STIMULATION OF MICROTUBULE DYNAMIC TURNOVER IN LIVING CELLS TREATED WITH OKADAIC ACID

We have examined the effects of okadaic acid, an inhibitor of protein phosphatases type 1 and 2A, on the dynamic instability behavior of ind ividual microtubules in living cells. Addition of 1 mu M okadaic acid to PtK1 epithelial cells induced ruffling of lamellar regions; after 5 0 min in okadaic acid, many cells were observed to round up. ConfocaI microscopy of okadaic acid-treated cells stained with an antibody to t ubulin showed that microtubules were more densely packed near the peri phery of the rounded cells, and in many cells, a reduction in the dens ity of microtubules near the microtubule-organizing center was observe d. The dynamic behavior of individual microtubules in cells previously injected with rhodamine-labeled tubulin was quantified by tracking in dividual microtubules from image sequences. Microtubule dynamic turnov er was markedly stimulated in cells treated with 1 mu M okadaic acid f or 50-60 min: The average rates of both microtubule growing and shorte ning increased, and the average duration of pause, or attenuation, a p hase in which neither growth nor shortening could be detected, was sig nificantly decreased. Further, okadaic acid induced an approximately t wo-fold increase in the frequency of catastrophe transitions and a thr eefold decrease in the frequency of rescue transitions. Dynamicity, a measure df the net gain and loss of polymer at microtubule plus ends, increased nearly threefold in okadaic acid-treated cells. These result s demonstrate that microtubule turnover is stimulated in okadaic acid- treated cells and suggest that phosphorylation of molecules which inte ract with microtubules may result in increased microtubule dynamic tur nover in vivo. (C) 1996 Wiley-Liss, Inc.