordance between the effects of Trio and Rac, the phenotypes produced by altering Trio activity are opposite to those from manipulation of the Abl tyrosine kinase pathway. This was surprising in light of prior work showing that the effects of Trio mimic those of Abl in axonal development, and that led to the suggestion that Trio is a core component of the Abl pathway. Two hypotheses could account for this discrepancy. First, it could be that the Trio-Rac module should be thought of as an adjunct to the Abl signaling network, with a variable and context-dependent relationship to Abl, rather than as itself being a core element of that pathway. Such a relationship would allow the Trio-Abl interaction to produce different morphological outcomes in different developmental settings. Alternatively, the possibility cannot be ruled out that the relationship of Trio to Abl at the molecular level is the same in dendrites as in axons, but it manifests in opposite morphological consequences due to the complexities of the relationship between signaling, cytoskeletal dynamics and morphology. Indeed, there are many examples of a cytoplasmic signaling protein producing seemingly opposite effects in different developmental contexts. In the current setting, however, this interpretation is not favored since such non-linear effects of signaling proteins in other systems typically lead to observation of contradictory phenotypes upon manipulating the activity of a gene across a wide dynamic range. In the case of Trio, in contrast, all of the gain- and loss-of function manipulations give a consistent set of effects on dendritic branching. Additional experiments will be required, however, to distinguish fully between these hypotheses (Shivalkar, 2012).

The data reported in this study show that Trio, like its effector Rac, regulates dendritic arborization in Drosophila sensory neurons. The data also suggest that the relationship of Trio to the Abl tyrosine kinase signaling network may be more nuanced than was previously appreciated. It seems likely that the interplay of these signaling modules channels the molecular machinery of morphogenesis in a variety of ways to help produce the vast range of neuronal shapes (Shivalkar, 2012).


In later larval and pupal stages, Abl protein levels are also highest in differentiating muscle and neural tissue including the photoreceptor cells of the eye. Abl protein is localized subcellularly to the axons of the central nervous system and the apical cell junctions of the imaginal disk epithelium. Abl protein is detected in the epithelial cells of leg, wing and eye-antennal discs. The Abl protein is present throughout the cytoplasm, but is concentrated within the apical cortical region of the cells, a region containing actin-rich adherens-type junctions. Abl protein is found in adepithelial cells that give rise to much of the musculature of the adult thorax. In the leg disc, only the adepithelial cells that have migrated into the more distal folds of the leg pouch, and have begun morphological changes, show an increased level of Abl protein. In pupal leg and wing discs there is a higher level of staining in the proximal region where the leg and wing imaginal discs have fused to form the notum. The Abl protein is present at high levels in developing muscle (Bennett, 1992).

Low levels of Abl protein are detected in undifferentiated cells ahead of the morphogenetic furrow in the developing eye disc. Higher levels of protein are detected in developing photoreceptor cells. The higher levels of Abl protein are detected simultaneously in photoreceptor cells R2, R5 and R8. Later Abl is detected in cells R3 and R4, followed by R1 and R6, and finally R7, coinciding with the order of photoreceptor differentiation. The Abl protein is present throughout the photoreceptor cell bodies and axons, and is concentrated in the apical portions of the cells. Abl protein is detected in neurons of the developing adult CNS, but is not found in neuroblasts. Abl protein is present in the developing neuropil of the brain (Bennett, 1992).

Enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons

The golgi apparatus is optimized separately in different tissues for efficient protein trafficking, little is known of how cell signaling shapes this organelle. This study finds that the Abl tyrosine kinase signaling pathway controls the architecture of the golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-golgi in developing PRs. Overexpression or loss-of-function of Ena increases the number of cis and trans-golgi cisternae per cell, and Ena overexpression also redistributes golgi to the most basal portion of the cell soma. Loss of Abl, or of its upstream regulator, the adaptor protein Disabled, lead to the same alterations of golgi as does overexpression of Ena. The increase in golgi number in Abl mutants arises in part from increased frequency of golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, it was demonstrated that the effects of Abl signaling on golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion (Kannan, 2014).

The Abl tyrosine kinase signaling pathway controls golgi morphology and localization in Drosophila photoreceptors through its regulation of the actin cytoskeleton. Ena, the main effector of Abl in morphogenesis, is associated with the cis-golgi compartment, and it regulates golgi localization and dynamics under the control of Abl and its interacting adaptor protein, Dab. Reducing the levels of Abl or Dab, or overexpressing Ena, led to similar defects in golgi fragmentation state and subcellular distribution. During golgi biogenesis, Abl increases the frequency of fusion of golgi cisternae, and decreases fission events. Abl evidently controls golgi organization through its regulation of actin structure, as the effect of Abl signaling on golgi could be blocked by modulating actin structure genetically or pharmacologically. Collectively, these data reveal an unexpected link between a fundamental tyrosine kinase signaling pathway in neuronal cells and the structure of the golgi compartment (Kannan, 2014).

The data reported here suggest that the Abl signaling pathway controls golgi morphology and localization through its control of actin structure. This is consistent with previous reports that altering the levels of actin modulators perturbs the structure and function of the golgi apparatus. A variety of proteins that modulate actin dynamics have been localized to golgi. Ultra-structural studies established the association of actin filaments with golgi membranes and the association of β and γ actin with the golgi. In cultured cell models, including neurons, actin depolymerization leads to golgi compactness, fragmentation and altered subcellular distribution. It is noted, moreover, that the reported golgi-associated signaling proteins include several that have been linked to Abl signaling, including the Abl target Abi, the Abi binding partner WAVE, and various effectors of Rac GTPase including ADF/cofilin, WASH and Arp2/3. Thus, for example, Abi and WAVE have been implicated in actin dependent golgi stack reorganization and in scission of the golgi at cell division to allow faithful inheritance of golgi complex to daughter cells in Drosophila S2 cell cycles (Kondylis, 2007). These data reinforce the importance of actin-regulating signaling pathways for controlling golgi biogenesis (Kannan, 2014).

Two lines of evidence suggest that the increase observed in golgi number in Abl pathway mutants is due primarily to net fragmentation of pre-existing golgi cisternae and not to de novo synthesis of golgi. First, live imaging of golgi dynamics in neurons of the Drosophila eye disc reveals that the steady-state number of golgi cisternae reflects an ongoing balance of fusion and fission events, much as observed previously in yeast. Quantification of these events in wildtype vs Abl mutant tissue demonstrated directly that loss of Abl significantly increased the frequency of fission events, and reduced the frequency of fusions. Second, the absolute volume of cis-golgi in Abl mutant photoreceptors was not substantially greater than that in wildtype, as judged by direct measurement of the volume of GM130- immunoreactive material in deconvoluted image stacks of photoreceptor clusters. While a small apparent increase was observed in golgi volume in the mutants (~55%, based on pixel counts), it is noted that golgi cisternae are small on the length scale of the point spread function of visible light, such that the fluorescent signal from a single cisterna extends into the surrounding cytoplasm. The increase in apparent golgi volume is therefore within the range expected due simply to fluorescence 'spillover' from the three-fold greater number of separate golgi cisternae in the mutants (Kannan, 2014).

It is striking that both increase and decrease of Ena led to net fragmentation of golgi. Why might this be? It is known that both fission and fusion of membranes requires actin dynamics: at scission, polymerization provides force for separating membranes, while in fusion, actin polymerization is essential for bringing membranes together and for supplying membrane vesicles, among other things. As a result, altering actin dynamics is apt to change the probabilities of multiple aspects of both fission and fusion events, making it impossible to predict a priori how the balance will be altered by a given manipulation, just as either increase or decrease of Ena can inhibit cell or axon motility, depending on the details of the experiment, due to the non-linear nature of actin dynamics. Indeed, this study also observed net golgi fragmentation when actin was stabilized with jasplakinolide, just as was done from depolymerization with cytochalasin or latrunculin. More direct experiments will be necessary to fully understand this dynamic, however. deficits selectively disrupt dendritic morphogenesis but not axogenesis, and perhaps consistent with this, Abl/Ena function is essential for dendrite arborization in these cells but has not been reported to affect their axon patterning. Finally, in some contexts, neuronal development requires local translation of guidance molecules in the growth cone rather than translation in the cell soma. It is likely that the need for actin dynamics to target different subcellular compartments in different cell types will be reflected in different patterns of Abl/Ena protein localization (Kannan, 2014).

This study reports the role of Abl/Ena-dependent regulation of actin structure on overall golgi structure and localization but there may be more subtle effects on golgi function as well. For example, recent evidence supports a role for actin-dependent regulation of the specificity of protein sorting in the golgi complex. Preferential sorting of cargos is achieved by nucleation of distinct actin filaments at the golgi complex. In Hela cells, for example, Arp2/3 mediated nucleation of actin branches at cis-golgi regulates retrograde trafficking of the acid hydroxylase receptor CI-MPR, while Formin family mediated nucleation of linear actin filaments at golgi regulates selective trafficking of the lysosomal enzyme cathepsin D. Similarly, the actin-severing protein ADF/cofilin, the mammalian ortholog of Drosophila twinstar, sculpts an actin-based sorting domain at the trans-golgi network for selective cargo sorting. It will be important to investigate whether the effects of Abl/Ena on golgi morphology have functional consequences on bulk secretion or protein sorting (Kannan, 2014).

Protein trafficking and membrane addition in neurons need to be coordinated with the growth requirements of the axonal and dendritic plasma membranes, but the mechanisms that do so have been obscure. Abl pathway proteins associate with many of the ubiquitous guidance receptors that direct axon growth and guidance throughout phylogeny, including Netrin, Roundabout, the receptor tyrosine phosphatase DLAR, Notch and others. The data therefore suggest a potential link between the regulatory machinery that senses guidance information and the secretory machinery that helps execute those patterning choices. Indeed, preliminary experiments suggest that some of the axonal defects of Abl pathway mutants may arise from alterations in golgi function. Beyond this, Abl signaling is essential in neuronal migration, epithelial polarity and integrity, cell adhesion, hematopoiesis and oncogenesis, among other processes The data reported in this study now compel a reexamination of the many functions of Abl to ascertain whether some of these effects arise, at least in part, from regulation of secretory function (Kannan, 2014).

Abl tyrosine kinase: Biological Overview | Evolutionary Homologs | Regulation | Effects of Mutation | References

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