CHARACTERIZATION OF GAMMA-TUBULIN IN ARTEMIA - ISOFORM COMPOSITION AND SPATIAL-DISTRIBUTION IN POLARIZED CELLS OF THE LARVAL EPIDERMIS

Microtubule arrangement is influenced by gamma-tubulin, a soluble prot ein of the eukaryotic cell cytosol and a component of microtubule-orga nizing centers. In this study, affinity purified antibodies to gamma-t ubulin were prepared and their specificity demonstrated by immunostain ing of Western blots and in competitive ELISAs. When employed to label mouse fibroblasts, one or two brightly stained dots appeared in each cell, a pattern characteristic of centrosomes. Antibody 9, raised to a conserved amino-terminal peptide of gamma-tubulin, was used with TU-3 0 (from P. Draber) to characterize gamma-tubulin in the crustacean, Ar temia franciscana. Cell-free protein extracts from Artemia contained g amma-tubulin and it purified with alpha/beta-tubulin through several p reparative steps. Probing of Western blots prepared from two-dimension al gels yielded a single isoform of gamma-tubulin in Artemia with a pI of about 5.6. Immunostaining with TAT, a general antibody to alpha-tu bulin, demonstrated that Artemia possess two morphological types of im mune blood cells (hemocytes) with distinctive microtubule arrays. Both the compact spherical hemocytes and the flatter, spreading cells exhi bited fluorescent dots, often in pairs, when labelled with antibodies to gamma-tubulin. Microtubules in polarized cells of the epidermis wer e also brightly stained with antibody to alpha-tubulin, revealing inte rphase arrangements, anastral mitotic spindles and midbodies. Antibody 9 and TU-30 gave punctate staining patterns in interphase epidermal c ell layers and they occasionally labelled midbodies. Unexpectedly, gam ma-tubulin was seen only rarely at both poles of mitotic spindles in e pidermal cells. The complete absence of asters and the apparent lack o f gamma-tubulin at all but a small number of poles indicate that forma tion and structure of the mitotic spindle in epidermal cells of Artemi a are unusual. Cell Motil. Cytoskeleton 40:331-341, 1998. (C) 1998 Wil ey-Liss, Inc.