Dynamics of chromosomes and microtubules visualized by multiple-wavelength fluorescence imaging in living mammalian cells: Effects of mitotic inhibitors on cell cycle progression

Background: Mitotic events are accomplished by a coordinated interaction of molecular components within a cell. The continuous observation of specific molecular components in individual living cells provides a unique opportunity to examine the temporal and spatial coordination of these components. In order to be able to observe dynamic events in living mammalian cells, we have developed a computerized fluorescence microscope system. Results: Using this fluorescence microscope system, we observed the dynamic behaviour of chromosomes and microtubules in Living HeLa cells throughout an entire cell cycle. Chromosomes and microtubules were stained with Hoechst 33342 and rhodamine-conjugate tubulin, respectively. Microinjected rhodamine-tubulin was incorporated into microtubules at all stages of the cell cycle. In addition, rhodamine-tubulin stained the centrosomes during the G2 phase of the cell cycle and allowed us to observe centrosome movement. We also examined the effects of mitotic inhibitors on cell cycle progression. In a living cell treated with an inhibitor of type II DNA topoisomerase (ICRF-193) chromosomes formed an aberrant metaphase plate and failed to segregate, However, following mitotic spindle disassembly, the chromosomes decondensed normally. While the cell began to divide into two portions, cell division was eventually aborted and the daughter cells, still connected by a cytoplasmic bridge, rejoined into a single cell, These results demonstrate how a polyploid nucleus is generated in the presence of ICRF-193. On the other hand, microtubule polymerization inhibitors, such as colchicine, nocodazole and vinblastine, arrested the cell in the metaphase with condensed chromosomes; cytochalasin D did not affect the mitotic behaviour of either chromosomes or microtubules. Conclusions: Our observations of living cells demonstrate that the failure of chromosome segregation in and of itself does not prevent an exit from the metaphase, whereas the inhibition of microtubule assembly arrests cells at metaphase, Thus, a metaphase checkpoint is monitored by functional spindle microtubules but not by the completion of chromosome segregation.