Skip to main content

Use Endosidin2 to Study Plant Exocytosis and Vacuolar Trafficking

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1789))

Abstract

Membrane trafficking is essential for multiple aspects of plant growth and development. Some small molecules have been widely used to study the mechanisms of membrane trafficking in plants. For example, short-term treatment with small molecules combined with live cell imaging has been shown to be very useful in understanding the dynamic processes of membrane trafficking. Small molecule Endosidin2 (ES2) has been found to target Exo70 protein and inhibit exocytosis and promote vacuolar trafficking in plants. Here we describe the method of using short-term ES2 treatment combined with live cell imaging to study plant exocytosis and vacuolar trafficking in Arabidopsis seedlings expressing a cargo protein PIN2:GFP.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Jackson CL, Casanova JE (2000) Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell Biol 10:60–67

    Article  CAS  PubMed  Google Scholar 

  2. Mansour SJ, Skaug J, Zhao X-H, Giordano J, Scherer SW, Melançon P (1999) P 200 ARF-GEP1: a Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A. Proc Natl Acad Sci U S A 96:7968–7973

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Morinaga N, Adamik R, Moss J, Vaughan M (1999) Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors. J Biol Chem 274:17417–17423

    Article  CAS  PubMed  Google Scholar 

  4. Peyroche A, Paris S, Jackson CL (1996) Nucleotide exchange on ARF mediated by yeast Gea1 protein. Nature 384:479–481. https://doi.org/10.1038/384479a0

    Article  PubMed  CAS  Google Scholar 

  5. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, Gälweiler L, Palme K, Jürgens G (1999) Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286:316–318

    Article  CAS  PubMed  Google Scholar 

  6. Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112:219–230

    Article  CAS  PubMed  Google Scholar 

  7. Grebe M, Xu J, Möbius W, Ueda T, Nakano A, Geuze HJ, Rook MB, Scheres B (2003) Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes. Curr Biol 13:1378–1387

    Article  CAS  PubMed  Google Scholar 

  8. Lippincott-Schwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klausner RD (1991) Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 67:601–616

    Article  CAS  PubMed  Google Scholar 

  9. Paciorek T, Zazímalová E, Ruthardt N, Petrásek J, Stierhof YD, Kleine-Vehn J, Morris DA, Emans N, Jürgens G, Geldner N, Friml J (2005) Auxin inhibits endocytosis and promotes its own efflux from cells. Nature 435:1251–1256. https://doi.org/10.1038/nature03633

    Article  PubMed  CAS  Google Scholar 

  10. Pedrazzini E, Komarova NY, Rentsch D, Vitale A (2013) Traffic routes and signals for the tonoplast. Traffic 14:622–628. https://doi.org/10.1111/tra.12051

    Article  PubMed  CAS  Google Scholar 

  11. Robinson DG, Jiang L, Schumacher K (2008) The endosomal system of plants: charting new and familiar territories. Plant Physiol 147:1482–1492. https://doi.org/10.1104/pp.108.120105

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Zhang C, Brown MQ, van de Ven W, Zhang ZM, Wu B, Young MC, Synek L, Borchardt D, Harrison R, Pan S, Luo N, Huang YM, Ghang YJ, Ung N, Li R, Isley J, Morikis D, Song J, Guo W, Hooley RJ, Chang CE, Yang Z, Zarsky V, Muday GK, Hicks GR, Raikhel NV (2016) Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc Natl Acad Sci U S A 113:E41–E50. https://doi.org/10.1073/pnas.1521248112

    Article  PubMed  CAS  Google Scholar 

  13. Tamura K, Shimada T, Ono E, Tanaka Y, Nagatani A, Higashi SI, Watanabe M, Nishimura M, Hara-Nishimura I (2003) Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J 35:545–555

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Purdue University Provost start-up fund to C.Z.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Chunhua Zhang .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Huang, L., Zhang, C. (2018). Use Endosidin2 to Study Plant Exocytosis and Vacuolar Trafficking. In: Pereira, C. (eds) Plant Vacuolar Trafficking. Methods in Molecular Biology, vol 1789. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7856-4_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7856-4_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7855-7

  • Online ISBN: 978-1-4939-7856-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics