Regulation of MACERATOR during Cytokinesis

Alyssa M. Parish

doi:10.7273/000007078

Plant cytokinesis is driven by the expansion of a plant-specific microtubule array the phragmoplast. The mechanism behind phragmoplast expansion remains poorly understood. Furthermore, the majority of regulatory proteins of microtubules in the phragmoplast are conserved in animals, which use a different cytokinetic mechanism. Limited information about plant-specific regulators of microtubules in the phragmoplast hinders our progress in understanding the expansion process. One of the very few plant-specific microtubule associated proteins in the phragmoplast is MACERATOR4 (MACET4). It has been shown that MACET4 promotes microtubule nucleation in vitro. Our group demonstrated that MACET4 is expressed through all stages of mitosis but binds microtubules during cytokinesis. The major goal of this research is to determine how MACET4 activity is regulated during cytokinesis and why association of MACET4 with microtubules of the mitotic spindle is inhibited. The central hypothesis of this work is MACET4 activity is regulated through interaction with other proteins and by phosphorylation. MACET4 phosphorylation sites are identified using open source prediction tools. Phospho-mimetic mutants were designed for testing the effect of phosphorylation on MACET4 activity in vitro and in vivo. To determine whether MACET4 is regulated through interaction with other proteins, pull-down assays and affinity chromatography were performed and results were analyzed by tandem mass spectrometry. The interactions were verified by bimolecular fluorescence complementation and microtubule biochemical assays. MACET4 interactions were verified with AUGMIN7 and γ-tubulin. The MACET4 protein interactions were analyzed in planta by crossing established Arabidopsis mutant lines. Novel marker lines were utilized to study both MOZART1a and KATANIN in the phragmoplast. The genetic interaction between Arabidopsis MACET4 and MACET5 with CLASP and AUGMIN7 were analyzed by crossing clasp-1 and/or augmin7-1 into macet4-1macet5. This work identified MACET4 as a tethering factor of AUGMIN7 subunit, part of the microtubule nucleation factor Augmin Complex. Additionally, the phosphorylation sites responsible for microtubule binding of MACET4 were identified. The experimental data presented here demonstrates that phosphorylation regulates MACET4 in cell division and reveals the role of MACET4 and MACET5 in branching microtubule nucleation during plant cytokinesis. In conclusion, this research contributes to advancing our understanding of plant-specific cytokinetic mechanisms and the role of microtubule-associated proteins in phragmoplast expansion.

Regulation of MACERATOR during Cytokinesis

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