MAP65 coordinate microtubule growth during bundle formation.

Microtubules (MTs) are highly dynamical structures that play a crucial role in cell physiology. In cooperation with microtubule-associated proteins (MAPs), MTs form bundles endowing cells with specific mechanisms to control their shape or generate forces. Whether the dynamics of MTs is affected by the lateral connections that MAPs make between MTs during bundle formation is still under debate. Using in vitro reconstitution of MT bundling, we analyzed the dynamics of MT bundles generated by two plant MAP65 (MAP65-1/4), MAP65-1 being the plant ortholog of vertebrate PRC1 and yeast Ase1. MAP65-1/4 limit the amplitude of MT bundle depolymerization and increase the elongation phases. The subsequent sustained elongation of bundles is governed by the coordination of MT growth, so that MT ends come in close vicinity. We develop a model based on the assumption that both MAP65-1/4 block MT depolymerization. Model simulations reveal that rescue frequencies are higher between parallel than between anti-parallel MTs. In consequence the polarity of bundled MTs by MAP65 controls the amplitude of bundle's growth. Our results illustrate how MAP-induced MT-bundling, which is finely tuned by MT polarity, robustly coordinates MT elongation within bundles.

Arabidopsis kinetochore fiber-associated MAP65-4 cross-links microtubules and promotes microtubule bundle elongation.

The acentrosomal plant mitotic spindle is uniquely structured in that it lacks opposing centrosomes at its poles and is equipped with a connective preprophase band that regulates the spatial framework for spindle orientation and mobility. These features are supported by specialized microtubule-associated proteins and motors. Here, we show that Arabidopsis thaliana MAP65-4, a non-motor microtubule associated protein (MAP) that belongs to the evolutionarily conserved MAP65 family, specifically associates with the forming mitotic spindle during prophase and with the kinetochore fibers from prometaphase to the end of anaphase. In vitro, MAP65-4 induces microtubule (MT) bundling through the formation of cross-bridges between adjacent MTs both in polar and antipolar orientations. The association of MAP65-4 with an MT bundle is concomitant with its elongation. Furthermore, MAP65-4 modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, and thereby supports the progressive lengthening of MT bundles over time. These properties are in line with its role of initiating kinetochore fibers during prospindle formation.