Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies.

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Abstract
Microtubules were isolated from unfertilized eggs of the sea urchin with the use of the anti-tumor drug taxol. In addition to tubulin, prominent high-MW (205,000-350,000) microtubule-associated proteins (MAP) were identified as well as MAP species of MW 77,000, 100,000 and 120,000. The microtubules were covered with short periodic arms and longer filamentous arms, both classes of which appeared to crosslink the microtubules into bundles. Monoclonal antibodies were prepared to an unfractionated MAP preparation. Clonal hybridoma lines producing antibodies to tubulin and to 4 nontubulin proteins of MW 235,000, 205,000, 105,000 and 37,000 were isolated. All antibodies strongly and specifically stained the mitotic spindle of dividing sea urchin eggs. All 4 of the immunoreactive, nontubulin species behaved as MAP during microtubule isolation. Thus, a variety of sea urchin MAP were identified by biochemical, ultrastructural and immunochemical means. The immunochemical experiments demonstrated that 4 of these proteins are microtubule-associated components of the mitotic spindle. Those proteins that were observed as cross-bridges between the isolated microtubules may be either structural or functional components of the spindle.