BIOLOGICAL OVERVIEW

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 abl^M; 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 abl^M 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 abl^M 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 abl^M mutants, Dia and Arp2/3 mislocalization may not. In fact, reduction of the dose of Dia enhanced the abl^M phenotype. Dia normally promotes actin polymerization lining the furrows. In abl^M 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 abl^M phenotype. The abnormal recruitment of Dia to the apical regions in abl^M 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 abl^M 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 Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase

In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. This study shows that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at all and fails to send out its axon projection. Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, the data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. It was also found that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. The results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl (Zhu, 2011).

Several studies have suggested that Hem dynamically regulates polymerization of F-actin. Hem can play a crucial role in linking extracellular signals to the cytoskeleton. On the other hand, Hem is also part of the WAVE complex and it may regulate the activity of the WAVE complex to promote polymerization of F-actin. The result that the migration defect in Hem mutants can be completely rescued by expression of WAVE from a transgene indicates that Hem regulates neuronal migration via WAVE (Zhu, 2011).

How Hem regulates WAVE is controversial. It has been argued that Hem (together with PIP212) inhibits WAVE in the WAVE complex. Upon activation by Rac1 or Nck, the WAVE complex dissociates releasing an active WAVE-HSPC300 to mediate actin nucleation. This conclusion was also supported by the findings that loss-of-function for Hem leads to an excess of F-actin in the cytosol. Moreover, a reduction in the WAVE gene dosage suppressed axon guidance defects in Hem mutant embryos. But, in vitro studies using Drosophila tissue culture cells argue that Hem protects WAVE from proteasome-mediated degradation. The current in vivo results are consistent with these studies and show that WAVE is protected by Hem and the above alternate model may be incorrect (Zhu, 2011).

The WAVE protein was first identified in Dictyostelium discoideum as a suppressor of mutations in the cAMP receptor (SCAR) but it is present in flies to humans. All WAVEs contain a N-terminal WHD/SHD (WAVE/SCAR homologue domain), a central proline-rich region and a C-terminal VCA domain. WAVE protein regulates actin polymerization by mediating the signal of Rac to Arp2/3 in lamellipodia. It is involved in forming branched and cross-linked actin networks. Unlike WASp proteins, which are intrinsically inactive by autoinhibition and activated by directly binding to Cdc42, PIP2 etc., WAVE appears to be intrinsically active, at least in vitro.However, the majority of WAVE is in the 'WAVE complex' with four other proteins: Hem, Sra-1/PIR121/CYFIP, Abi and HSPC300/Brk1 (Zhu, 2011).

In the WAVE-complex, direct association between WAVE, Abi and HSPC300 represents the backbone of the complex. Hem binds to Sra-1 forming a sub-complex, which is able to bind to Rac through Sra-1. The interaction between Abi and Hem is what binds Hem and Sra-1 into the complex. Hem and Sra-1 are sequentially recruited to the WAVE complex. Free subunits and assembly intermediates of the WAVE-complex are usually not detected but supposedly degraded. Also, previous studies suggest that depletion of one component leads to degradation of others. Indeed, the current results, that in Hem mutants, the level of WAVE protein, but not the WAVE gene transcription, is drastically reduced supports this contention. Perhaps in the absence of Hem, WAVE complex is either not formed or partially formed, resulting in the degradation of WAVE and phenotypes such as mis-migration of neurons. When the levels of WAVE are supplemented using a WAVE transgene (UAS-WAVE), the migration defect in Hem mutants is promptly rescued (Zhu, 2011).

While a complete lack of WAVE (or Hem) function causes an arrest in the migration of RP2, a reduction in the levels of WAVE due to a reduction in the levels of Hem causes abnormal migration. For example, the lowest level of WAVE is seen in the Hem allele that has the strongest penetrance. Moreover, since this mis-migration defect is rescued by expressing WAVE from a transgene, it can be concluded that this mis-migration is also due to an effect on WAVE. It has been suggested that the WAVE-complex exists cytoplasmically and in membrane-bound forms. Through an interaction with Rac, WAVE gets recruited to the lamellipodia where actin polymerization required for membrane protrusion is initiated and regulated. The integrity of the complex is critical for its proper localization since removal of either WAVE or Abi prevents its translocation to the leading edge of the lamellipodia. It is possible that a reduction in the levels of WAVE in Hem mutant embryos causes non-translocation of the WAVE complex to the membrane, causing a non/mis-migration of RP2 (Zhu, 2011).

These results show that WAVE protein exists as three different molecular weight forms. Treatment of the extract with phosphatase collapses these three forms into a single band, indicating that WAVE protein is phosphorylated, with varying degrees of phosphorylation to yield different molecular weight species. Whether there are any changes in the three different forms with respect to their relative contributions in Hem and Abl mutants was examined. However, no changes were found in their relative contributions and the levels of all the forms were reduced in Hem mutants. Therefore, it may be that the reduction in all the forms, or that the reduction in one or two of the forms is responsible for the migration defect. In Abl mutants, the level of WAVE is modestly increased, which is the opposite to that of the effect of Hem on WAVE. Thus, it seems more likely that the activity of WAVE is affected in Abl mutants. Being a protein kinase, it was possible that Abl phosphorylates WAVE, thus affecting either its activity or level. However, no significant changes in were seen in the relative levels of the different phosphorylated forms of WAVE in Abl mutants. It has been shown in vitro that Abl is recruited to WAVE by Abi following cell stimulation, triggering the translocation of Abl together with the WAVE complex to the leading edge of the membrane. Thus, Abl might affect WAVE activity, either directly or indirectly, via the translocation of the WAVE complex to the membrane of an actively migrating RP2. It is also possible that Abl affects migration in a pathway that does not involve WAVE (Zhu, 2011).

In contrast, the effect of loss-of-function for Abl on WAVE levels is more pronounced in older embryos. These results indicate that Abl directly or indirectly regulates the levels of WAVE. Furthermore, though modest, ectopic expression of Abl does down-regulate WAVE. Interestingly, the results also show that Hem regulation of WAVE levels is downstream of the Abl regulation of WAVE since the Hem; Abl double mutants had the same levels of WAVE as Hem single mutants. It seems likely that in the absence of Hem, WAVE protein gets degraded, resulting in the loss of migration or abnormal migration. Whereas in Abl mutants, the most likely scenario is that the activity of WAVE is affected, resulting in the same migration defect (Zhu, 2011).

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 and Canoe/Afadin mediate mechanotransduction at tricellular junctions

Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. This study identified a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling (Yu, 2020).

This study shows that Canoe and Abl function in a mechanotransduction pathway that regulates dynamic changes in cell adhesion at tricellular junctions during epithelial remodeling. Canoe localization to tricellular junctions is acutely disrupted by laser ablation, demonstrating that Canoe localization is rapidly modulated by mechanical perturbation. Canoe mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine (Y1987) in the Canoe actin-binding domain, and Canoe localization to tricellular junctions is required to stabilize tricellular adhesion. Conversely, constitutively anchoring Canoe at tricellular junctions arrests cell rearrangement, indicating that Canoe levels can tune the rate of junctional remodeling. These results demonstrate that the mechanosensitivity of this critical junctional regulator is modulated by phosphorylation of a single tyrosine residue and reveal an essential role of Canoe in coupling tricellular adhesion with mechanical forces during epithelial remodeling (Yu, 2020).

Mechanical forces trigger a cascade of molecular events in cells that translate biophysical signals into altered cellular behaviors. However, the mechanosensors that directly change conformation under tension in vivo are not well defined. Cell surface receptors are well positioned to detect forces generated by neighboring cells, but Canoe localization and function at tricellular junctions do not require the PDZ domain that mediates its interaction with known receptors. One possibility is that the Canoe protein itself could act as a mechanosensor. Canoe is predicted to be anchored to the membrane through interactions with Rap1 and the actin cytoskeleton. Thus, cytoskeletal tension could stretch the Canoe protein and expose tyrosine 1987 to phosphorylation by Abl. Alternatively, Abl or its upstream activators could be regulated by tension during this process, because the activity of Abl and other tyrosine kinases such as Src and FAK has been shown to be regulated by tension in vitro. In a third possibility, the regulation of Canoe by Abl could allow Canoe to detect force-induced changes in other molecules at tricellular junctions. Because tyrosine 1987 is in the Canoe actin-binding domain, phosphorylation at this site could allow Canoe to recognize distinct actin structures at tricellular junctions, force-induced conformational changes in the cadherin-catenin complex, or other specialized features of tricellular junction composition or geometry. Once recruited to tricellular junctions, Canoe could stabilize adhesion at these sites by reinforcing the connection between adherens junctions and the actomyosin cytoskeleton (Yu, 2020).

Abl tyrosine kinases influence many structural changes that are driven by mechanical forces in tissues, including epithelial remodeling, tissue invagination, axon guidance, and cell migration. Abl has been shown to regulate a number of proteins that act at tension-bearing structures in cells in addition to Canoe/Afadin recruitment to tricellular junctions, including vinculin localization and β-catenin recycling at bicellular junctions and regulation of membrane curvature by BAR-domain proteins. Together, these results raise the possibility that Abl could transduce mechanical forces into a wide range of structural changes within cells. For example, during cell rearrangement, Abl could simultaneously stabilize tricellular adhesion by recruiting Canoe and destabilize bicellular adhesion by enhancing β-catenin turnover, which could allow bicellular junctions to complete contraction before tricellular junctions are remodeled. Tricellular junctions serve many important roles in epithelial development and homeostasis, including modulating cell rearrangement, orienting mitotic spindles, balancing stem cell proliferation and differentiation, and maintaining epithelial barrier function. An understanding of how mechanical inputs affect the conformation, localization, activity, and interactions of proteins at tricellular junctions will provide insight into how these structures sense and integrate mechanical forces in epithelial tissues (Yu, 2020).

Htt is a repressor of Abl activity required for APP-induced axonal growth

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. This study asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. htt mutations were found suppress axon outgrowth defects of alphabeta neurons in Appl mutant MB by derepressing the activity of Abl. Abl is required in MB alphabeta neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth (Marquilly, 2021).

Neurodegenerative disease (ND) encompasses a large and heterogeneous group of maladies, including many that are associated with accumulation of specific misfolded proteins. Despite their variety, however, these diseases share a number of cellular pathologies, raising the question of whether different ND-associated genes might function in shared genetic pathways. Several of these disease genes, moreover, have been implicated in neurodevelopmental processes, suggesting that studies of development may be an effective strategy to reveal initially cryptic connections among genes implicated in ND (Marquilly, 2021).

Huntington's disease (HD) is a progressive, autosomal dominant, neurodegenerative disorder. It is a monogenic disease caused by the expansion of a polyglutamine (polyQ) tract at the N-terminus of a large cytoplasmic protein (3144 a.a.), Huntingtin (Htt). Several studies indicate that an alteration of wild-type Htt function might also contribute to disease progression. Consistent with this, numerous biochemical and in vitro studies have suggested that Htt functions in mammalian neuronal development, synaptic function and axonal trafficking. While HD has been characterized as a neurodegenerative disease, a recent study indicates it is also required for normal human brain development (Marquilly, 2021).

Drosophila has been useful as a model to examine the effects of polyQ-expanded human huntingtin transgenes on neuronal form and function. The fly Huntingtin protein (Htt, 3583 a.a.), although lacking a polyQ tract, is similar to the human Htt protein, with four regions of high sequence homology clustered along the protein in the N-, central- and C-terminal regions. Fly Htt is expressed ubiquitously at low level in embryos, larval and adult tissues, with no specific pattern of expression. Fly Htt is found predominantly in the cytoplasm, even when overexpressed. Despite htt being highly conserved across all Drosophila species, indicating an essential role for biological fitness, null htt mutants display no gross developmental defects, although brains from Drosophila Htt mutants have reduced axon complexity. This suggests that it could be necessary to alter the expression of another gene (or genes) during development to be able to detect a htt loss of function phenotype. Therefore, it was asked whether mutant htt modifies a brain axon growth defect present when another neurodegeneration-related protein is lacking, i.e., in a sensitized genetic background (Marquilly, 2021).

In contrast to HD, Alzheimer's Disease (AD) is highly genetically complex. Like HD, AD is also viewed as a proteinopathy, since it is associated with accumulation of amyloid fibrils derived from the Amyloid Precursor Protein (APP). APPs have therefore been investigated intensely, however their normal function in the brain remains unclear and controversial. Drosophila encodes a single APP homologue, called Appl, that is expressed in all neurons throughout development. It has been shown that Appl is a conserved neuronal modulator of a Wnt planar cell polarity (Wnt/PCP) pathway. This signaling pathway is essential for proper axon outgrowth in the learning and memory center of the fly, a bilaterally symmetric pair of structures called the Mushroom Bodies (MB). In this context, it has been proposed that Appl is part of the membrane complex formed by the core PCP receptors, and further that it promotes phosphorylation of the Dishevelled (Dsh) cytoplasmic adaptor protein. Dsh is a core component required for all known Wnt pathways, including the Wnt/PCP pathway. Specifically, Appl recruits a non-receptor protein tyrosine kinase, called Abl, to the PCP receptor complex and positively modulates its phosphorylation of Dsh (Soldano, 2013). Consistent with this view, it has been shown that a 50% reduction of Abl leads to enhancement of the Appl mutant phenotype. Conversely, overexpression of wild-type Abl+, but not a kinase-dead version, in the MB neurons led to a strong rescue of the Appl mutant phenotype. Together with accompanying biochemical experiments, these data suggested that Appl promotes the phosphorylation of Dsh by Abl kinase, and further showed that this mechanism is conserved in mammals (Soldano, 2013). Thus, Abl is a key downstream effector of Appl required for it to stimulate MB axon outgrowth (Marquilly, 2021).

The Abl family of non-receptor tyrosine kinases includes human ABL1 and ABL2 as well as Drosophila Abl. Each Abl protein shares a conserved domain structure consisting of a SH3-SH2-TK (Src homology 3-Src homology 2-tyrosine kinase) domain cassette which confers autoregulated kinase activity. A carboxy-terminal F (F-actin-binding) domain ties Abl-dependent phosphoregulation to actin filament reorganization. ABL1 has been implicated in a range of cellular processes including actin dynamics and cell migration. Abl was discovered as a cellular proto-oncogene that is constitutively active in human chronic myelogenous leukemia and acute lymphocytic leukemia. Kinase activity of Abl in vivo is limited both by intramolecular interactions and by cellular inhibitors, such as Pag/Msp23. After removal of inhibition, Abl acquires substantial catalytic activity that is further enhanced by primary and secondary (auto)phosphorylation. The Abl kinases have also been shown to play a crucial role in the development of the nervous system. Overexpression of active Abl in adult mouse neurons results in neurodegeneration and neuroinflammation and activation of Abl has been shown to occur in human neurodegenerative disease. In contrast to mammalian Abl, Drosophila Abl has not been shown to directly cause tissue hyperplasia or cell fate transformation in vivo, but rather is essential for cell adhesion and morphogenetic processes such as axonogenesis and growth cone motility. Several studies have shown that the precise level of Abl activity is critical to its axonal function, with loss- and gain-of-function both leading to severe defects in neural patterning. Recent experiments revealed the cell biological and biophysical basis of this relationship, showing that either increased or decreased levels of Abl activity induce disorder in growth cone actin and thereby greatly augment the frequency of stochastic errors in growth and guidance. Of particular relevance to this study, Abl has been implicated in axonal arborization and growth in the Drosophila brain, including the MBs though the precise role of Abl in normal MB development has not been documented (Marquilly, 2021).

The MBs, together with the central complex, form the core of the adult central brain of Drosophila. Due to extensive study, they offer an exceptionally powerful system for analyses of genetic and molecular mechanisms of development and function. The MBs are two bilaterally symmetric structures that are required for learning and memory. Each MB is comprised of 2000 neurons that arise from 4 identified neuroblasts. Three types of neurons appear sequentially during development: the embryonic/early larval β', the larval α'β' and the late larval/pupal αβ. Each αβ neuron projects an axon that branches to send an α branch dorsally, which contributes to the formation of the α lobe, and a β branch medially, which contributes to the formation of the β lobe. Both lobes require the PCP mechanism for efficient axon extension. The PCP genes, however, do not act cell-autonomously to promote MB axon growth. Rather, PCP produces a 'community effect' that coordinates the growth decisions of large groups of MB axons, and that overrides the effect of mutations in single PCP components in any single axon or cluster of axons. Appl is required for this PCP-dependent 'community effect'. In β-branches, Appl is evidently also required for some other mechanism that acts cell-autonomously, in addition to its contribution to the non-cell-autonomous PCP mechanism. The molecular nature of this second autonomous Appl function remains unknown (Marquilly, 2021).

This study investigated the genetic and functional interactions of Htt, Appl, Abl and the core PCP gene dsh in Drosophila. Whtt mutations suppress the MB axonal outgrowth defects observed in Appl mutants. Since Abl is known to act downstream of Appl it seemed a potential target for htt mutant-induced suppression of the Appl phenotype. Therefore this study characterized the role of Abl in normal MB development. Using analysis of Abl loss-of function (LOF) alleles in single-cell MARCM clones it was shown that Abl is required for axonal growth in the developing αβ neurons of the MBs and that it is expressed in these neurons. Importantly, the overexpression of Abl in these neurons also leads to axonal growth defects, suggesting the possible existence of cellular proteins that negatively control neuronal Abl activity. Finally, this study demonstrates that Htt acts as a cellular inhibitor of Abl activity, both genetically, as it functions antagonistically to Abl in MB axon growth, and biochemically, as FRET measurement of Abl kinase activity in vivo reveals that is derepressed by reducing Htt expression. These results indicate that Appl and htt, whose human homologs are central players in neurodegeneration, regulate Abl kinase in opposite directions to maintain its activity in the narrow range necessary for normal axon outgrowth in MB αβ neurons (Marquilly, 2021).

Tumorigenesis and neurodegeneration may be two sides of the same coin. Indeed, defining the overlap of molecular pathways implicated in cancer and neurodegeneration may open the door to novel therapeutic approaches for both groups of disorders. Correlative studies have highlighted a decreased cancer incidence in the population with the neurodegenerative disorder Huntington's disease and both wild-type and mutant huntingtin (Htt) have been implicated in tumor progression. Interestingly, it has been proposed that, in the normal physiological situation, the neurodegeneration-related Amyloid precursor protein (APP) recruits the oncogenic Abelson (Abl) kinase in order to promote axonal outgrowth (Soldano, 2013). It is, therefore, tempting to propose that different neurodegenerative diseases (ND) may share components and mechanisms and that Abl may also have a role in ND (Marquilly, 2021).

This study has shown that Abl is required for axonal growth in the MBs, a brain structure that is involved in memory. Furthermore, both Abl overexpression and lack of expression in the MBs result in similar phenotypes, indicating the need to tightly regulate Abl activity during MB axon outgrowth. This raises the question of how Abl activity is normally negatively regulated during MB axon outgrowth. This study confirmed the previous observation that overexpression of Abl rescues the Appld MB phenotypes (Soldano, 2013). Furthermore, Abl overexpression also rescues the dsh1 MB phenotype. These two results support the model that Appl activates Abl, which in turn phosphorylates Dsh. At the genetic level, it was expected that an increase of Abl activity in an individual bearing a loss-of-function mutation of a putative Abl repressor. It was therefore hypothesized that reducing the levels of an Abl repressor would result in suppression of the Appl and dsh mutant MB phenotype. Htt is such an inhibitor of Abl activity in the MBs. The loss of one dose of htt increased the activity of the wild-type Abl still present in Abl2/+ individuals and therefore prevented the enhancement of the MB mutant phenotype. While a number of studies have concluded that Htt deficiency results in significant alterations to kinase signaling pathways, this study is the first to implicate the crucial tyrosine kinase, Abl. Together, these data demonstrate the power that neurodevelopmental studies have to reveal close functional relationships between genes implicated in different forms of neurodegenerative disease (Marquilly, 2021).

It was a surprise that null htt mutants show no obvious developmental defects in Drosophila although strong defects could have been expected from the lack of such a conserved protein. One possible hypothesis is that, due to its fundamental importance, some functional redundancy has been selected to buffer against variation in the production of the Htt protein. It was reasoned that altering the levels of another protein, particularly another protein known to be implicated in neurodegeneration, could reveal cryptic phenotypes of htt mutation during brain development. Following this reasoning, a null Appl mutant was combined with heterozygous null htt mutations in double mutant individuals. Unexpectedly, it was found that mutant htt suppressed the Appl MB axonal outgrowth defect (Marquilly, 2021).

Abl is a key component of the Appl signaling pathway required for axonal arborization and growth in the fly brain and the functional relationship between these two proteins is likely conserved in mammals. While the role of APP-mediated signaling has been shown most clearly in Drosophila, a number of lines of evidence suggest that mammalian APP also fulfills a signaling role. Importantly, and in line with its proto-oncogenic role, Abl tyrosine kinase activity is tightly regulated by intramolecular inhibition. Although Abl is clearly required as a downstream effector of Appl in the MB axon growth, its precise role and regulation in the MBs has not been described previously (Marquilly, 2021).

The three Abl alleles used in this study have all been shown to result in truncated proteins. While Abl1 retains the SH3, SH2 and TK domains of Abl, Abl2 is mutated within the TK domain and only retains the SH3 and SH2 domains, while Abl4 is mutated in the SH2 domain, and only retains an intact SH3 domain. It therefore seems likely that very little or no residual Abl function remains in Abl2/Abl4 and Abl4/Abl1 individuals and may explain why no rescue was observed when one dose of htt was removed in these genetic backgrounds. In contrast, the Abl1/Abl2 allelic combination is likely less severe than the other two genotypes and Abl1/Abl2 animals do accumulate truncated Abl proteins. Indeed, while significant amounts of truncated Abl1 and Abl2 mutant proteins are detectable, only faint protein bands are observed in Abl4 pupae. Therefore, some functionally significant kinase activity could remain in Abl2/Abl1 individuals and the loss of one dose of htt might increase the activity of the remaining kinase activity. Finally, removing one dose of htt in animals overexpressing Abl would result in even more Abl activity, which in turn would exacerbate the mutant phenotype. Contrarily, over-expressing Htt, as in the UAS-Abl, UAS-htt doubly overexpressing individuals, would inhibit Abl function when compared to the UAS-Abl overexpression alone which thus might explain the observed rescue (Marquilly, 2021).

There are three different levels at which Htt might act to modulate Abl activity. First, Htt could either directly or indirectly affect Abl mRNA levels. Although fly Htt has been described as a cytoplasmic protein, htt has been shown to be a suppressor of position-effect variegation, suggesting a possible role in chromatin organization. This hypothesis is unlikely for the MB phenotype described in this study since qRT-PCR analysis of third instar brains did not reveal a significant differences in Abl mRNA levels between httint/+ and control individuals. Second, Htt could play a role regulating Abl protein level in the MBs. This is also considered unlikely since the quantity of the endogenous Abl is unchanged in httint/+ relative to control individuals. Third, Htt could influence the kinase activity of Abl itself. Taking advantage of a FRET biosensor enabling Abl kinase activity to be assayed directly in the MBs, a significant increase in active Abl was seen in httint/+ versus control individuals. Therefore, a model is favored of Htt acting as an inhibitor of Abl kinase activity during normal MB axonal growth. Abl activity in axons needs to be maintained within rather narrow limits. These two recent studies show that either increase or decrease of Abl activity cause disorganization of actin structure in the growth cone and prevent the orderly oscillation of growth cone actin that is the motor for growth cone advance and thus axon extension. Those papers also explain why Abl gain and loss can result in superficially similar mutant axon patterning phenotypes even though the molecular effects of Abl increase versus decrease are opposite (Marquilly, 2021).

The HEAT repeat domains of Htt are thought to function as a solenoid-like structure that acts as a scaffold and mediates inter- and intra-molecular interactions. It is therefore tempting to propose that a scaffolding role of Htt could elicit repression of Abl kinase activity. At least in the MBs, a balance seems to exist in the activity of Abl, positively regulated by the membrane complex formed by the core PCP proteins and Appl, and suppressed by Htt and possibly other proteins, as well. In one case, if Appl is absent, Abl is not optimally activated leading to defects in MB axon growth. Conversely, decrease of Htt to 50% of wild type levels leads to de-repression of Abl kinase activity, which in turn compensates for its sub-optimal level of activation in the absence of Appl. This unexpected apparent balance of activation and inhibition of Abl by Appl and htt, whose mutant orthologs are central players in human neurological disease, may define a conserved functional interaction to maintain Abl activity in the relatively narrow window to appropriately effect axon outgrowth (Marquilly, 2021).

Glial-secreted Netrins regulate Robo1/Rac1-Cdc42 signaling threshold levels during Drosophila asymmetric neural stem/progenitor cell division

Asymmetric stem cell division (ASCD) is a key mechanism in development, cancer, and stem cell biology. Drosophila neural stem cells, called neuroblasts (NBs), divide asymmetrically through intrinsic mechanisms. This study shows that the extrinsic axon guidance cues Netrins, secreted by a glial niche surrounding larval brain neural stem cell lineages, regulate NB ASCD. Netrin-Frazzled/DCC signaling modulates, through Abelson kinase, Robo1 signaling threshold levels in Drosophila larval brain neural stem and progenitor cells of NBII lineages. Unbalanced Robo1 signaling levels induce ectopic NBs and progenitor cells due to failures in the ASCD process. Mechanistically, Robo1 signaling directly impinges on the intrinsic ASCD machinery, such as aPKC, Canoe/Afadin, and Numb, through the small GTPases Rac1 and Cdc42, which are required for the localization in mitotic NBs of Par-6, a Cdc42 physical partner and a core component of the Par (Par-6-aPKC-Par3/Bazooka) apical complex (de Torres-Jurado, 2022).

A precise regulation of ASCD is critical in development, tissue homeostasis, and tumorigenesis. Extrinsic signals from specialized microenvironments, the niches, importantly contribute to that regulation by promoting the stem cell fate in the daughter cell that receives those signals, whereas the other daughter cell enters a differentiation program. Intriguingly, the ASCD of some stem cells, including the Drosophila CNS stem cells called NBs, the subject of this study, seems to depend exclusively on intrinsic regulatory mechanisms. Then, are those stem cells completely independent of their surrounding environment? Also, how general is the requirement of niches and the signals secreted from them for maintaining the stem cell fate in an ASCD (de Torres-Jurado, 2022)?

This study shows that the ASCD of Drosophila NBs, a traditional paradigm for studying intrinsic ASCD regulatory mechanisms, do also depend on extrinsic cues secreted by a glial niche, which are in close contact with those neural stem cell lineages in the larval brain. However, these extrinsic signals are not required to maintain the stem cell fate. They ultimately impact on the regulation of intrinsic factors to induce differentiation in one daughter cell, repressing the self-renewal 'basal state' in this cell. Different studies have shown the possibility of growing in culture isolated larval NBs, which are able to form crescents and divide asymmetrically without any additional extrinsic signal. However, most of these experiments were performed using central brain type I NB lineage (NBI) NBs, which do not require Fra and Robo1 signaling for their correct development, or included type II NB lineage (NBII,) but only particular markers were analyzed (i.e., Baz and Pon). This is relevant as, for example, it was observed that the localization of some ASCD regulators were not affected without extrinsic signals (i.e., Insc, Mira, and Brat). In the experiments in culture, some of the latter regulators are frequently used. It is also key to properly quantify the cases of crescent formation at metaphase, as the phenotypes are never fully penetrant and can be even totally rescued at telophase. For example, in the system used in this study, aPKC showed about 50% localization failures at metaphase, implying that there are NBIIs that show aPKC crescents. Nevertheless, finding that glia-secreted cues are specifically required in NBII lineages was indeed very intriguing. NBII lineages are larger than NBI, as they undergo an additional proliferation phase through INPs and hence are more prone to induce tumor-like overgrowth when ASCD fails. Thus, additional levels of regulation might have evolved in these lineages to ensure the correct division of the NB and INPs to avoid overgrowths. This issue will be further examined in the future (de Torres-Jurado, 2022).

This work has unveiled a novel function for the axon guidance cues Netrins and their Fra/DCC-like receptor in regulating self-renewal versus differentiation in neural stem and progenitor cells of larval NBII lineages. The cortex glial niche that surrounds those lineages secretes Netrins, which modulate Robo1 signaling threshold levels through Fra and Abl kinase in stem and progenitor cells. Whereas Robo1 signaling is activated by its ligand Slit, also secreted by the glial niche, Abl kinase represses this signaling, and these balanced Robo1 signaling levels appear to be critical for the cell-fate commitment of the daughter cell prone to differentiate. The cortex glia dynamically undergoes remodeling through larval stages; only at late third instar larval stages (L3) the glia chamber enwrapping each NB lineage forms completely. This study already observed the presence of the secreted ligands Slit and NetA in the cortex glia at L2, suggesting that these cues are being secreted from the glia since the reactivation of dormant NBs at early L2 (de Torres-Jurado, 2022).

Slit-Robo signaling regulates progenitor cell proliferation in the mammalian CNS and promotes the terminal asymmetric division of a differentiation-committed cell in the Drosophila CNS. Likewise, in mammary stem cells, Robo1 favors their asymmetric mode of cell division. Slit-Robo signaling is also required in other stem or progenitor cells to regulate their lineage specification, identity, or their adhesion/anchoring to the niche. No role for Netrin-Fra/DCC signaling has been previously described in all those contexts (de Torres-Jurado, 2022).

Ultimately, a transcriptional control has been pointed out as the most common way of action by which Robo signaling regulate the above-mentioned cellular processes. This study shows a novel, transcription-independent mechanism by which Robo1 signaling regulates ASCD. Robo1 signaling would be required to activate the small GTPases Rac1 and Cdc42 by repressing its inhibitor RhoGAP93B as well as by recruiting the Dock-Pak complex, which, through Pak, can also bind activated forms of both Rac1 and Cdc42 Rac1 and Cdc42 downregulation directly impacted on the intrinsic machinery that modulate ASCD in neural stem and progenitor cells. Specifically, compromising those small GTPases led to defects in the localization of the ASCD regulators, Par-6, aPKC, Cno, and Numb, and the concomitant formation of ectopic NBs (eNBs) within brain neural lineages, a phenotype that recapitulates that of Slit-Robo1 signaling impairment. The overexpression of robo1 caused similar defects than the loss of robo1. It was also a similar phenotype than for the loss of fra, the downregulation of Abl, or the expression of a kinase dead form of Abl (unable to repress Robo1). Hence, based on all those experiments, a working model proposes that Netrin-Fra signaling would be modulating, through Abl kinase, the Robo1 signaling threshold levels necessary to regulate in turn the correct activity of the small GTPases Rac1 and Cdc42. In fact, and according to this, the expression of Rac1V12 within NBII lineages caused the formation of eNBs and led to defects in the localization of aPKC in NB and progenitor cells, a similar phenotype than that observed after overexpressing robo1 in these NBII lineages. It would be interesting to determine whether this novel function of Netrin-Fra/DCC signaling regulating ASCD is also conserved in vertebrates, and whether Robo/Rac1-Cdc42 signaling threshold levels in the above-mentioned contexts are also critical for and dependent on Netrin-DCC signaling, as was have found in Drosophila (de Torres-Jurado, 2022).

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. This study shows that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, these data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions (Koca, 2023).

This study demonstrates that dAbl regulates cell motility during OR. Although loss of Abl function interferes with multiple aspects of photoreceptor development and morphogenesis, overexpression of dAbl in developing ommatidial clusters in eye discs affects specifically OR, suggesting that dAbl has a defined function in rotation. During OR, dAbl appears to have an inhibitory role, as ommatidial clusters with increased dAbl levels under-rotate, whereas dAbl mutant ommatidia tend to rotate faster (Koca, 2023).

The localization pattern of dAbl posterior to the MF provides further insight about its role in OR. dAbl becomes apically localized in photoreceptors R8, R2/R5, and R4, following a steady phase of rotation, at the time when clusters slow down and refine their motility until the completion of the 90° angle. Prominent Abl localization within the apical plane of specific photoreceptors suggests that Abl is likely to have a local function in the apical junctional domain. Under-rotation features observed upon dAbl overexpression are consistent with the notion that dAbl becomes apically localized in specific R cells, toward the later stages of OR, to slow down the process. Interestingly, there is a differential localization of dAbl between R3 and R4 in the apical junctional domain. Considering the role of the R3/R4 pair and associated signaling pathways in OR, it is tempting to speculate that this differential dAbl localization is comparable to the requirement of the Nmo kinase within R3/R4, with Nmo providing a directional impulse to rotation in R433 and dAbl regulating its slowing down. The data argue that dAbl activity within R3/R4 pairs is indeed important for fine-tuning rotation. Knockdown and overexpression of dAbl in R3/R4 pairs lead to over-rotation and under-rotation, respectively, during the active rotation process in eye discs, suggesting that Abl activity negatively regulates rotation. Specifically, knockdown of Abl in R3/R4 leads to over-rotation of ommatidia, which, taken together with the WT localization of Abl being restricted to the R4 apical junctional domain, suggests that Abl is required in R4 within the apical region to slow down rotation. In the case of under-rotation caused by m Δ0.5>Abl overexpression, apical dAbl was detected in both cells of the R3/R4 pair and, importantly, temporally earlier in this background compared with WT, suggesting that early dAbl expression in both cells causes an under-rotation phenotype by interfering with rotation. Taken together, these observations are consistent with the hypothesis that the timing and specificity of apical localization of dAbl in R4 is critical for its normal function in OR (Koca, 2023).

Notably, Abl overexpression does not appear to affect ommatidial chirality and the localization of PCP factors, as Fmi expression and localization remain intact. Furthermore, Abl overexpression causes a specific and severe under-rotation defect, unlikely resulting from deregulation of core PCP factors, which are commonly associated with random ommatidial chirality and rotation. It is most likely that Abl overexpression, under sev- or m Δ0.5-Gal4 drivers, is temporally too late to interfere with Fz/PCP signaling-mediated R3/R4 cell fate decisions, and thus specifically affects OR (Koca, 2023).

Fz/PCP signaling appears dispensable for the R4-specific apical dAbl localization, as the pattern is maintained in core PCP mutant ommatidia. Yet dAbl does synergize with Fmi, when co-overexpressed in the R3/R4 pair, in a rotation specific manner. This OR-associated functional interaction of Abl with membrane-associated core PCP factors, along with the localization pattern of Abl in the apical domain further suggests that dAbl activity is important in R4 in the apical junctional domain. The results identify Notch and Notch signaling in R4 as critical for apical dAbl localization. Notch over-activation within the R3/R4 pair (via expression of stable isoforms of the receptor) induces apical dAbl localization in both cells of the pair. In contrast, expression in R3/R4 pairs of a version of Notch deficient in Delta binding, the key Notch ligand in the eye, and thus interference with ligand induced Notch activation, leads to a loss of apical dAbl in R4. Similarly, reduction of Notch levels in R3/R4 cells (via RNAi-mediated knockdown) also causes a marked decrease in apical dAbl levels in R4. As Notch-dependent transcription is still active in these backgrounds, the combination of these results suggests that Notch-mediated dAbl apical localization is rather direct, and not via a secondary mechanism through transcriptional regulation. This conclusion is corroborated by the co-immunoprecipitation experiments (Koca, 2023).

Several experimental lines support the hypothesis that the Notch receptor physically recruits dAbl to the membrane. In salivary glands, Notch overexpression augments junctional dAbl localization, leaving total dAbl levels unaffected. dAbl co-immunoprecipitates with Notch in third-instar larval eye disc extracts, supporting a membrane-associated Notch-Abl interaction in vivo, independent of nuclear Notch signaling activity. The sev>Abl GOF rotation phenotype is markedly suppressed upon removal of one copy of Notch, further supporting the idea that a functional N-Abl signaling module in the apical domain of R4 regulates OR (Koca, 2023).

dAbl localization appears to be within the apical region and not restricted to the apical membrane ring. There may be multiple reasons for this. As the Notch receptor is cleaved upon ligand binding and its intracellular domain is released to the cytoplasm, distribution of Abl molecules in the apical region may be broader than restricted to the transmembrane fraction of Notch. Abl-Notch interactions likely last after Notch cleavage, considering efficient Abl co-immunoprecipitation with the Notch ICD. Abl can also interact with actomyosin cytoskeletal elements, which are apically enriched in R cells (Koca, 2023).

As the apical diameter of R cells in this region is less than 2 μm, the imaging resolution does not separate the membrane Abl signal from the juxta-membrane cytoplasmic signal. Notably, in Notch overexpression contexts, Abl signal is often detected as a ring at the apical membrane, likely attributable to the presence of more uncleaved membrane-associated Notch. Furthermore, it is possible to detect and quantify Abl at junctions in salivary glands, and thus document the increased levels of membrane-associated Abl upon higher Notch levels. All these data are consistent with the notion that Abl is specifically recruited to the apical junctional membrane domain by Notch (Koca, 2023).

In Drosophila, dAbl has been suggested to act downstream of Notch during axonal pathfinding in embryos. Compelling evidence suggests that a non-canonical Notch signaling branch, which does not entail nuclear Notch activity, instructs axonal pathfinding and axon-guidance-specific genetic interactions between dAbl and Notch argue that a non-canonical Notch signaling pathway via dAbl may be at work in this context (Koca, 2023).

The results are in accordance with these observations and provide further evidence for a non-canonical Notch-Abl signaling module during morphogenesis. Recently, a non-canonical Notch pathway has been reported in the regulation of adherens junction organization during human vascular barrier formation, with the transmembrane domain of Notch forming complexes with the tyrosine phosphatase LAR, vascular endothelial cadherin, and Rac1GEF Trio to confer barrier function in human engineered microvessels. The Notch transmembrane domain requires the cleavage of the Notch extracellular and intracellular domains in this context. The data during OR indicate that apical dAbl recruitment in R4 similarly requires Notch activation by Delta. Whether the transmembrane domain of Notch is an essential component of dAbl recruitment and/or regulation remains to be confirmed. There is a growing body of evidence that Notch uses alternative downstream signaling events to regulate cellular morphogenesis and organization, besides canonical transcriptional target gene regulation. (Koca, 2023).

Abl appears to affect junctional N-cad and Arm levels in the R3/R4 pair. N-cad mutants show OR defects. Although the mechanism of N-cad involvement remains unclear, N-cad and/or Arm at the R3/R4 boundary could mediate the communication between these cells to determine relative force generation or other directional behavior to give the rotation direction or impulse/force. Such mechanisms have been suggested in border cell migration through E-cad (Koca, 2023).

N-cad mutant ommatidia appear to over-rotate unlike Abl-overexpressing ommatidia (in which N-cad is downregulated at the R3/R4 border). Although this seems like a discrepancy, Abl overexpression by m Δ0.5-Gal4 (unlike N-cad mutations) is spatially and temporally restricted to R3/R4s, possibly accounting for the differences observed in these backgrounds. Furthermore, Abl likely affects OR via regulating several downstream effectors, including cytoskeletal regulators, in parallel to N-cad and thus has a more complex impact on OR than N-cad alone (Koca, 2023).

The observation that the non-phosphorylatable isoform of Arm/β-catenin, ArmY667F, rescues the Abl GOF defects, supports the idea that Arm is a key and direct target of dAbl in the OR context. dAbl is involved in the regulation of multi-cellular reorganization in the context of Drosophila germband elongation through the phosphorylation of Arm/β-catenin on tyrosine 667 (Y667), by which it controls adherens junction turnover to promote convergent extension cell movements (Koca, 2023).

The data argue that dAbl may similarly be involved in regulating Arm/β-catenin dynamics through the same residue during the OR process. The under-rotation phenotype associated with the dAbl GOF (sev>Abl) showed a trend toward rescue by co-(over)expression of Arm-WT and ArmY667E, which is likely due to the fact that exogenously overexpressed Arm isoforms compete with endogenous Arm for dAbl binding. Further experiments will be needed to test these hypotheses (Koca, 2023).

The requirement of Abl in R4 for accurate rotation suggests that it acts antagonistically to Nemo which is enriched at junctions in R4 early via core PCP factors and its function is to promote rotation (Koca, 2023).

There is a temporal sequence of apical plane enrichment of factors in R4 with Nemo first to initiate rotation, and Abl a few hours later to slow it down. It was originally proposed that OR is a two-step process, with an initial fast rotation to 45° and a subsequent slower step to achieve the full 90°. However, this idea goes back to the identification of the original allele of nemo, which is a hypomorph, and only affected the rotation process partially (Koca, 2023).

Recent live imaging studies documenting OR dynamics have established that rotation is continuous with comparable speed throughout. Similarly, there is growing evidence that for rotation to occur correctly, adherens junctions need to be dynamically regulated at the interface between all photoreceptors and the non-rotating inter-ommatidial cells, and possibly between individual inter-ommatidial cells. It is thus very likely that Abl overexpression with m Δ0.5 and sev drivers interferes with rotation by affecting adherens junction regulation and dynamics in all or multiple R cells, like Nemo (Koca, 2023).

Localization of Abl within the apical plane of R4, as well as R2/R5, is detected at late stages of rotation (from rows 7 and 8 onward), when rotation needs to be slowed down and stopped at 90°, indicating that Abl has a role at the late phases of the process, to terminate rotation. There are additional cues that appear to signal within ommatidia to stop rotation. For example, EGFR signaling via Argos (the original allele of argos being 'roulette/rlt') certainly feeds into slowing down rotation, as without the inhibitory EGFR ligand, argos^rlt mutant clusters rotate beyond 90° (as the name 'roulette' indicates). Similarly, Scabrous (Sca), a secreted fibrinogen-like factor, has been suggested to regulate the properties of the extracellular matrix to create a barrier to rotation (Koca, 2023).

Although the mechanism of Sca function remains unknown, a direct involvement of the ECM in rotation has been reported with a specific link of Integrin signaling and ECM in the OR process. A model is thus emerging that suggests the degree of rotation depends on an interplay between multiple signaling pathways, including Notch-Abl signaling, and their regulatory input to cell adhesion and cytoskeletal elements (Koca, 2023).

Notch signaling in R3/R4 pairs is critical to coordinate OR via its feeding into the transcriptional regulation of argos, with Notch signaling directly promoting the transcription of argos, the inhibitory ligand to EGFR, required to fine-tune EGFR signaling activity during OR. This study shows that Notch signaling regulates OR via apical junctional recruitment of dAbl in R4, linking Notch activity to non-canonical, Abl-mediated Notch signaling and associated local cellular processes, with Abl modulating cadherin/β-catenin-based junctional complexes. Involvement of Notch signaling in cellular morphogenesis has been suggested in various contexts, including Drosophila oogenesis and neuronal pathfinding, zebrafish sensory organ development and human vascular barrier formation among others (Koca, 2023).

Besides the reported Notch signaling-mediated transcriptional inputs into adhesion and cytoskeletal dynamics a direct link from the Notch receptor to cell adhesion has been revealed (Koca, 2023).

This work also suggests a direct input from Notch signaling to cell adhesion dynamics. Many regulators of OR show conservation across developmental processes in vertebrates. The role of Notch signaling in OR suggests a potential involvement for Notch in PCP-mediated morphogenetic events in vertebrates, which has not been reported thus far. Similarly, Abl kinase may have a role in such processes in its interaction with PCP and Notch signaling pathways. Strikingly, the mouse abl^-/- arg-/- double mutants exhibit defects in neurulation and delays in neural tube closure, a process generally requiring PCP-regulated features (Koca, 2023).

The work described here provides insight into Notch-Abl signaling in a tissue remodeling, cell motility process. Although all data are consistent with the proposed model, this model is generated by inference from analyses of static fixed tissue samples, genetics, and biochemical studies. As it involves a cell motility process, it would be desirable to analyze the respective mutant genotypes via live imaging in vivo, including studies applying FRAP and other technologies. This would allow a more complete understanding of how Abl affects junctional dynamics during OR. Future studies will be needed to provide insight into the mechanistic details of how Notch and Abl cooperate in regulating junctional complexes and their dynamics during OR and other morphogenetic developmental and disease processes (Koca, 2023).

Previous treatment of Abl in The Interactive Fly

The Philadelphia chromosomal translocation is responsible for the fusion of two genes, ABL and BCR. Recognized as a frequently occuring aberrant chromosome in acute lymphoblastic leukemia, it was understood more than a decade ago that the Philadelphia chromosome occurs as a result of a recombination between two genes: the cellular ABL gene on chromosome 9, and BCR (breakpoint cluster region) gene located on chromosome 22. The Drosophila Abelson (Abl) protein is a homolog of mammalian c-Abl the wild-type gene coding for the ABL sequence found in the BRC/ABL hybrid protein. The BCR/ABL oncogene product, derived from this specific chromosomal translocation is present in Philadelphia chromosome-postive acute lymphoblastic leukemia. Despite its homology to c-Abl, the wild type mammalian protein, Drosophila ABL protein shows distinct properties and functional differences from c-Abl that suggest the two proteins are not strictly comparable. Notably, the Drosophila protein shows no nuclear localization; this suggests that the functions listed below, found for the mammalian protein, are absent in Drosophila:

• association with nuclear cell cycle proteins
• phosphorylation of the C-terminal domain of RNA polymerase II
• association with proteins involved in DNA repair
• a presumed DNA binding function

Assuming that no other Abl type proteins are found in Drosophila, and no nuclear function can be found for Drosophila Abl, Abl can be used to illustrate the extent of an evolutionary divergence of nuclear function.

Nevertheless, the cytoplasmic functions of Abl appear to be conserved. Both fly and mammalian proteins have a conserved actin interaction domain and both proteins show genetic or physical interaction with a conserved protein known as Disabled (Dab). The Drosophila Disabled protein colocalizes with Abl in cell bodies and axons of embryonic CNS neurons. Dab is essential for normal CNS development, even in the presence of Drosophila Abl. Dab is tyrosine phosphorylated in insect cells and given its co-localization with Abl in the CNS, it has been suggested that Dab may be a physiological substrate of Abl. Identification of Abl substrates is important in building a picture of the role of Abl in the CNS. Drosophila Abl is required in disabled heterozygotes after the time of cell fate specification and during the time of axonogenesis in the embryonic CNS (Gertler, 1993). Murine Disabled is expressed in certain neuronal and hematopoietic cell lines and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, murine Disabled is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion, but when nerve tracts are established, murine Disabled lacks detectable phosphotyrosine. The properties of murine Disabled and genetic analysis of Disabled in Drosophila suggest that these molecules function in key signal transduction pathways involved in a function downstream of Abl in axonogenesis (Howell, 1997). The involvement of Abl in axonogenesis does not require Abl's kinase domain (Henkemeyer, 1990).

The importance of Abl in neurogenesis is also evidenced by Abl's interaction with another conserved protein, Enabled. Enabled is a member of the rapidly expanding VASP family of cytoskeletal associated proteins. The well studied migration of neuronal growth cones serves as a model for the actin-driven formation of membrane protrusions. Establishment of proper connections in the central nervous system depends on the ability of neuronal growth cones to guide neurites to their final targets. The ABL-ENA-Profilin pathway is implicated in the process of axonal outgrowth and fasciculation. Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identify alleles of only one gene, enabled. The ENA protein contains proline-rich motifs and binds to ABL and Src SH3 domains. ENA is (as Disabled has been suggested to be) also a substrate for the Abl kinase. Tyrosine phosphorylation of ENA is increased when it is coexpressed in cells with human or Drosophila Abl, and endogenous ENA tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos show defects in axonal architecture. Therefore, it has been concluded that a critical function of Drosophila ABL is to phosphorylate and negatively regulate ENA protein during neural development (Gertler, 1995).

Drosophia Abl interacts with four other fly genes: prospero, failed axon connections, and Fasciclin 1 and Fasciclin 2, two genes coding for cell adhesion proteins. The interaction with prospero has not been explored further since the non-uniform cytoplasmic distribution of Prospero protein was discovered. The action of Inscuteable, a cytoskeleton associated protein, suggests an involvement of cytoskeleton in providing cues for Prospero and Numb subcellular localization. Cytochalasin D, which disrupts Actin filaments, eliminates Inscuteable crescents and results in incorrect Prospero crescent positioning (Kraut, 1996). Since the highest levels of Abl are not in neuroblasts but in postmitiotic neurons (Bennett, 1992), the relation between Prospero and Abl is currently unknown.

The interaction of Abl with Fas1, Fas2, and failed axon connections suggests the involvement of Abl in a feedback mechanism in which the cytoskeleton of the cell is given information about cell adherence events occurring during axonogenesis. This might be the Drosophila function of Abl with greatest relevance to vertebrate neurobiology. Abl also seems to be involved in signaling in early embryonic development both in Drosophila and Xenopus. The nature of this involvement has not yet been documented.

GENE STRUCTURE

Exons - 10

Bases in 3' UTR - 1611

PROTEIN STRUCTURE

Amino Acids - 1520

Structural Domains

Abelson (Abl) gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type lb 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region is expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues is observed. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development (Henkemeyer, 1988). There are five different 3' ends to ABL mRNA sequences, due to multiple polyadenylation sites separated from one another by as much as one kb (Telford, 1985).

The amino-terminal region of Abl tyrosine kinase related proteins shares several common features with Src-family members. These include a myristoylation site, and the arrangement of Src-homology domains in the primary sequence. Characteristic of the Abl-family members, however, is a large C-terminal extention beyond the kinase domain. The C-terminal segments are not well conserved among Abl-family members. For example, Drosophila Abl and the mammalian c-Abl are between 16-32% identical in this region. The divergent C-terminal segments are not interchangeable between Drosophila Abl and c-Abl, while the kinase domains are interchangeable. These results indicate that the C-terminal segments are important in specifying the biological functions of the Abl tyrosine kinases, and suggest that Drosophila Abl and c-Abl may perform different functions. Nevertheless, a C-terminal actin binding domain appears to be conserved. A presumed DNA binding domain and nuclear transport sequence is present in a region in c-Abl that is not conserved in Drosophila Abl (Wang, 1993 and references).

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

date revised: 1 June 2024 

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