TPX2 Dynamically Binds to Microtubules and Reduces the Velocity of the Kinesin EG5

Proper assembly of a mitotic spindle requires the concerted efforts of many molecular motors and microtubule associated proteins (MAPs). TPX2 is a multifunctional MAP that binds microtubules, activates the mitotic kinase Aurora A, regulates the localization and activity of the mitotic kinesin Eg5 and is required for the dynein dependent, minus end directed transport of the kinesins Eg5 and Xklp2. Although TPX2 has been shown to regulate Eg5, the mechanism of action is still unknown. To address this question, we studied the behavior of the two molecules using in vitro reconstitution assays. In bulk assays, full length TPX2, purified from insect cells, binds to microtubules with a sub-micromolar affinity. Subtilisin treatment of the microtubules does not prevent this binding, suggesting that the tubulin E-hook is not required for the interaction. In single molecule TIRF assays, mCherry TPX2 dynamically binds to microtubules with an average dwell time of 61.4sec. At lower concentrations, TPX2 binds to both GDP-like and GTP-like microtubules equally well and at higher concentrations TPX2 shows a preferential binding to the GTP-like microtubules. We show that TPX2 slows the unidirectional processive walking of Eg5 molecules on single microtubules.