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Plasmodesmata Ultrastructure Determination Using Electron Tomography.

Jules D Petit1,2, Marie Glavier1, Lysiane Brocard3

  • 1Laboratoire de Biogenèse Membranaire, UMR5200 CNRS, Université de Bordeaux, Villenave d'Ornon, France.

Methods in Molecular Biology (Clifton, N.J.)
|March 29, 2022
PubMed
Summary
This summary is machine-generated.

This protocol details high-resolution transmission electron tomograms of plant plasmodesmata (PD). It enables detailed 3D visualization of these crucial cell-to-cell communication channels.

Keywords:
CryofixationElectron tomographyPlasmodesmataSegmentationUltrastructure

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Area of Science:

  • Plant Biology
  • Cell Biology
  • Microscopy

Background:

  • Plasmodesmata (PD) are vital intercellular channels for plant communication, growth, and stress response.
  • Their small size and complex structure hinder detailed 3D visualization and functional correlation.
  • Previous electron microscopy studies revealed PD diversity but lacked nanometer-resolution 3D detail.

Purpose of the Study:

  • To provide a detailed protocol for generating high-resolution transmission electron tomograms of plant plasmodesmata (PD).
  • To enable detailed three-dimensional structural analysis of PD at nanometer resolution.
  • To facilitate linking PD structure to cellular function.

Main Methods:

  • High-pressure cryofixation and cryo-substitution for sample preparation.
  • Ultramicrotomy for sectioning and filmed grid preparation.
  • Transmission electron tomography (TET) data acquisition and IMOD-based tomogram reconstruction.

Main Results:

  • A complete, step-by-step protocol for producing high-resolution transmission electron tomograms of PD.
  • Detailed methods for sample preparation, sectioning, tilt-series acquisition, and tomogram reconstruction.
  • Guidelines for segmenting reconstructed tomograms for further analysis.

Conclusions:

  • This protocol overcomes the limitations of visualizing PD structure in 3D at nanometer resolution.
  • It provides a powerful tool for detailed structural analysis of PD and understanding their role in plant biology.
  • Enables advanced research into PD function and cell-cell communication.