Monoclonal Antibodies Specific for an Acetylated Form of a-Tubulin Recognize the Antigen in Cilia and Flagella from a Variety of Organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of a-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic o~-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only a-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal a-tubulin isoform acetylated in vivo on the ~-amino group of lysine(s) (L'Hernault, S. W., and J. L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal atubulin, unassembled o~-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic a-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated ~-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of a-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated a-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo. Microtubules are implicated in a variety of cellular functions including mitosis, cytokinesis, intracellular transport, the maintenance of cell shape, and the formation of motile systems such as eukaryotic cilia and flagella (5). In many cases, microtubules involved in different functions are organized into morphologically different arrays. Although the structure of a variety of microtubule frameworks has been described in great detail, the molecular mechanisms that specify the assembly of morphologically.and functionally different microtubule arrays have yet to be determined. The diversity among microtubular structures may be generated by the association of microtubule components with accessory proteins localized in different parts of the cell (37), by the co-polymerization of different aand #-tubulin subunits, or by a combination of these mechanisms. Structurally different tubulin subunits have been identified as the products of different genes (for review, see reference 4) and as the result of posttranslational modifications. At least three modifications oftubulin subunits THE JOURNAL OF CELL BIOLOGY VOLUME 101 DECEMBER 1985 2085-2094 2085 © The Rockefeller University Press 0021-9525/85/12/2085/10 $1.00 on A ril 4, 2017 D ow nladed fom Published December 1, 1985