Identification of a minus end-specific microtubule-associated protein located at the mitotic poles in cultured mammalian cells

A mitosis-specific centrosomal component was studied with a human autoantibody, SP-H, which immunostained mitotic poles and interphase nuclei, and a single polypeptide with an apparent molecular mass of 200 to 230 kDa in various lines of cultured cells. Early mitotic PtK₁ cells treated with 10 pg/ml taxol contained short bundles of parallel microtubules around the nuclei and cell periphery. At the time of nuclear envelope breakdown, the nuclear staining by SP-H disappeared, and the antigen relocated a tone end of the parallel microtubules. Determination of the microtubule polarity demonstrated that the peripheral bundles of microtubules were arranged with their minus ends directed to the cell periphery, and the SP-H antigen was specifically localized at this end. Parallel microtubules were further rearranged first into a fan-like shape, and then into completely radial structures as observed by De Brabander et al. (Int. Rev. Cytol. 101, 215-274 (1986)). The SP-H antigen was always detected at the minus end domain of such microtubule-containing structures during the transformation process. When microtubules were depolymerized by nocodazole treatment, the SP-H antigen appeared as discrete cytoplasmic foci, suggesting that the antigen may self-associate, forming multimeric structures. The antigen in mitotic HeLa cell extracts co-sedimented in vitro with exogenous brain microtubules. The microtubule-associated SP-H antigen was insensitive to ATP extraction, but was removed from microtubules by treatment with 0.5 M NaCl. Thus the 200 to 230 kDa centrosomal component could be a novel microtubule-associated protein with affinity for the minus end of microtubules, and it might play an essential role in the organization of spindle poles during mitosis.