ch-TOGp is required for microtubule aster formation in a mammalian mitotic extract.
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.
Dionne
M A
MA
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Sanchez
A
A
Compton
D A
DA
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011108:Polymers / Q000378:metabolism; D008941:Spindle Apparatus / Q000502:physiology*
C454180:CKAP5 protein, human; D008869:Microtubule-Associated Proteins; D011108:Polymers