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© The Rockefeller University Press, 0021-9525/2000//357 $5.00
The Journal of Cell Biology, Volume 149, Number 2, , 2000 357-368

Mek and Cdc2 Kinase Are Sequentially Required for Golgi Disassembly in Mdck Cells by the Mitotic Xenopus Extracts

^a Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan
^b Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-Ku, Kyoto 606-8502, Japan
^c Department of Physiology, Kansai Medical University, Osaka 570-8506, Japan
^d CREST, Japan Science and Technology Corporation, Japan
Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan.181-564-52-7913181-564-55-7815

mmurata{at}nips.ac.jp

At the onset of mitosis, the Golgi apparatus, which consists of several cisternae, disperses throughout the cell to be partitioned into daughter cells. The molecular mechanisms of this process are now beginning to be understood. To investigate the biochemical requirements and kinetics of mitotic Golgi membrane dynamics in polarized cells, we have reconstituted the disassembly of the Golgi apparatus by introducing Xenopus egg extracts into permeabilized Mardin-Darby canine kidney (MDCK) cells. We used green fluorescence protein (GFP)-tagged galactosyltransferase-expressing MDCK cells to analyze the morphological changes of the Golgi membrane in the semi-intact system. Analyses by fluorescence and electron microscopies showed that the Golgi disassembly can be dissected into two elementary processes morphologically. In the first process, the perinuclear Golgi stacks break into punctate structures, intermediates, which are comprised of mini-stacks of cisternae associating with apical microtubule networks. In the second process, the structures fragment more thoroughly or substantially relocate to the ER. Our analyses further showed that cdc2 kinase and mitogen-activated protein kinase kinase (MAPKK = MEK) are differently involved in these two processes: the first process is mainly regulated by MEK and the second mainly by cdc2.

Key Words: Golgi complex • mitosis • semi-intact cells • GFP • kinase

© 2000 The Rockefeller University Press

Abbreviations used in this paper: BL, butyrolacotone I; ERK, extracellular signal-regulated kinase; GT-GFP, GFP-tagged mouse galactosyltransferase; GFP, green fluorescence protein; IC, Cdc2-activated interphase extracts; IS, MEK-activated interphase extracts; ISC, cdc2-activated interphase extracts; M, mitotic; MEK, mitogen-activated protein kinase kinase; NRK, normal rat kidney; PD, PD98059; PDI, protein disulfide isomerase; r.t., room temperature; SB, SB203580; SLO, streptolysin O; SS, staurosporine; TB, transport buffer; TEM, transmission electron microscopy; TRITC-phalloidin, tetramethylrhodamine B isothiocyanate–labeled phalloidin.

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