Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH-terminus of alpha-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells: most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.
Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.
