"Intracellular transport plays a key role in many cellular processes. Cells rely on a well-regulated, two-way transport system that consists of molecular motors and cytoskeletal filaments for their correct functioning. In nerve cells, breakdown of transport is tightly linked to neurodegenerative diseases.Understanding what goes wrong with intracellular transport during disease requires knowledge of how motors work inside cells to transport cargo. While much effort has been devoted to understanding motor mediated intracellular transport, our knowledge of motor-cargo and motor-microtubule interactions in the cellular environment is still very rudimentary. The small size of motors, the complex architecture of their microtubule tracks, and the inherently dynamic nature of transport have made these interactions virtually inaccessible to observation in living cells until recently. With new advances in fluorescence microscopy, in particular with the development of ground-breaking methods that surpass the diffraction limit, we can finally begin to address this challenging problem.In this ambitious proposal we will study, at an unprecedented level of detail, the nanoscale organization and stoichiometry of motor proteins on their cargo and interactions of motor proteins with microtubules in living cells. We will achieve this goal by using a multidisciplinary approach that combines cutting-edge biophysical tools such as single particle tracking, quantitative single molecule counting and super-resolution nanoscopy with novel genetic manipulation and fluorescence labeling methods. Using these unique set of tools we will unravel the molecular mechanisms that regulate motors to achieve efficient transport. The results obtained in this proposal will provide, for the first time, a detailed picture of how motors function inside living cells, greatly enhancing our knowledge of a fundamental cell biological process and of its implications in disease."