Characterization of γ-tubulin inArtemia: Isoform composition and spatial distribution in polarized cells of the larval epidermis

Abstract

Microtubule arrangement is influenced by γ‐tubulin, a soluble protein of the eukaryotic cell cytosol and a component of microtubule‐organizing centers. In this study, affinity purified antibodies to γ‐tubulin were prepared and their specificity demonstrated by immunostaining 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 γ‐tubulin, was used with TU‐30 (from P. Dráber) to characterize γ‐tubulin in the crustacean, Artemia franciscana. Cell‐free protein extracts from Artemia contained γ‐tubulin and it purified with α/β‐tubulin through several preparative steps. Probing of Western blots prepared from two‐dimensional gels yielded a single isoform of γ‐tubulin in Artemia with a pI of about 5.6. Immunostaining with TAT, a general antibody to α‐tubulin, demonstrated that Artemia possess two morphological types of immune blood cells (hemocytes) with distinctive microtubule arrays. Both the compact spherical hemocytes and the flatter, spreading cells exhibited fluorescent dots, often in pairs, when labelled with antibodies to γ‐tubulin. Microtubules in polarized cells of the epidermis were also brightly stained with antibody to α‐tubulin, revealing interphase arrangements, anastral mitotic spindles and midbodies. Antibody 9 and TU‐30 gave punctate staining patterns in interphase epidermal cell layers and they occasionally labelled midbodies. Unexpectedly, γ‐tubulin was seen only rarely at both poles of mitotic spindles in epidermal cells. The complete absence of asters and the apparent lack of γ‐tubulin at all but a small number of poles indicate that formation and structure of the mitotic spindle in epidermal cells of Artemia are unusual. Cell Motil. Cytoskeleton 40:331–341, 1998. © 1998 Wiley‐Liss, Inc.