The microtubule-nucleating factor MACERATOR tethers AUGMIN7 to microtubules and governs phragmoplast architecture.

The plant cytokinetic microtubule array, called the phragmoplast, exhibits higher microtubule dynamics in its center (midzone) than at the periphery (distal zone). This behavior is known as the axial asymmetry. Despite being a major characteristic of the phragmoplast, little is known about regulators of this phenomenon. Here we address the role of microtubule nucleation in axial asymmetry by characterizing MACERATOR (MACET) proteins in Arabidopsis thaliana and Nicotiana benthamiana with a combination of genetic, biochemical, and live-cell imaging assays, using photo-convertible microtubule probes, and modeling. MACET paralogs accumulate at the shrinking microtubule ends and decrease the tubulin OFF rate. Loss of MACET4 and MACET5 function abrogates axial asymmetry by suppressing microtubule dynamicity in the midzone. MACET4 also narrows the microtubule nucleation angle at the phragmoplast leading edge and functions as a microtubule tethering factor for AUGMIN COMPLEX SUBUNIT 7 (AUG7). The macet4 macet5 double mutant shows diminished clustering of AUG7 in the phragmoplast distal zone. Knockout of AUG7 does not affect MACET4 localization, axial asymmetry, or microtubule nucleation angle, but increases phragmoplast length and slows down phragmoplast expansion. The mce4-1 mce5 aug7-1 triple knockout is not viable. Experimental data and modeling demonstrate that microtubule nucleation factors regulate phragmoplast architecture and axial asymmetry directly by generating new microtubules and indirectly by modulating the abundance of free tubulin.

Quantification of Microtubule-Bundling Activity of MAPs Using TIRF Microscopy.

Cross-linking of microtubules by microtubule-associated proteins (MAPs) results in the formation of microtubule bundles. It has been shown that a majority of microtubules in interphase plant cells are bundled. Bundling can contribute to maintaining structural stability and sustaining spatial organization of microtubule arrays. While bundling can be readily detected by an electron or fluorescent microscope, quantifying this activity remains technically challenging. Here we describe a method for quantifying microtubule-bundling in vitro using green and red stable microtubules. Furthermore, this method distinguishes between different types of microtubule-microtubule interactions: bundling, annealing, and branching. Our technique can be used to compare bundling activity of different MAPs and generate parameters for modeling their contribution to organization and dynamics of microtubule arrays.