Protocol to reconstitute motor protein clustering on vesicle membranes using a DNA scaffold-based system in vitro

Cytoskeletal motor proteins drive long-range intracellular transport, but how their organization on vesicle membranes affects motility remains poorly understood. Here, we present a protocol to reconstitute kinesin-1 clustering on synthetic liposomes using a DNA-based scaffold. We describe steps for functionalizing liposomes with kinesin-1, inducing DNA-mediated motor clusters, and analyzing liposome motility on surface-immobilized microtubules. This protocol enables quantitative investigation of motor organization in vesicle transport and is adaptable to other cytoskeletal motors. For complete details on the use and execution of this protocol, please refer to Jiang et al. 1 • Steps for assembling and isolating kinesin-functionalized liposomes • Procedures for inducing and verifying motor clustering on liposome surfaces • Guidance on quantifying motor number and comparing motility of clustered motors Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Cytoskeletal motor proteins drive long-range intracellular transport, but how their organization on vesicle membranes affects motility remains poorly understood. Here, we present a protocol to reconstitute kinesin-1 clustering on synthetic liposomes using a DNA-based scaffold. We describe steps for functionalizing liposomes with kinesin-1, inducing DNA-mediated motor clusters, and analyzing liposome motility on surface-immobilized microtubules. This protocol enables quantitative investigation of motor organization in vesicle transport and is adaptable to other cytoskeletal motors.