The effect of cytochalasin J on kinetochore structure in PtK1 cells is mitotic cycle dependent

Mitotic PtK1 cells were arrested in mitosis with nocodazole to determine the effect of cytochalasin J (CJ) on kinetochore structure in arrested and nocodazole-released cells. In previous studies it was shown that CJ had a more pronounced effect on alteration of kinetochore structure and spindle microtubule (MT) architecture when applied during prophase or prometaphase. In this study, mitotic cells were treated at preanaphase for 10 min with 1 microg/ml nocodazole, or in 1 microg/ml nocodazole and 10 microg/ml CJ to allow for the advancement of the 'mitotic clock'. Thus it can be determined if either changes in the timing of mitosis, the maturation of the kinetochore, and/or the lack of MT connection to the kinetochore affects the ability of CJ to detach or alter the attachment of chromosomes to the developing spindle. Preanaphase cells treated with 1 microg/ml nocodazole for 10 min and released into 10 microg/ml CJ showed significant changes in MT organization and kinetochore structure. MTs nucleated at the centrosome are fragmented and kinetochore structure was significantly altered showing only two laminae with few MTs inserted into this structure. Preanaphase cells treated with 1 microg/ml nocodazole and 10 microg/ml CJ for 10 min and released into 10 microg/ml CJ showed similar, but more pronounced, effects on kinetochore structure and spindle MT organization. We interpret these results to suggest that CJ treatment has a greater effect on MT attachment and kinetochore structure in nocodazole pre-treated cells, where the kinetochore structure is mature and the mitotic cycle has been advanced.