Autoregulation and dual stepping mode of MYA2, an Arabidopsis myosin XI responsible for cytoplasmic streaming

Takeshi Haraguchi,Kohji Ito,Nao Shoji,Kohei Yoshimura,Takamitsu Morikawa,Motoki Tominaga,Mitsuhiro Iwaki,Atsushi Kimura

doi:10.1038/s41598-022-07047-0

Abstract

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining total internal reflection fluorescence microscopy (TIRFM) and the fluorescence imaging with one-nanometer accuracy (FIONA) method, revealed that the MYA2 processively moved on actin with three different step sizes: − 28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes; hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2–3 pN). These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

Highlights

Arabidopsis thaliana has 13 genes belonging to the myosin XI family
Arabidopsis myosin XI is composed of a motor domain (MD), a neck domain with six IQ motifs to which calmodulin or calmodulin-like proteins bind, a coiled-coil region, and a globular tail domain (GTD) (Fig. 1a)[9,19,20]
Exogenous addition of the GTD inhibited the actin-activated ATPase activities of heavy meromyosin which lacked GTD (HMM) and 6IQ in a concentration-dependent manner, but not that of MD, suggesting that GTD inhibits ATPase activity via the IQ motif (Fig. 2b). Such GTD suppression was not observed in full-length tobacco 175 kDa myosin XI purified from tobacco BY-2 c ells[11]

Summary

Introduction

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in Arabidopsis cells. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD). Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2–3 pN) These results indicated that MYA2 has different transport properties from that of the myosin V responsible for vesicle transport in animal cells. Such properties may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells. We found that MYA2 has different motile and regulatory properties than those of tobacco 175 kDa myosin XI

Methods

Results

Conclusion