Nucleated assembly of mitotic microtubules in living PTK2 cells after release from nocodazole treatment.

The reassembly of microtubules is described in mitotic cells after release from nocodazole-induced block. The formation of microtubules was followed by light microscopic immunocytochemical staining using the PAP method, combined with toluidine blue staining of the chromatin. The light microscopic observations on whole cells were compared with ultrastructural observations on thin sections. This step is essential to ascertain complete destruction of microtubules during the nocodazole treatment and to correlate immunocytochemical staining with the presence of microtubules. Removal of nocodazole (10 or 1 micrograms/ml) after a sufficiently long incubation to induce a complete disappearance of microtubules resulted in the appearance of tubulin staining specifically associated with the centromeres and with one or two isolated points in the cytoplasm. Electron microscopy confirmed that the staining was due to the massive accumulation of small microtubules at the kinetochores and centrosomes. Kinetochore nucleation was seen only in association with condensed metaphase-stage chromosomes and not with the less-condensed prophase chromosomes. In a second type of experiment cells were allowed to enter mitosis in the presence of an incompletely active concentration of nocodazole (0.1 microgram/ml). The construction of the mitotic spindle was arrested; however, short microtubules were assembled at the kinetochores and centrosomes. These experiments demonstrate that in living mitotic PTK2 cells the kinetochores, as well as the centrosomes, exert a nucleating action on tubulin assembly. The further elongation of microtubules after removal of nocodazole was seen to occur preferentially along axes between the centrosomes and the kinetochores. This resulted in the construction of normal metaphases that evolved through anaphase and telophase. We have attempted to formulate a hypothesis that may explain the oriented assembly that seems to be essential in the construction of the spindle.

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