Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens

Mai L Tran,Magdalena Bezanilla,Thomas W Mccarthy,Charles T Anderson,Joanna H Norris,Alison W Roberts,Luis Vidali,Hao Sun,Shu-Zon Wu

doi:10.1038/s41598-017-18994-4

Abstract

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane. This differs from Arabidopsis, in which DCB causes CSC accumulation in the plasma membrane and a different cellulose synthesis inhibitor, isoxaben, clears CSCs from the plasma membrane. In this study, live cell imaging of the moss Physcomitrella patens indicated that DCB and isoxaben have little effect on protonemal growth rates, and that only DCB causes tip rupture. Live cell imaging of mEGFP-PpCESA5 and mEGFP-PpCESA8 showed that DCB and isoxaben substantially reduced CSC movement, but had no measureable effect on CSC density in the plasma membrane. These results suggest that DCB and isoxaben have similar effects on CSC movement in P. patens and Arabidopsis, but have different effects on CSC intracellular trafficking, cell growth and cell integrity in these divergent plant lineages.

Highlights

Cellulose is composed of β-1,4-glucan chains that are hydrogen-bonded together to form microfibrils, which are major contributors to the strength of plant cell walls
Complementation was tested for gametophore phenotypes, both PpCESA5 and PpCESA8 are expressed in protonema[44]
In preparation for testing the effect of cellulose synthesis inhibitors DCB and isoxaben on mEGFP-PpCESA particle movement, we examined their effect on P. patens protonemal filaments by live cell imaging to determine sensitivity relative to the related species Funaria hygrometrica[35]

Summary

Introduction

Cellulose is composed of β-1,4-glucan chains that are hydrogen-bonded together to form microfibrils, which are major contributors to the strength of plant cell walls. These microfibrils are synthesized by Cellulose Synthase (CESA) proteins that reside in the plasma membrane within Cellulose Synthase Complexes (CSCs). The study of CESAs and CSCs entered a new era with the development of methods for tagging CESAs with fluorescent proteins (FPs), facilitating live cell imaging of CSC movement behaviors[3]. Because ppcesa[5] KO and ppcesa[8] KO lines have clear phenotypes, the functionality of mEGFP-PpCESA fusion proteins can be determined by testing transformed lines for complementation of these phenotypes

Methods

Results

Conclusion