Reconstitution of Neuronal Motor Traffic on Septin-Associated Microtubules

Reconstitution of intracellular transport in cell-free in vitro assays enables the understanding and dissection of the molecular mechanisms that underlie membrane traffic. Using total internal reflection fluorescence (TIRF) microscopy and microtubules, which are immobilized to a functionalized glass surface, the kinetic properties of single kinesin molecules can be imaged and analyzed in the presence or absence of microtubule-associated proteins. Here, we describe methods for the in vitro reconstitution of the motility of the neuronal kinesin motor KIF1A on microtubules associated with heteromeric septin (SEPT2/6/7) complexes. This method can be adapted for various neuronal septin complexes and kinesin motors, leading to new insights into the spatial regulation of neuronal membrane traffic by microtubule-associated septins.