Kinesin-1 is a tetramer composed of two ATP-dependent motor-bearing heavy chains (Kif5) and two light chains (KLC). When not engaged in transport, it is kept in an autoinhibited state. While transport of many cellular cargoes requires the regulatory KLCs that can recognise various adaptor proteins ultimately resulting in relief of autoinhibition and anterograde movement along MTs, not all cargoes are transported in a KLC-dependent manner. Mitochondria, for example, whose distribution within cells is necessary to match local energy demands, are transported by kinesin-1 independently of KLCs. The latter in fact negatively impact the association between Kif5s and the Milton-Miro complex required for mitochondrial anchoring. These observations raise questions about possible mechanisms of KLC sequestration that enable KLC-independent transport along MTs. Over the years the Steiner group (UniPD and King’s College London) has contributed important structural discoveries that defined general and isoform-specific mechanisms of cargo recognition by KLCs. Using cellular and in vivo work, the UniMI unit has recently shown that dynamin-related protein 1 (Drp1), a key player in the process of mitochondrial and peroxisomal division, hijacks KLCs promoting mitochondrial transport along MTs in skeletal muscle of Drp1-overexpressing mice. The UniBA unit provides expertise in molecular docking, dynamics, and virtual screening to investigate existing and novel KLC small-molecule modulators. Overall, this project will employ an integrated and comprehensive approach ranging from biophysics to a mouse model to dissect the molecular mechanisms by which Drp1 hijacks KLCs ultimately affecting mitochondrial motility. This project will significantly advance the fundamental understanding of kinesin-1 regulation and its functional modulation with relevance to the healthy and disease states.