PROJECT SUMMARY/ABSTRACT Damage to the peripheral nervous system (PNS) can be incurred through a wide spectrum of conditions including trauma, metabolic imbalances, and chemotherapy. Neuronal injuries occur when their long and fragile processes (i.e. axons) are damaged, for example by stretching or severing. Compared to PNS neurons, the regenerative capacity of injured central nervous system (CNS) neurons is extremely limited, typically resulting in life-long disability. Regenerative growth of axons can occur in the injured peripheral nervous system, however, restoration of function is often incomplete, resulting in loss of motor and sensory function and frequently the development of neuropathic pain. Regeneration of an injured axon is thought to involve changes in microtubule dynamics and post- translational modifications (PTMs). Microtubules are dynamic polymers of α,β-tubulin and are the core structural element of axons and dendrites. Microtubules also serve as tracks for molecular motor proteins (kinesin, cytoplasmic dynein) that transport various cargos between synaptic terminals and the neuronal cell soma. Following injury, microtubules undergo rapid fragmentation, a process known as Wallerian degeneration. In developing neurons and during axon regeneration, microtubules in the neuronal growth cone and axon shaft play key roles in axon guidance and axon elongation. Regulation of MT function is largely achieved by the differential expression of α- and β-tubulin isotypes, and by PTMs of tubulin subunits within the microtubule polymer. Studies of microtubule isotypes and PTMs inherently rely on antibodies that require fixation and permeabilization of the cell or tissue, which severely limits our understanding of the spatiotemporal component of tubulin PTMs. We have developed a pipeline that allows us to generate live-cell probes for tubulin PTMs. Here, we will generate probes for tubulin isotypes and PTMs relevant to neuronal degeneration and regeneration and use them to map the spatiotemporal patterns of changes to the microtubule cytoskeleton following injury to the PNS and CNS.