Molecular Mechanisms of Axon Growth and Regeneration

Neurons are cells with unique and extremely polarized morphologies. The axon allows communication between the cell soma and the distantly located synaptic terminal and can extend up to one meter in humans. This exceptional cellular structure therefore has specialized biological processes dedicated to its growth, maintenance and regeneration. The structure that carries out axon elongation during development and regeneration is the growth cone, which features a cytoskeletal structure that is both highly dynamic yet consistent in overall organization. The growth cone coordinates a leading edge of dynamic actin with the microtubules of the growing axon, enabling directed outgrowth while maintaining the structural integrity of the axon. In the axon, microtubules are oriented uniformly with their plus-ends facing away from the cell body, providing directionality for cargo transport streams. Axonal transport is critical during growth and after the axon has reached its target to ensure proper distribution of organelles and other necessary components of the axonal biology machinery. Axon transport is especially important because the axon is capable of independently carrying out tasks ranging from protein synthesis to synaptic transmission. Therefore a thorough understanding of microtubule biology and axon transport is critical for understanding how axons grow during development and regeneration. I first investigated the role of a neuron-specific molecular motor adaptor, JIP3, in axon elongation. JIP3 had been previously demonstrated to bind both the anterograde motor kinesin-1 and the retrograde motor complex dynein/dynactin, generating cargo-specific directional transport in the axon. Furthermore, our lab had also demonstrated that JIP3 was capable of binding directly and activating dimeric kinesin heavy chain (KHC) for motility. While the function of dimeric KHC is still debated, tetrameric kinesin-1 is known as the primary anterograde motor in axons. I therefore investigated the role of JIP3 in regulating tetrameric kinesin-1 motility, and the functional significance of this interaction in axons. I used a total internal reflection fluorescence (TIRF)-based single molecule imaging assay to demonstrate that JIP3 binds to the kinesin-1 tetramer in at least a 2:1 ratio. I also used this TIRF assay to show that JIP3 binding to the cargo-adapting kinesin light chain (KLC) activated tetrameric kinesin-1 for microtubule binding, while JIP3 binding to KHC activated tetrameric kinesin-1 motility along microtubules. Furthermore, while the JIP3/KLC interaction is dispensable for axon growth and regeneration in neurons in vitro, the JIP3/KHC interaction is necessary for both these activities. This work demonstrated that a single molecule can activate tetrameric kinesin-1 for microtubule binding and motility. Furthermore, this work demonstrated that regulation of molecular motor activity supports axon growth and regeneration. I next investigated the small GTPase Ran and its role in axon elongation. Ran is best known for its canonical function as a nuclear transport protein, but it can also stabilize microtubules during mitotic spindle formation and is located in the axon. While Ran has a proposed role in regulating a retrograde injury signaling complex in adult axons, it is unknown whether Ran also regulates cytoskeletal dynamics in developing axons. I used Ran knockdown to demonstrate that Ran promotes axon elongation on growth-promoting substrates, and restricts axon elongation on growth-inhibiting substrates. This effect is at least partially mediated by microtubule dynamics, as microtubule imaging using a cell-permeable pan-tubulin marker showed decreased microtubule dynamics in Ran knockdown cells on growth-promoting substrates. Furthermore, I demonstrated that a Ran activating protein, RanBP10, is enriched in growth cones and promotes axon elongation, and that both of these phenomena are Ran-dependent. This work raises the exciting possibility that Ran controls microtubule dynamics in the growth cone, distinct from its canonical role in nucleocytoplasmic trafficking