Advertisement

Immunogold Labeling and Electron Tomography of Plant Endosomes

  • Alexandra Chanoca
  • Marisa S. OteguiEmail author
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1209)

Abstract

High-resolution imaging of endosomal compartments and associated organelles can be achieved using state-of-the-art electron microscopy techniques, such as the combination of cryofixation/freeze-substitution for sample processing and electron tomography for three-dimensional (3D) analysis. This chapter deals with the main steps associated with these imaging techniques: selection of samples suitable for studying plant endosomes, sample preparation by high-pressure freezing/freeze-substitution, and electron tomography of plastic sections. In addition, immunogold approaches for identification of subcellular localization of endosomal and cargo proteins are also discussed.

Key words

Electron tomography Endosomes Trans-Golgi network Multivesicular body Cryofixation Immunolabeling 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

This work was supported by NSF grant MCB1157824 to M.S.O.

References

  1. 1.
    Reyes FC, Buono R, Otegui MS (2011) Plant endosomal trafficking pathways. Curr Opin Plant Biol 14:666–673PubMedCrossRefGoogle Scholar
  2. 2.
    Dettmer J, Hong-Hermesdorf A, Stierhof YD, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18:715–730PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Lam SK, Siu CL, Hillmer S, Jang S, An G, Robinson DG, Jiang L (2007) Rice SCAMP1 defines clathrin-coated, trans-Golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells. Plant Cell 19:296–319PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Viotti C, Bubeck J, Stierhof YD, Krebs M, Langhans M, van den Berg W, van Dongen W, Richter S, Geldner N, Takano J, Jurgens G, de Vries SC, Robinson DG, Schumacher K (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22:1344–1357PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Kang BH, Nielsen E, Preuss ML, Mastronarde D, Staehelin LA (2011) Electron tomography of RabA4b- and PI-4Kbeta1-labeled trans Golgi network compartments in Arabidopsis. Traffic 12:313–329PubMedCrossRefGoogle Scholar
  6. 6.
    Chow C-M, Neto H, Foucart C, Moore I (2008) Rab-A2 and Rab-A3 GTPases define a trans-Golgi endosomal membrane domain in Arabidopsis that contributes substantially to the cell plate. Plant Cell 20:101–123PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA (2006) The proteolytic processing of seed storage proteins in Arabidopsis embryo cells starts in the multivesicular bodies. Plant Cell 18:2567–2581PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Hanton S, Matheson L, Chatre L, Rossi M, Brandizzi F (2007) Post-Golgi protein traffic in the plant secretory pathway. Plant Cell Rep 26:1431–1438PubMedCrossRefGoogle Scholar
  9. 9.
    Henne William M, Buchkovich Nicholas J, Emr Scott D (2011) The ESCRT pathway. Developmental Cell 21:77–91PubMedCrossRefGoogle Scholar
  10. 10.
    Haas TJ, Sliwinski MK, Martínez DE, Preuss M, Ebine K, Ueda T, Nielsen E, Odorizzi G, Otegui MS (2007) The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19:1295–1312PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Spitzer C, Reyes FC, Buono R, Sliwinski MK, Haas TJ, Otegui MS (2009) The ESCRT-related CHMP1A and B proteins mediate multivesicular body sorting of auxin carriers in Arabidopsis and are required for plant development. Plant Cell 21:749–766PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Scheuring D, Viotti C, Kruger F, Kunzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K (2011) Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis. Plant Cell 23:3463–3481PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Mastronarde DN (1997) Dual-axis tomography: an approach with alignment methods that preserve resolution. J Struct Biol 120:343–352PubMedCrossRefGoogle Scholar
  14. 14.
    Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51PubMedCrossRefGoogle Scholar
  15. 15.
    Mastronarde DN (2008) Correction for non-perpendicularity of beam and tilt axis in tomographic reconstructions with the IMOD package. J Microsc 230:212–217PubMedCrossRefGoogle Scholar
  16. 16.
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76PubMedCrossRefGoogle Scholar
  17. 17.
    Donohoe BS, Kang BH, Staehelin LA (2007) Identification and characterization of COPIa- and COPIb-type vesicle classes associated with plant and algal Golgi. Proc Natl Acad Sci U S A 104:163–168PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Giddings TH (2003) Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples. J Microsc 212: 53–61PubMedCrossRefGoogle Scholar
  19. 19.
    McEwen BF, Frank J (2001) Electron tomographic and other approaches for imaging molecular machines. Curr Opin Neurobiol 11:594–600PubMedCrossRefGoogle Scholar
  20. 20.
    Marsh BJ (2005) Lessons from tomographic studies of the mammalian Golgi. Biochim Biophys Acta 1744:273–292PubMedCrossRefGoogle Scholar
  21. 21.
    Otegui MS, Austin JR II (2007) Visualization of membrane-cytoskeletal interactions during plant cytokinesis. Methods Cell Biol 79:221–240PubMedCrossRefGoogle Scholar
  22. 22.
    O’Toole ET, Giddings TH, JR., Dutcher SK (2007) Understanding microtubule organizing centers by comparing mutant and wild-type structures with electron tomography. In: McIntosh JR (ed) Cellular Electron Tomography, vol 79. Methods Cell Biol, 2007/03/01 edn. Elsevier, pp 125–143Google Scholar
  23. 23.
    Otegui MS, Staehelin LA (2004) Electron tomographic analysis of post-meiotic cytokinesis during pollen development in Arabidopsis thaliana. Planta 218:501–515PubMedCrossRefGoogle Scholar
  24. 24.
    Segui-Simarro JM, Austin JR II, White EA, Staehelin LA (2004) Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing. Plant Cell 16:836–856PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Ladinsky MS, Mastronarde DN, McIntosh JR, Howell KE, Staehelin LA (1999) Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J Cell Biol 144: 1135–1149PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Otegui MS, Mastronarde DN, Kang BH, Bednarek SY, Staehelin LA (2001) Three-dimensional analysis of syncytial-type cell plates during endosperm cellularization visualized by high resolution electron tomography. Plant Cell 13:2033–2051PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of BotanyUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.Department of GeneticsUniversity of Wisconsin-MadisonMadisonUSA

Personalised recommendations

Cite protocol

Buy options