Quantitative characterization of filament dynamics by single-molecule lifetime measurements

Single-molecule measurements provide a powerful tool for obtaining quantitative information on biological process. However, properly interpreting these measurements can be challenging. Here we present a framework for understanding the turnover dynamics of single molecules in cytoskeletal filaments. We show that the single-molecule lifetime distribution is equivalent to the distribution of first-passage times of the filaments ends. We calculate expected lifetime distributions for a number of models of filament dynamics. We also describe methods to measure single-molecule turnover of tubulin molecules in Xenopus egg extract spindles. We show that analyzing the shape of the lifetime distribution gives insight into the mechanism of microtubule turnover in these spindles and can be used to quantify the effect of molecular perturbations on microtubule stability. Similar methods should prove useful in studying cytoskeletal filaments in other contexts in vitro and in vivo.