Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence...

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.