Taxol-induced assembly and characterization of microtubule proteins from developing brine shrimp (Artemia)

Abstract

Rafiee, P., MacKinlay, S. A. & MacRae, T. H. (1986) Taxol-induced assembly and characterization of microtubule proteins from developing brine shrimp (Artemia). Biochem. Cell Biol. 64, 238-249 Incubation of Artemia cell-free extracts with taxol, followed by centrifugation through sucrose cushions, yielded pellets composed of short, morphologically normal microtubules which exhibited a tendency to fray at their ends. Immunological staining of protein blots with polyclonal or monoclonal antibodies revealed that the major pellet protein is tubulin and that bovine neural tubulin and Artemia tubulin are antigenically distinct. By several criteria, but prinicipally by their taxol-induced coassembly with tubulin, many of the nontubulin pellet proteins are microtubule-associated proteins (MAP). In spite of extensive morphogenesis, hatching, and the eventual resumption of mitosis during development, no new MAP appear, with reduction in the number of MAP after hatching the only observable change in these proteins. We have yet to demonstrate a function for Artemia MAP but have shown that the rate and extent of assembly of Artemia tubulin, which polymerizes readily in vitro in the absence of MAP, are stimulated by bovine MAP. Electrophoretic analysis revealed that the taxol-assembled microtubules were composed of several isotubulins, these being identical to the isoforms in biochemically purified Artemia tubulin. In addition, a new Artemia a-tubulin was observed, and it was shown that the isotubulin population does not change during the period of development examined. Maintenance of identical isotubulin populations in developing organisms for extended periods, which suggests that all tubulins are functional, in concert with the lack of change in tubulin during cell differentiation, runs counter to the proposal that chemically distinct isotubulins are required for assembly of functionally specific microtubules. Rafiee, P., MacKinlay, S. A. & MacRae, T. H. (1986) Taxol-induced assembly and characterization of microtubule proteins from developing brine shrimp (Artemia). Biochem. Cell Biol. 64, 238-249L'incubation d'extraits acellulaires d'Artemia avec le taxol et la centrifugation a travers des coussins de sucrose donnent des culots composCs de microtubules courts, morphologiquement normaux, qui manifestent une tendance 2 1 s'effilocher leurs extrtmitts. La coloration immunologiques des calques (blot) de prottines avec des anticorps polyclonaux ou monoclonaux rtvkle que la principale prottine des culots est la tubuline et que la tubuline neurale bovine et la tubuline d'Artemia sont antigtniquement distinctes. De nombreux critkres, principalement le coassemblage induit par le taxol avec la tubuline, suggkrent que plusieurs des autres prottines des culots sont des protCines assocites aux microtubules (MAP). En dCpit d'une morphogCn2se importante, de l'tclosion et de la reprise tventuelle des mitoses durant le dtveloppement, aucune nouvelle MAP n'apparait malgrC la rkduction du nombre des MAP aprks I'Cclosion, le seul changement o...

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

“…Coomassie blue staining revealed well resolved isoforms of ␣and ␤-tubulin ( Fig. 4a), similar to those in previous work [Rafiee et al, 1986b]. Membranes obtained by blotting of two-dimensional gels were stained by the ECL procedure with antibody 9, yielding a single spot which comigrated with the more acidic isoform of ␤-tubulin (Fig.…”

“…Artemia are unusual because they are capable of extensive postgastrula development in the absence of cell division, producing free-swimming larvae [Rafiee et al, 1986a;Olson and Clegg, 1978). Moreover, brine shrimp have a small number of tubulin genes and products thereof, suggesting that isoform diversity has little influence on microtubule function in Artemia [Xiang and MacRae, 1995;Langdon et al, 1991aLangdon et al, ,b, 19901986b]. Our analysis of microtubule organization in Artemia has now been expanded through study of ␥-tubulin.…”

Production and utilization of detyrosinated tubulin in developingArtemialarvae: evidence for a tubulin-reactive carboxypeptidase

“…Coomassie blue staining of two-dimensional gels and hybridization analysis of restriction digested DNA revealed a limited number of tubulin isoforms and genes in Artemia (Langdon et al 1990(Langdon et al , 1991aRafiee et al 1986;MacRae and Ludueiia 1984). Complexity of the a-tubulin family is augmented by posttranslational changes, including acetylation and detyrosination.…”

Artemia tubulin genes and mRNA

“…We showed, by electrophoresis on two-dimensional gels, that brine shrimp contain three a-and two 8-tubulins. The isotubulins, in contrast to the situation in most eukaryotes, do not change during development (MacRae & Luduena, 1984;Rafiee et al, 1986). To determine the origins of the Artemiu isotubulins we analysed Artemia tubulin genes and mRNA, using cloned tubulin genes from other organisms as probes.…”

“…The 3'-end is the most variable region of tubulin genes (Cleveland & Sullivan, 1985), encoding a peptide domain enriched in negatively charged aminoacyl residues and which acts as a major microtubuleassociated protein (MAP) binding site (Maccioni et al, 1985). The lack of 3'-end hybridization may reflect properties of Artemia tubulin leading, in contrast to neural tubulin, to its ready assembly in the absence of MAP (MacRae & Luduena, 1984;Rafiee et al, 1986).…”