Characterization of Cytokinetic Mutants Using Small Fluorescent Probes

Methods Mol Biol. 2016;1370:199-208. doi: 10.1007/978-1-4939-3142-2_15.

Abstract

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

Keywords: Cell plate; Cell wall; Cytokinesis; DNA; Fluorescent probes; Lipids; Mutants.

MeSH terms

  • Arabidopsis / cytology*
  • Arabidopsis / genetics
  • Arabidopsis / ultrastructure
  • Cell Wall / genetics
  • Cell Wall / ultrastructure
  • Cytokinesis*
  • DNA, Plant / analysis*
  • DNA, Plant / genetics
  • Fluorescent Dyes / analysis*
  • Indoles / analysis
  • Microscopy, Fluorescence / methods
  • Mutation
  • Propidium / analysis
  • Pyridinium Compounds / analysis
  • Quaternary Ammonium Compounds / analysis
  • Staining and Labeling / methods*

Substances

  • DNA, Plant
  • FM 4-64
  • Fluorescent Dyes
  • Indoles
  • Pyridinium Compounds
  • Quaternary Ammonium Compounds
  • Propidium
  • DAPI