Directional cell expansion requires NIMA-related kinase 6 (NEK6)-mediated cortical microtubule destabilization

Shogo Takatani,Taku Takahashi,Takashi Hotta,Noriyoshi Yagi,Hiroyasu Motose,Yuichiro Takahashi,Takashi Hashimoto,Shinichiro Ozawa

doi:10.1038/s41598-017-08453-5

Abstract

Plant cortical microtubules align perpendicular to the growth axis to determine the direction of cell growth. However, it remains unclear how plant cells form well-organized cortical microtubule arrays in the absence of a centrosome. In this study, we investigated the functions of Arabidopsis NIMA-related kinase 6 (NEK6), which regulates microtubule organization during anisotropic cell expansion. Quantitative analysis of hypocotyl cell growth in the nek6-1 mutant demonstrated that NEK6 suppresses ectopic outgrowth and promotes cell elongation in different regions of the hypocotyl. Loss of NEK6 function led to excessive microtubule waving and distortion, implying that NEK6 suppresses the aberrant cortical microtubules. Live cell imaging showed that NEK6 localizes to the microtubule lattice and to the shrinking plus and minus ends of microtubules. In agreement with this observation, the induced overexpression of NEK6 reduced and disorganized cortical microtubules and suppressed cell elongation. Furthermore, we identified five phosphorylation sites in β-tubulin that serve as substrates for NEK6 in vitro. Alanine substitution of the phosphorylation site Thr166 promoted incorporation of mutant β-tubulin into microtubules. Taken together, these results suggest that NEK6 promotes directional cell growth through phosphorylation of β-tubulin and the resulting destabilization of cortical microtubules.

Highlights

Previous studies showed that cortical microtubules possess self-organizing properties
We identified five β-tubulin target sites that undergo NIMArelated kinase 6 (NEK6)-mediated phosphorylation in vitro, and found that Thr[166] is the most important phosphorylation site for depolymerization of cortical microtubules when expressed in mutant forms in vivo
Ectopic outgrowths formed on elongating epidermal cells during hypocotyl growth (Fig. 1b,c), implying that ectopic outgrowth is due to a defect in the directional growth of epidermal cells

Summary

Introduction

Previous studies showed that cortical microtubules possess self-organizing properties. A loss-of-function Arabidopsis nek[6] mutant exhibits ectopic protrusions and aberrant cortical microtubule arrays in the epidermal cells of the hypocotyl and petiole, indicating that NEK6 suppresses ectopic outgrowth by modulating microtubule organization[22,23,24]. Both the kinase activity and localization to microtubules of NEK6 are required for its function in directional growth[22]. We identified five β-tubulin target sites that undergo NEK6-mediated phosphorylation in vitro, and found that Thr[166] is the most important phosphorylation site for depolymerization of cortical microtubules when expressed in mutant forms in vivo. We propose a novel regulatory mechanism for cortical microtubule arrays in plants in which NEK6 functions to remove aberrant microtubules through phosphorylation of β-tubulin at specific residues

Methods

Results

Conclusion