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...
Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.
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