Identification and characterization of a land-plant specific microtubule nucleation factor, MACET4

Abstract

An embryophyte-specific protein MACERATOR (MACET4) binds microtubules in vitro and in vivo; promotes microtubule polymerization at sub-critical tubulin concentrations; decreases the lag phase in microtubule bulk polymerization assays; and co-localizes with microtubule nucleation sites. Furthermore, MACET4 forms oligomers, which induce aster formation in vitro in the manner that is similar to centrosomes and TPX2. MACET4 expresses during cell division and accumulates at the microtubule nucleation regions of the plant-specific cytokinetic microtubule array, the phragmoplast. We found that MACET4 localises to the preprophase band and the cortical division zone, but not the spindle. MACET4 appears as cytoplasmic foci in vivo and forms octamers in vitro. Transient expression in tobacco leaf pavement cells results in labelling of shrinking plus and minus ends. MACET4 facilitates microtubule depolymerization by increasing the frequency of catastrophes in vivo and by suppressing rescues in vitro. Microtubules formed in the presence of MACET4 in vitro are shorter. Accordingly, MACET4 knockdown results in longer phragmoplasts. We conclude that direct activity of MACET4 is promoting nucleation. MACET4 also restricts microtubule elongation most likely by depleting the pool of free tubulin.