Microtubule orientation and spacing within bundles is critical for long-range kinesin-1 motility

Cells rely on active transport to quickly organize cellular cargo. How cells regulate transport is not fully understood. One proposed mechanism is that motor activity could be altered through the architecture of the cytoskeleton. This mechanism is supported by the fact that the cytoskeletal network is tightly regulated in cells and filament polarity within networks dictates motor directionality. For instance, axons contain bundles of parallel microtubules and all cargos with the same motor species will move in the same direction. It is not clear how other types of networks, such as antiparallel bundles in dendrites, can regulate motor transport. To understand how the organization of microtubules within bundles can regulate transport, we studied kinesin-1 motility on three bundle types: random-polarity bundles that are close-packed, parallel polarity bundles, and antiparallel polarity bundles that are spaced apart. We find that close-packed bundles inhibit motor motion, while parallel arrays support unidirectional motion. Spacing the microtubules with microtubule-associated proteins enhances run lengths. Our results indicate that microtubule bundle architecture dictates the motion of single motors and could have effects on cargo transport.