Macromolecular crowding acts as a physical regulator of intracellular transport

Eukaryotic cell processes depend on molecular motors to transport cargo along cytoskeletal filaments, and the response of these mechanoenzymes to external forces shapes their cellular function. These responses have been largely mapped out in dilute, in vitro media. The cytosol, however, is host to a high concentration of macromolecules, and this crowding can alter protein conformation, binding rates, reaction kinetics and therefore motor function. Here, we use live-cell and single-molecule imaging and optical tweezer force measurements to uncover the consequences of macromolecular crowding on cargo transport by kinesin-1 motors. Surprisingly, we find that crowding significantly slows transport by teams of motors, while having no effect on single-motor velocity. We find that this emergent property of kinesin teams results from the increased sensitivity of the individual motors to hindering load when in a crowded medium. We explain this increased sensitivity using a model where entropic forces due to crowding push the two kinesin heads together into a more compact configuration when the motor is poised to take a step. Our results highlight the importance of motor–motor interactions in cargo transport, explain the long-observed variability of cargo velocity and suggest the use of crowding as a control parameter to study kinesin’s mechanochemical cycle.