Transmission Electron Microscopy and Tomography on Plasma Membrane Sheets to Study Secretory Docking
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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 wordsPlasma membrane Transmission electron microscopy Protein distribution Lipid distribution Electron tomography Exocytotic site
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.
- 9.Gabel M, Delavoie F, Royer C, Tahouly T, Gasman S, Bader MF, Vitale N, Chasserot-Golaz S (2019) Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells. Biochim Biophys Acta, Mol Cell Res 2019:1207–1217Google Scholar