The fundamental roles of axonal actin during neuronal growth and longevity

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The actin cytoskeleton within axon shafts has long been neglected, but supra-resolution microscopy has now made it amenable to investigation. Here we will use Drosophila genetics to dissect the roles of axonal actin, building on promising pilot data which suggest important functions in axon growth and in maintaining axonal MTs. Axons are the cable-like protrusions of neurons which electrically wire the nervous system and are indispensable for its function. In spite of their importance, the fundamental mechanisms which underpin the formation and maintenance of axons remain poorly understood. Important understanding will come from work on the actin and microtubule (MT) cytoskeleton which is absolutely required for the growth and maintenance of axons. Actin and MTs are filamentous protein polymers which arrange into intracellular scaffolds maintaining cell architecture and mediating cell dynamics. So far, research has primarily been focussed on the prominent cytoskeletal networks of neurons: firstly, abundant actin networks in motile growth cones (GCs) which guide axon elongation during development; secondly, bundles of MTs which form the structural backbones of axons and are required to establish and then maintain axons for an organism's lifetime (i.e. decades in humans). Further important roles are likely to come from the actin cytoskeleton in axon shafts, but this actin has been difficult to visualise and has been widely neglected. Recently, supra-resolution microscopy of mouse neurons delivered a precise template for studies of axonal actin. These studies revealed bundles of short actin filaments arranged into periodically patterned rings which surround the axonal MT bundles, ideal to regulate their dynamics. Notably, we find similar repetitive patterns when using supra-resolution microscopy on neurons of the fruitfly Drosophila, suggesting that these structures are evolutionary conserved and functionally relevant. Such relevance is further supported by our experiments with two classes of genetic and drug manipulations: one class is expected to affect actin in axon shafts, the other to maintain actin rings, but both clearly remove actin from GCs. These treatments have differential effects on axon extension which strongly support a model in which axonal actin has growth-promoting roles. Such a role of axonal actin would introduce novel mechanistic concepts into models of axon growth, thus providing new opportunities to unravel this still unresolved, fundamental problem in neurobiology. Furthermore, our experiments with the two classes of actin manipulations suggest that axonal actin has a second role, which is to maintain axonal MTs. Thus, when MTs are destabilised through specific genetic manipulation, additional removal of axonal actin eliminates their proliferation and axons retract and eventually vanish. This surprising and novel finding likewise opens up new opportunities, and we believe that it will have potential implications not only for axon growth but also for axon degeneration and branching. To turn our pilot data on axonal actin into substantial understanding of axon biology, we will capitalise on the unique genetic and experimental opportunities provided by fly neurons, for which we have 10 years of experience. Thus, we have already investigated ~40 actin and MT regulators of Drosophila, alone or in combinations, during axon growth and GC regulation. This provides us with a solid and unique knowledge base for the research on this project. Here, we will 1) use supra-resolution microscopy in combination with our actin manipulations to functionally validate the model of axonal actin rings, 2) proof the growth-promoting roles using micro-fluid chambers, refined live imaging and traction fore microscopy, 3) unravel the underlying mechanisms by focussing on actin-dependent MT polymerisation and forward sliding of MTs, and 4) demonstrate the relevance of axonal actin in vivo.