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.

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Brachypodium distachyon MAP20 functions in metaxylem pit development and contributes to drought recovery.

Pits are regions in the cell walls of plant tracheary elements that lack secondary walls. Each pit consists of a space within the secondary wall called a pit chamber, and a modified primary wall called the pit membrane. The pit membrane facilitates transport of solutions between vessel cells and restricts embolisms during drought. Here we analyzed the role of an angiosperm-specific TPX2-like microtubule protein MAP20 in pit formation using Brachypodium distachyon as a model system. Live cell imaging was used to analyze the interaction of MAP20 with microtubules and the impact of MAP20 on microtubule dynamics. MAP20-specific antibody was used to study expression and localization of MAP20 in different cell types during vascular bundle development. We used an artificial microRNAs (amiRNA) knockdown approach to determine the function of MAP20. MAP20 is expressed during the late stages of vascular bundle development and localizes around forming pits and under secondary cell wall thickenings in metaxylem cells. MAP20 suppresses microtubule depolymerization; however, unlike the animal TPX2 counterpart, MAP20 does not cooperate with the γ-tubulin ring complex in microtubule nucleation. Knockdown of MAP20 causes bigger pits, thinner pit membranes, perturbed vasculature development, lower reproductive potential and higher drought susceptibility. We conclude that MAP20 may contribute to drought adaptation by modulating pit size and pit membrane thickness in metaxylem.