KINASE AND PHOSPHATASE INHIBITORS CAUSE RAPID ALTERATIONS IN MICROTUBULE DYNAMIC INSTABILITY IN LIVING CELLS

To examine whether microtubule dynamic instability can be rapidly regu lated during interphase, we used video-enhanced differential interfere nce contrast (DIG) microscopy to observe individual microtubules at th e periphery of living newt lung epithelial cells. Microtubules were ob served before and after perfusion with either the phosphatase inhibito r okadaic acid or the kinase inhibitors staurosporine or olomoucine. A ddition of these inhibitors caused rapid changes in dynamic instabilit y. Thirty to sixty seconds after perfusion with 0.2-1 mu M okadaic aci d, a 1.5-fold increase in elongation velocity and small increases in c atastrophe and rescue frequencies were observed. In contrast, treatmen t with 40-200 nM staurosporine decreased microtubule elongation and sh ortening velocities similar to 2-fold, and catastrophes were slightly more frequent. Olomoucine, at 100 mu M, had similar effects. Transitio n dynamics were further examined by probabilistic analysis, which show ed that microtubules become more likely to undergo catastrophe as they elongated and more Likely to undergo rescue as they shortened, an eff ect previously called microtubule ''memory.'' This memory effect for c atastrophes was observed in untreated and okadaic acid-treated cells b ut was abolished by staurosporine or olomoucine. In contrast, the memo ry effect for rescue was unaffected by these treatments, suggesting th at catastrophe and rescue proceed via distinct, multistep mechanisms. Overall, these results demonstrate that microtubule assembly regulator s can be altered rapidly by inhibition of either kinases or phosphatas es and suggest that, in the absence of inhibitors, these regulators ex ist in a dynamic equilibrium between phosphorylated and dephosphorylat ed states. (C) 1997 Wiley-Liss, Inc.