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.
Dixit
Ram
R
Department of Biology, Washington University, St. Louis, Missouri 63130, USA.
Ross
Jennifer L
JL
D000818:Animals; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D021621:Imaging, Three-Dimensional; D057230:Limit of Detection; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D055503:Protein Multimerization; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*
D005456:Fluorescent Dyes