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

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.

Authors:
 [1]; ORCiD logo [2];  [3];  [4];  [1];  [4];  [3]; ORCiD logo [2]; ORCiD logo [1]
  1. Univ. of Rhode Island, Kingston, RI (United States)
  2. Pennsylvania State Univ., University Park, PA (United States)
  3. Worcester Polytechnic Inst., Worcester, MA (United States)
  4. Univ. of Massachusetts, Amherst, MA (United States)
Publication Date:
Research Org.:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1500047
Grant/Contract Number:  
SC0001090
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Tran, Mai L., McCarthy, Thomas W., Sun, Hao, Wu, Shu-Zon, Norris, Joanna H., Bezanilla, Magdalena, Vidali, Luis, Anderson, Charles T., and Roberts, Alison W. Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens. United States: N. p., 2018. Web. doi:10.1038/s41598-017-18994-4.
Tran, Mai L., McCarthy, Thomas W., Sun, Hao, Wu, Shu-Zon, Norris, Joanna H., Bezanilla, Magdalena, Vidali, Luis, Anderson, Charles T., & Roberts, Alison W. Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens. United States. doi:10.1038/s41598-017-18994-4.
Tran, Mai L., McCarthy, Thomas W., Sun, Hao, Wu, Shu-Zon, Norris, Joanna H., Bezanilla, Magdalena, Vidali, Luis, Anderson, Charles T., and Roberts, Alison W. Mon . "Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens". United States. doi:10.1038/s41598-017-18994-4. https://www.osti.gov/servlets/purl/1500047.
@article{osti_1500047,
title = {Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens},
author = {Tran, Mai L. and McCarthy, Thomas W. and Sun, Hao and Wu, Shu-Zon and Norris, Joanna H. and Bezanilla, Magdalena and Vidali, Luis and Anderson, Charles T. and Roberts, Alison W.},
abstractNote = {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.},
doi = {10.1038/s41598-017-18994-4},
journal = {Scientific Reports},
issn = {2045-2322},
number = 1,
volume = 8,
place = {United States},
year = {2018},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Figures / Tables:

Figure 1 Figure 1: Expression of mEGFP-PpCESAs rescue the cognate mutant phenotypes. (a) Transformation of the gametophore defective ppcesa5KO-2 line with Ubi::GFP-PpCESA5 restores wild type gametophore development. (b) Transformation of the ppcesa8KO-5B line, which is characterized by weak midrib birefringence due to reduced secondary cell wall deposition, was partially restored to themore » wild type phenotype by transformation with Act1::GFP-PpCESA8. (c) Quantitative analysis of fluorescence intensity in midribs stained with S4B confirmed partial restoration of the wild type phenotype by expression of Act1::GFP-PpCESA8 the ppcesa8KO-5B line.« less

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