Anterograde Flux of an Accumulated Actin Distribution: A Novel Mechanism for Axon Extension and Guidance Regulated by the Non-Receptor Abelson Tyrosine Kinase
Open AccessPrecise formation of neural circuits depends upon the ability of neuronal growth cones to sense and respond to guidance cues in 3-dimensional space. While many molecular components of the growth cone sensory apparatus have been identified, we still do not understand the organization of signaling cascades or how they produce the cellular level effects of axonal growth, retraction or turning that are necessary for guidance decisions in complex tissues in vivo. The non-receptor Abelson tyrosine kinase (Abl) signaling network controls cell migration, epithelial organization, and other aspects of development, including axon patterning. While individual components of the network are known, the relationships among them have remained mysterious. Here we use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to define the structure of this network in Drosophila. Specifically, we show that Abl constitutes a bifurcating network, suppressing the activity of the actin regulatory factor Enabled, which extends linear actin filaments, while it simultaneously acts through the GEF Trio to stimulate the signaling activity of Rac GTPase, activating the actin branching factor, WAVE. These results suggest that the balancing of linear and branched actin networks by Abl is likely to be central to its regulation of axon patterning. To investigate this hypothesis, we have developed 4D live imaging and quantitative analytical methods to dissect the mechanism of growth of the TSM1 neuron, and its response to Abl, as it extends within the intact Drosophila wing. Multiplexed live imaging reveals that TSM1 axon extension relies on filopodia-like protrusions, and not on large, leading lamellipodia. Lamellipodia and other key features of the common growth cone model are almost devoid from these axons. Instead, we find that local accumulation of actin in the distal axon coincides with the primary site of filopodial localization and dynamics. During extension, this actin-rich zone displays net advance over time due to longitudinal fluctuations in the position of the leading and trailing edges of the zone along the axon. Coherent anterograde translocation of the actin mass within the axon, or into a filopodium with an appropriate orientation extends and guides the axon. Filopodial protrusions that are left behind the advance of accumulated actin are dismantled, and new protrusions emerge at the new more distal site of the actin bolus. Indeed, anterograde actin flux organizes the morphology of the TSM1 growth cone as the axon extends. We further find that Abl is required for normal TSM1 development, as Abl perturbation grossly disrupts the organization of anterograde actin flux in the axon, resulting in abnormal growth cone morphology and terminal TSM1 axon patterning defects. These data strongly suggest that organized flux of the accumulated actin distribution is critical for TSM1 axon extension and guidance. Qualitative comparison of TSM1 morphologies and cytoskeletal organizations to published in vivo imaging in other vertebrate and invertebrate preparations suggests that many features we observe in these Drosophila peripheral axons are conserved in axons growing on natural substrata in many species. Together, these data recast our understanding of growth cone morphology, the mechanisms underlying their motility, and the role of Abl at the molecular and cellular levels during axon growth and guidance
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