Abstract
Microtubules induced to polymerize with taxol in a mammalian mitotic extract organize into aster-like arrays in a centrosome-independent process that is driven by microtubule motors and structural proteins. These microtubule asters accurately reflect the noncentrosomal aspects of mitotic spindle pole formation. We show here that colonic-hepatic tumor-overexpressed gene (ch-TOGp) is an abundant component of these asters. We have prepared ch-TOGp-specific antibodies and show by immunodepletion that ch-TOGp is required for microtubule aster assembly. Microtubule polymerization is severely inhibited in the absence of ch-TOGp, and silver stain analysis of the ch-TOGp immunoprecipitate indicates that it is not present in a preformed complex and is the only protein removed from the extract during immunodepletion. Furthermore, the reduction in microtubule polymerization efficiency in the absence of ch-TOGp is dependent on ATP. These results demonstrate that ch-TOGp is a major constituent of microtubule asters assembled in a mammalian mitotic extract and that it is required for robust microtubule polymerization in an ATP-dependent manner in this system even though taxol is present. These data, coupled with biochemical and genetic data derived from analysis of ch-TOGp-related proteins in other organisms, indicate that ch-TOGp is a key factor regulating microtubule dynamics during mitosis.
Highlights
In somatic cells, the number of spindle poles is determined by the number of centrosomes
This result is consistent with the prior characterization of homologues of ch-TOGp acting to stimulate microtubule polymerization (19 –21) and organize the mitotic spindle [22, 35, 38, 39] and indicates that ch-TOGp plays an important role in regulating microtubule dynamics during spindle assembly in mitosis
To identify new protein components of microtubule asters assembled in a mammalian mitotic extract, we separated the asters from the soluble components of the extract by centrifugation through 20% sucrose
Summary
The number of spindle poles is determined by the number of centrosomes. Indirect immunofluorescence microscopy showed ch-TOGp to be distributed throughout the microtubule asters assembled in our cell-free extract (data not shown), and immunoblot analyses of the soluble and insoluble fractions obtained following microtubule aster assembly showed that ch-TOGp is highly enriched in the insoluble, astercontaining pellet fraction (Fig. 2C).
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