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Identification of Plasmodesmal Localization Sequences in Proteins In Planta
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A protocol to expand plant nuclei.

Ivona Kubalová1, Markéta Schmidt Černohorská2, Martina Huranová2

  • 1Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany.

Methods in Cell Biology
|January 22, 2021
PubMed
Summary
This summary is machine-generated.

Expansion microscopy (ExM) physically expands plant nuclei, enabling detailed visualization of chromatin structures. This technique, applied to barley nuclei, achieved high resolution, revealing ultrastructural insights into centromeric chromatin.

Keywords:
CENH3Expansion microscopyIsolated nucleiPlant chromatinRabl configurationStructured illumination microscopy

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Area of Science:

  • Plant Biology
  • Microscopy Techniques
  • Molecular Biology

Background:

  • Conventional light microscopy resolution is limited by light diffraction.
  • Expansion microscopy (ExM) is a novel technique that overcomes diffraction limits by physically expanding specimens.
  • ExM utilizes polymer chemistry and hydrogel swelling to enhance sample size.

Purpose of the Study:

  • To provide a detailed protocol for expansion microscopy of isolated plant nuclei.
  • To visualize plant chromatin ultrastructure and proteins using ExM.
  • To investigate the potential of ExM for studying plant chromatin dynamics and function.

Main Methods:

  • Isolation of barley (Hordeum vulgare) root tip nuclei.
  • Expansion microscopy (ExM) protocol involving hydrogel embedding and swelling (~4.2x expansion).
  • Indirect immunolabeling of centromere-specific histone H3 variant (CENH3) and DAPI staining.
  • Imaging with conventional wide-field (WF) microscopy and structured illumination microscopy (SIM).

Main Results:

  • ExM enabled detection of DAPI-labeled chromatin structures (~50-60nm resolution) with WF microscopy.
  • SIM imaging achieved higher resolution (~25-35nm), revealing finer chromatin details.
  • Visualization of CENH3 in expanded barley nuclei provided insights into centromeric chromatin organization.
  • Some distortion of centromeric chromatin ultrastructure was observed after expansion.

Conclusions:

  • Expansion microscopy is a viable method for enhancing the resolution of plant nuclear structures.
  • ExM combined with super-resolution microscopy offers new possibilities for studying plant chromatin.
  • The protocol facilitates detailed investigation of plant chromatin ultrastructure, dynamics, and function.