Quantification of Spatial Patterns of Microtubule Transport by Kinesin-1 Head and Tail

The conventional kinesin-1 is a plus-end-directed microtubule-dependent motor protein with distinct motor head, stalk, and tail domains. Along with the motor head, which binds and walks along microtubules in an adenosine 5'-triphosphate (ATP) dependent manner, kinesin also contains a C-terminal microtubule binding tail. Motor-driven collective motility is well characterized using in vitro gliding assays, which show uninterrupted, smooth trajectories of transport. However, gliding assays driven by the full-length Drosophila kinesin-1 with both head and tail resulted in the emergence of spontaneous spatial microtubule patterns and stop-and-go motion. This was reproduced by an equimolar ratio of the active head and passive tail. Here, we describe the detailed protocol to reconstitute these microtubule gliding assays using multiple motor types: the full-length kinesin-1, the motor head or tail, mixtures of both head and tail, and a rigor mutant of the kinesin. We provide details of the approach taken to acquire the image time-series, to then quantify the spatial patterns that result from these motor combinations. Our approach provides a framework to systematically characterize the spatiotemporal effects of molecular motor-driven collective microtubule transport. Key features • This protocol highlights the cloning and expression of two major Drosophila kinesin-1 constructs: the microtubule binding tail and isoleucine-alanine-lysine (IAK)-deleted kinesin-1 full length. • This protocol describes a typical gliding assay setup for the kinesin motor domain, alone as well as in combination with the kinesin tail. • We present a systematic framework for typical gliding assays, including experimental acquisition as well as quantitative analysis of microtubule collective transport. • This protocol gives a quantitative metric for microtubule spatial patterns, which enables systematic analysis of microtubule curvature, both in vitro and in vivo.