In-series vs. in-parallel: The role of cargo-motor connectivity in transport by ensembles of cytoskeletal motor proteins

The movement of the cytoskeletal molecular motor proteins dynein and kinesin on microtubule networks facilitates the complex intracellular organization of eukaryotic cells. Ensembles of these motors are a prevalent feature of cytoskeletal transport, indicating possible synergistic effects of their use for intracellular organization. While the force output of individual motors is well studied, the mechanisms governing emergent force generated by multiple motors acting as an ensemble remains unclear. Determining the fundamental mechanisms controlling the interaction and integration of the forces produced by these motors at the nanoscale is essential to understanding the broader system of intracellular transport. Using total internal reflection fluorescence (TIRF) microscopy, we are investigating the role of the spatial arrangement of motors in an ensemble with a particular emphasis on the connectivity between motors and their cargo. Using motors purified from yeast and the nanoscale construction techniques of DNA origami, we create programmable chassis structures in which multiple cytoplasmic dyneins or Kip2 kinesins are arranged with independent, “in parallel” connections to a resistive cargo load or, in contrast, are arranged with a single, “in series” shared cargo connection. We expect the use of these chassis will allow us to determine the mechanisms behind the structural integration of the piconewton forces produced by cytoskeletal molecular motors within ensembles, ultimately providing insight on how the individual connectivity of motors to one another and their cargo affects load-sharing, force integration, and the resultant emergent motility of the motor ensemble.