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Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking

  • Franck Delavoie
  • Cathy Royer
  • Stéphane Gasman
  • Nicolas Vitale
  • Sylvette Chasserot-GolazEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2233)

Abstract

To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.

Key words

Plasma membrane Transmission electron microscopy Protein distribution Lipid distribution Electron tomography Exocytotic site 

Notes

Acknowledgments

We acknowledge the microscopy facilities of  the Plateforme de microscopie électronique METi (Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS 31062, Toulouse, France) and the municipal slaughterhouse of Haguenau (France) to provide bovine adrenal glands. This work was supported by CNRS, Université de Strasbourg, Inserm, and FRM.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Franck Delavoie
    • 1
  • Cathy Royer
    • 2
  • Stéphane Gasman
    • 3
  • Nicolas Vitale
    • 3
  • Sylvette Chasserot-Golaz
    • 3
    Email author
  1. 1.Centre National de la Recherche Scientifique, Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie IntégrativeUniversité de ToulouseToulouseFrance
  2. 2.Plateforme Imagerie In Vitro, Neuropôle de StrasbourgStrasbourgFrance
  3. 3.Centre National de la Recherche ScientifiqueUniversité de Strasbourg, Institut des Neurosciences Cellulaires et IntégrativesStrasbourgFrance

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