The microtubule cytoskeleton plays crucial roles in regulating a vast range of biological processes in different cell types, in particular in neurons that strongly depend on intracellular transport for cargo delivery over long distances. Posttranslational modifications (PTMs) of tubulin recently emerged as modulators of microtubule properties and functions, which is expected to dynamically adapt them to specific cellular functions. Neuronal microtubules are strongly enriched in the PTM polyglutamylation, which was shown to affect the transport of different cargos. Strikingly, perturbed polyglutamylation causes neurodegeneration in mice and humans. How the impact of polyglutamylation on neuronal microtubules translates into such drastic physiological effects is, however, not understood. In my project I aim at determining how polyglutamylation affects physiological functions that are driven by microtubule-based transport in neurons. I will use primary neurons with different levels and patterns of tubulin polyglutamylation and a novel CRISPR/Cas9 technology to endogenously label cargos. Super-resolution microscopy techniques will be applied to precisely determine how this PTM temporally and spatially regulates cargo distribution to their target sites, and how this contributes to neuronal homeostasis. The proposed work will provide a mechanistic understanding of how tubulin PTMs control the physiological roles of the microtubule cytoskeleton at cellular level. This will expand our knowledge of the role of the tubulin code in regulating organism homeostasis, which might provide a mechanistic explanation for its role in neurodegeneration.