Pironetin Preferentially Inhibits Microtubule Minus-End Growth and Enables Polarity Marking

Microtubules are dynamic cytoskeletal polymers and major targets of anticancer drugs that modulate their assembly dynamics. Here, we characterized the effects of two tubulin-targeting ligands, pironetin and maytansine, on microtubule dynamics in vitro and developed a method for generating polarity-marked microtubules based on asymmetric growth inhibition. Using total internal reflection fluorescence (TIRF) microscopy, kymograph analysis, and kinesin-1 gliding assays, we found that maytansine efficiently suppressed polymerization at both microtubule ends, whereas pironetin preferentially inhibited minus-end growth, while exerting only a minor effect on plus-end polymerization. This pronounced end-selectivity enabled the generation of two-color segmented microtubules with reliably identifiable polarity. After optimization, the pironetin-based protocol achieved performance comparable to that of the established N-ethylmaleimide (NEM)-based method while preserving the biochemical integrity of the microtubule lattice owing to the well-defined binding site of pironetin on α-tubulin. Our results identify pironetin as a promising tool for generating polarity-marked microtubules for in vitro studies of microtubule dynamics and interactions with end-specific microtubule-associated proteins.