Project Summary Tubulin plays fundamental roles in axon formation, maintenance, and dysfunction. Despite this, how tubulin is delivered to axons remains highly controversial, and how tubulin is removed from axons is entirely unknown. This is due to technical challenges in visualizing tubulin movement, that stem from the fact that tubulin is highly abundant in immobile polymers and that when movement occurs, it is extremely slow. We recently overcame similar obstacles to the visualization of spectrin delivery to axons. Here we propose to modify, adapt and validate our methodology in order to generate a new probe for visualizing the axonal delivery and removal of endogenous tubulin, in vivo, with single-axon resolution. Successful completion of this project will reveal how tubulin is delivered and removed from axons, and how delivery and removal are balanced to generate a steady state cytoskeletal mass under different physiological conditions. This knowledge and tools will move the field forward by opening the way for more mechanistic and disease-focused studies. Furthermore, the approach could be adapted to visualize the axonal delivery and removal of other slow axonal transport cargo.