Protocol for Reconstituting Adaptor‐Mediated Activation of Full‐Length Kinesin‐1

Kinesin-1 is a member of the kinesin superfamily that plays an essential role in intracellular cargo transport. In the absence of cargo, Kinesin-1 exhibits low motor activity due to autoinhibition. Multiple studies have demonstrated that adaptor proteins, which link cargos to Kinesin-1, can activate Kinesin-1 by releasing the autoinhibition. To elucidate the molecular mechanism of adaptor-mediated activation, in vitro reconstitution of the Kinesin-1 complex has proven to be a powerful approach. We have shown that the binding of an adaptor protein, Nesprin-4, is sufficient to activate Kinesin-1 motility in vitro. Here, we present protocols for the observation and assessment of adaptor-mediated Kinesin-1 activation. Using C. elegans and human Kinesin-1 as examples, we describe steps and critical considerations for preparing the Kinesin-1 complex. We also detail how to mix it with an adaptor protein and visualize the resulting motility using total internal reflection fluorescence (TIRF) microscopy. Finally, we describe analytical methods for assessing motor activation. This protocol will be valuable for future studies aiming to evaluate the activation of Kinesin-1 by known or unknown activator molecules.