Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes

Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.