Reconstitution and quantification of dynamic microtubule end tracking in vitro using TIRF microscopy

Abstract

Several microtubule-associated proteins localize in living cells selectively to an extended region at the growing microtubule plus ends. Over the last years, these plus-end-tracking proteins, also called +TIPs, have attracted considerable interest because they are involved in a large variety of essential intracellular processes. GFP-labeled versions of EB proteins are also often used as markers for intracellular microtubule organization and dynamics. The mechanism of selective +TIP binding to the end region of growing microtubule was unknown. Recently, the phenomenon of end tracking was reconstituted in vitro from purified proteins, which allowed the identification of EB proteins as the minimal core of the plus-end-tracking system and the dissection of the molecular mechanism of end tracking by these proteins. This in vitro reconstitution has started to be widely used for several +TIPs and promises to provide mechanistic insight into the functioning of the dynamic +TIP network at growing microtubule ends. Here, we describe the purification of EB1 and CLIP-170, the total internal reflection fluorescence microscopy assay to observe dynamic end tracking in vitro, and the quantitative analysis of fluorescent +TIP comet shape and of single +TIP molecule turnover at growing microtubule ends.