Stimulation of microtubule dynamic turnover in living cells treated with okadaic acid

Abstract

We have examined the effects of okadaic acid, an inhibitor of protein phosphatases type 1 and 2A, on the dynamic instability behavior of individual microtubules in living cells. Addition of 1 microM okadaic acid to PtK1 epithelial cells induced ruffling of lamellar regions; after 50 min in okadaic acid, many cells were observed to round up. Confocal microscopy of okadaic acid-treated cells stained with an antibody to tubulin showed that microtubules were more densely packed near the periphery of the rounded cells, and in many cells, a reduction in the density of microtubules near the microtubule-organizing center was observed. The dynamic behavior of individual microtubules in cells previously injected with rhodamine-labeled tubulin was quantified by tracking individual microtubules from image sequences. Microtubule dynamic turnover was markedly stimulated in cells treated with 1 microM okadaic acid for 50-60 min: The average rates of both microtubule growing and shortening increased, and the average duration of pause, or attenuation, a phase in which neither growth nor shortening could be detected, was significantly decreased. Further, okadaic acid induced an approximately twofold increase in the frequency of catastrophe transitions and a threefold decrease in the frequency of rescue transitions. Dynamicity, a measure of the net gain and loss of polymer at microtubule plus ends, increased nearly threefold in okadaic acid-treated cells. These results demonstrate that microtubule turnover is stimulated in okadaic acid-treated cells and suggest that phosphorylation of molecules which interact with microtubules may result in increased microtubule dynamic turnover in vivo.