Studying plus-end tracking at single molecule resolution using TIRF microscopy

The highly dynamic microtubule plus-ends are key sites of regulation that impact the organization and function of the microtubule cytoskeleton. Much of this regulation is performed by the microtubule plus-end tracking (+TIP) family of proteins. +TIPs are a structurally diverse group of proteins that bind to and track with growing microtubule plus-ends in cells. +TIPs regulate microtubule dynamics as well as mediate interactions between microtubule tips and other cellular structures. Most +TIPs can directly bind to microtubules in vitro; however, the mechanisms for their plus-end specificity are not fully understood. Cellular studies of +TIP activity are complicated by the fact that members of the +TIP family of proteins interact with each other to form higher-order protein assemblies. Development of an in vitro system, using minimal components, to study +TIP activity is therefore critical to unequivocally understand the behavior of individual +TIP proteins. Coupled with single molecule imaging, this system provides a powerful tool to study the molecular properties that are important for +TIP function. In this chapter, we describe a detailed protocol for in vitro reconstitution of +TIP activity at single molecule resolution using total internal reflection fluorescence microscopy.