Neurons are the building blocks of the brain. The ability of neurons to receive, process and transmit information depends on their polarized organization into axons and dendrites. To build such a highly polarized cell, cellular components synthesized in the cell body are differentially transported to either axons or dendrites. Polarized transport is driven by three families of cytoskeletal motor proteins, which can walk in different directions over the actin or microtubule cytoskeleton. Many subfamilies of motor proteins exist, but how each of these motor proteins contributes to selective cargo delivery is unknown.I have recently developed an approach to probe specific motor activity inside cells, which revealed that many microtubule-based motors selectively target axons. However, the molecular mechanisms behind this remarkable selectivity are unknown. In addition, it is well-established that most cargos are transported by a combination of different motors, but how the activity of different types of motors on the same cargo is integrated has remained unclear.The aim of this proposal is to understand how motor proteins navigate the neuronal cytoskeleton. We will take a multidisciplinary approach and combine neurobiology, molecular engineering, advanced microscopy, and mathematical modelling to study the origin of motor selectivity as well as the collective activity of dissimilar motor teams. We will employ and expand our unique methodology to: 1) Study how the spatial organization and post-translational modifications of the microtubule cytoskeleton facilitate selective transport. 2) Perform well-controlled intracellular multi-motor assays to understand the functional interplay between different types of motors.Successful achievement of these objectives will uncover key mechanisms of polarized transport, which will be relevant for understanding transport-associated neurodegenerative diseases.