Over the past year, we have made two key advances on this project. 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 been submitted for publication (Kannan, et al.). Our live-imaging of single Drosophila axons extending in their native environment have forced a radical reinterpretation of our fundamental ideas of how axons grow. Based on a large body of work done in cell culture, it has been widely assumed that the growth cone, the motile structure at the growing tip of an axon, is a broad, flat structure whose motility employs a mechanism akin to the adhesion clutch model observed at the leading edge of migrating fibroblasts. However, when we image fly growth cones extending on their native substratum in the developing wing, we find that they have essentially none of the physical structures that are thought to provide the motive force for growth. Re-examination of the published literature, moreover, reveals that this is a common feature of axons in vivo: in nearly all the cases we could find where live imaging was performed in vivo, rather than in culture, the axons have a morphology and mode of growth similar to what we observe in the fly wing. We have therefore been forced to develop a new model for axon growth, employing an entirely new set of parameters for describing its features and dynamics, in order to interpret our video images. We find that: - the growth cones of wing axons are defined by a localized accumulation of actin in the distal portion of the axon shaft - the axon grows by coherent translocation of the actin mass into a leading filopodium and turns by directed translocation of the actin mass into an off-axis filopodium - actin translocation occurs by biased oscillations of the actin mass along its major axis - Abl tyrosine kinase controls the speed of axon advance, in part, by modulating the bias in those oscillations, specifically the relative magnitudes of forward vs rearward displacements of the actin peak. This is an entirely new way to think about how axons extend and navigate, and will require reconsideration of the fundamental mechanisms that underlie neural wiring.
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