Initiation of ensemble kinesin-3 motility is regulated by the rigidity of cargo-motor attachment

Intracellular cargo transport is powered by molecular motors that move on their respective filamentous tracks. A key component in this process is the tether between cargo and motor, which is often connected by long slender coiled-coils. Several studies have identified mechanisms that regulate cargo transport and can be broadly categorized into regulation of the motor ATPase activity by autoinhibition, cargo adapters and modifications in the cytoskeletal tracks. The regulatory effects of cargo-motor linkers have been described in kinesin-3 subfamily motors. However, the effects of cargo-motor linker rigidity on ensemble cargo transport has not been explored. Here we have built a DNA origami scaffold, which can be tethered with multiple kinesin-3 motors using either single or double-stranded DNA linkages, mimicking rigid versus flexible cargo-motor linkages. Using this system, we show that regardless of the motor numbers attached to the cargo, only linkers with a lesser degree of freedom allow motors to engage with microtubule tracks. Together, our work identifies that the rigidity of cargo-motor linkages influences motor motility. This opens up the possibilities to identify new factors that can influence the rigidity of cargo-motor linkages that in turn can regulate intracellular cargo transport.