Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes. Hamm-Alvarez, Sarah F., Xinhua Wei, Norbert Berndt, and Maria Runnegar. University of Southern California Center for Liver Diseases and Department of Pharmaceutical Sciences, University of Southern California School of Pharmacy, Los Angeles CA 90033, Department of Medicine, University of Southern California School of Medicine, Los Angeles CA 90033 and Division of Hematology and Oncology, Children's Hospital, Los Angeles CA 90027
APStracts 3:0084C, 1996.
In order to investigate the regulation of microtubule-based vesicle transport and the interphase microtubule array in hepatocytes, we have used okadaic acid and microcystin, two toxins which inhibit serine-threonine protein phosphatases 1 and 2A, to alter cellular phosphorylation. Video-enhanced DIC microscopy analysis revealed that both toxins inhibited the frequency, velocity and run length of microtubule-dependent vesicle movements dose-dependently between 50 -500 nM. At our maximum dose of 500 nM, both toxins significantly decreased protein phosphatase 2A activity (okadaic acid to 45+/-12% and microcystin to 57+/-2%), whereas protein phosphatase 1 was inhibited only by microcystin. Since no additional effects on vesicle movements were caused by microcystin over the changes caused by okadaic acid, these data implicate protein phosphatase 2A in the regulation of microtubule-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the microtubule array, the effects of these toxins on microtubule distribution were examined by immunofluorescence microscopy. Although lower doses of okadaic acid produced no effects, treatment with 500 nM okadaic acid altered microtubule organization and also caused fragmentation and loss of acetylated (stable) microtubules. In contrast, microcystin concentrations up to 500 nM elicited no changes in microtubule organization in general, or in the acetylated (stable) array. From these findings we conclude that inhibition of microtubule-dependent vesicle movement by the protein phosphatase inhibitors, microcystin and okadaic acid, in hepatocytes cannot result from changes or disruption in the microtubule array. Since okadaic acid (an inhibitor of protein phosphatase 2A only in our system) at high doses caused loss of stable microtubules, while microcystin (an inhibitor of both protein phosphatases 1 and 2A) did not, we conclude that the control of the preservation of the stable microtubule array in hepatocytes is complex. Stable microtubules require active protein phosphatase 2A for maintenance, but the disruption of the array through inhibition of protein phosphatase 2A can be prevented if protein phosphatase 1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of microtubule stability. 

Received 3 November 1995; accepted in final form 8 March 1996.
APS Manuscript Number C664-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 27 March 96