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Visual Mapping of Cell Wall Biosynthesis

  • Yumiko Sakuragi
  • Morten H. H. Nørholm
  • Henrik V. Scheller
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 715)

Abstract

Biosynthesis of pectin and hemicelluloses occurs in the Golgi apparatus and is thought to involve spatial regulations and complex formation of biosynthetic enzymes and proteins. We have demonstrated that a combination of heterologous expression of recombinant proteins tagged with fluorescent proteins and live cell imaging with confocal laser scanning microscopy (CLSM) allows efficient visualization of biosynthetic enzymes and proteins in subcellular compartments. We have also successfully utilized bimolecular fluorescence complementation (BiFC) for in situ visualization of protein–protein interactions of pectin biosynthetic enzymes and for the determination of their membrane topology in the Golgi apparatus.

Key words

Bioimaging Cell wall biosynthesis Uracil-excision cloning Bimolecular fluorescence complementation Protein–protein interaction Subcellular localization Glycosyltransferase Pectin 

Notes

Acknowledgements

This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy; by the Danish Villum Kann Rasmussen Foundation through the VKR centre Pro-active Plants; and by the Danish Agency for Science, Technology and Innovation.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Yumiko Sakuragi
    • 1
  • Morten H. H. Nørholm
    • 1
  • Henrik V. Scheller
    • 2
  1. 1.The Department of Plant Biology and BiotechnologyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Joint Bioenergy Institution, Feedstocks DivisionLawrence Berkeley National LaboratoryEmeryvilleUSA

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