A gamma-tubulin antibody against a plant peptide sequence localises to cell division-specific microtubule arrays and organelles in plants

Micron. 2001 Oct;32(7):671-8. doi: 10.1016/s0968-4328(00)00064-0.

Abstract

Gamma tubulin (gamma-tubulin) is involved in microtubule initiation in the eukaryotes. In animal cells it is localised to centrosomes and to other, non-centrosomal sites of microtubule initiation. In addition, cytoplasmic complexes containing gamma-tubulin (gamma-TuRCs; gamma-somes) have been described, which are multiprotein complexes involved in microtubule initiation. Most localisations of gamma-tubulin in plants have previously been achieved using an antibody directed towards a conserved peptide sequence found in animal cells, showing co-localisation with all microtubule arrays throughout the cell cycle. Because different antibodies may give various patterns of subcellular localisation, in the present study we raised a polyclonal antibody ('Hayley') to the plant peptide sequence EDFATQGGDRKDVFFY (bold letters indicate plant-specific amino acids) to further investigate the subcellular distribution in plants. Immunoblotting using wheat root tip protein extracts revealed a 58 kDagamma-tubulin-like peptide as has been described before. Immunofluorescence microscopy of wheat root-tip cells, however, revealed localisation of gamma-tubulin to a subset of mitotic microtubule arrays and the cytokinetic phragmoplast, but not to interphase cortical arrays or the preprophase band of microtubules. This lack of labelling may be caused by a restriction of antibody access during interphase, but more likely by a cell division-specific conformational change in the gamma-tubulin molecule. Our antibody also gave an organelle-like labelling, not described before, which may represent storage forms or precursors of gamma-tubulin, perhaps related to plastid-based microtubule initiation in hepatics and hornworts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / immunology*
  • Cell Division / physiology
  • Centrosome / chemistry
  • Insect Proteins*
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Microtubule-Associated Proteins / analysis
  • Microtubule-Associated Proteins / chemistry
  • Microtubule-Associated Proteins / immunology
  • Microtubule-Organizing Center / chemistry
  • Microtubule-Organizing Center / ultrastructure
  • Microtubules / chemistry*
  • Microtubules / ultrastructure
  • Molecular Sequence Data
  • Organelles / chemistry*
  • Organelles / ultrastructure
  • Peptides / chemistry
  • Plant Roots / chemistry*
  • Plant Roots / ultrastructure
  • Rabbits
  • Triticum / chemistry*
  • Tubulin / analysis*
  • Tubulin / chemistry
  • Tubulin / immunology*

Substances

  • Antibodies
  • Insect Proteins
  • Microtubule-Associated Proteins
  • Peptides
  • Tubulin
  • microtubule-organizing center-associated polypeptide, insect