NANOMOLAR CONCENTRATIONS OF NOCODAZOLE ALTER MICROTUBULE DYNAMIC INSTABILITY IN-VIVO AND IN-VITRO

Previous studies demonstrated that nanomolar concentrations of nocodaz ole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-in duced stabilization of microtubules. We tested this hypothesis by exam ining the effects of nanomolar concentrations of nocodazole on microtu bule dynamic instability in interphase cells and in vitro with purifie d brain tubulin. Newt lung epithelial cell microtubules were visualize d by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentrat ion from 4 to 400 nM. Microtubules showed a loss of the two-state beha vior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and s hortening velocities, increased catastrophe, and an overall decrease i n microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microsc opy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechan isms responsible for changes in dynamic instability, we examined the e ffects of 4 nM to 12 mu M nocodazole on the assembly of purified tubul in from axoneme seeds. At both microtubule plus and minus ends, perfus ion with nocodazole resulted in a dose-dependent decrease in elongatio n and shortening velocities, increase in pause duration and catastroph e frequency, and decrease in rescue frequency. These effects, which re sult in an overall decrease in microtubule turnover after nocodazole t reatment, suggest that the mitotic block observed is due to a reductio n in microtubule dynamic turnover. In addition, the in vitro results a re similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increase s tubulin GTPase activity, we propose that nocodazole acts by generati ng TuD subunits that then alter dynamic instability.