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

Abstract

Microtubule arrangement is influenced by gamma-tubulin, a soluble protein of the eukaryotic cell cytosol and a component of microtubule-organizing centers. In this study, affinity purified antibodies to gamma-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 gamma-tubulin, was used with TU-30 (from P. Dráber) to characterize gamma-tubulin in the crustacean, Artemia franciscana. Cell-free protein extracts from Artemia contained gamma-tubulin and it purified with alpha/beta-tubulin through several preparative steps. Probing of Western blots prepared from two-dimensional 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-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 gamma-tubulin. Microtubules in polarized cells of the epidermis were also brightly stained with antibody to alpha-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, gamma-tubulin was seen only rarely at both poles of mitotic spindles in epidermal cells. The complete absence of asters and the apparent lack of gamma-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.

Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications

“…The Western blots contained cell-free extract from bovine brain (MacRae and Ludueña 1984) and synchronized instar II Artemia larvae (Walling et al 1998). The larvae were homogenized in a Dounce homogenizer in Pipes buffer containing a cocktail of proteolytic enzyme inhibitors, followed by centrifugation at 4°C (Campbell et al 1989).…”

“…Protein concentrations were determined by using bovine serum albumin (Sigma) as a standard (Lowry et al 1951). Bovine brain cell-free extract (3 µg protein/lane) and Artemia cell-free extract (10 µg protein/lane) were electrophoresed in 10% SDS polyacrylamide gels (MacRae and Ludueña 1984), blotted onto nitrocellulose (Protan, Schleicher and Schuell, Mandel Scientific, SaintLaurent, Quebec, Canada) and immunostained by the enhanced chemiluminescence technique (Renaissance, DuPont NEM, Boston, Mass., USA; Walling et al 1998).…”