Over the past year we have made several key advances on this project 1. We have completed experiments to determine the functional architecture of the Abl signaling network. Abl is the central signaling pathway downstream of nearly all the major families of axon guidance receptors, including Notch. The core components of the pathway have been known for years, but the relationships among those proteins have been quite obscure, making it impossible to develop specific, testable models for the mechanism by which guidance receptors control axon growth. We have now ordered the Abl pathway by deploying fluorescent in vivo reporters for the activity of its two key axonal outputs, Abl kinase itself and Rac GTPase. We have shown that Abl is not a single pathway but a branched network, with Abl suppressing activity of Enabled, a direct regulator of actin polymerization, but in parallel activating Rac, a regulator of actin branching. We showed further that Abl stimulates Rac by acting on the Rac guanine exchange factor, Trio, and we described the mechanism by which it does so. This pair of functions means that the organization of the Abl pathway intrinsically balances the two major forms of actin structures in the cell, linear actin bundles and branched actin networks. We propose that this dual function of Abl accounts for why it has so frequently been targeted by evolution as a regulator of morphology and motility. These data have now been published (Kannan, et al., 2017a; Kannan et al., 2017b). 2. A major motivation for ordering the Abl pathway was to allow us to dissect the mechanism by which Notch regulates Abl to control axon growth and guidance. This required, however, that we uncover the mechanism by which Notch regulates Abl activity. We have now completed those experiments. In brief, two key upstream activators of Abl signaling, the adaptor protein Disabled and the Rac GEF Trio, are bound to the intracellular domain of Notch prior to ligand activation. More specifically, they are bound to a specialized population of Notch protein molecules that are tyrosine-phsophorylated. Upon activation by ligand, Notch is cleaved at the extracellular S2 protease cleavage site and the intramembranous S3 (Presenilin) cleavage site, releasing the Notch intracellular domain from the plasma membrane. Since Disabled and Trio remain bound to Notch(ICD), but their targets, Abl and Rac, remain membrane-tethered by fatty acyl groups, this has the effect of disassembling Abl signaling complexes and thus silencing Abl signaling. These data have been submitted for publication and reviewed, and the revised manuscript has been resubmitted. We expect acceptance for publication shortly (Kannan, et al., 2017c). 3. Live imaging of a single Drosophila pioneer axon (TSM1), growing in situ in the fly wing, has suggested a fundamental reconsideration of the cell biology of axon growth. We find that this axon uses a non-adherent, protrusive mechanism of growth, rather than the adhesive mechanism that has been described for axons growing in culture. Examination of data from the literature shows that this is a common mechanism of pioneer axon growth across phylogeny, though its significance has not been recognized previously. We find that the growth cone is defined, not by its morphology, but by localized accumulation of actin, and that axon growth occurs by a forward-biased oscillation of that leading actin mass, driving an 'inchworm' style of axon growth and guidance. Our data further suggest that Abl tyrosine kinase regulates the essential oscillations of actin organization, and that it does so by controlling the ratio of linear to branched actin in the growth cone (see advance #1, above). The initial experiments describing this novel mode of motility are nearly complete and will be prepared for publication shortly.
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