Abstract
Semi-intact cells are cells with plasma membranes that have been permeabilized by bacterial pore-forming toxins or surfactants. The addition of mitotic Xenopus egg extract to semi-intact cells can reconstitute a number of intracellular events that occur specifically at the onset of mitosis. In this chapter, we describe methods for reconstituting the disassembly of the Golgi apparatus by introducing mitotic Xenopus egg extract into semi-intact Mardin-Darby canine kidney (MDCK) cells. The Golgi apparatus was visualized in the cells by expression of green fluorescence protein (GFP)-tagged galactosyltransferase, a marker of trans-Golgi cisternae. Xenopus egg extracts arrested at mitosis or interphase were then prepared and added to the semi-intact MDCK cells. Disassembly of the Golgi apparatus was induced by mitotic Xenopus egg extract. This system can be used not only to elucidate the factors that are involved in the reconstitution process, but also to dissect the process into several elementary steps morphologically and biochemically.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kano, F., Takenaka, K., Yamamoto, A., Nagayama, K., Nishida, E., and Murata, M. (2000) MEK and Cdc2 kinase are sequentially required for Golgi disassembly in MDCK cells by the mitotic Xenopus extracts. J. Cell Biol. 149, 357–368.
Kano, F., Sako, Y., Tagaya, M., Yanagida, T., and Murata, M. (2000) Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semiintact Chinese hamster ovary cells. Mol. Biol. Cell. 11, 3073–3087.
Kano, F., Nagayama, K., and Murata, M. (2000) Reconstitution of the Golgi reassembly process in semi-intact MDCK cells. Biophys. Chem. 84, 261–268.
Bhakdi, S., Tranum-Jensen, J., and Sziegoleit, A. (1985) Mechanism of membrane damage by streptolysin-O. Infect. Immunol. 47, 52–60.
Bhakdi, S., Weller, U., Walev, I., Martin, E., Jonas, D., and Palmer, M. (1993) A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med. Microbiol. Immunol. (Bed.) 182, 167–175.
Lafont, F., Burkhardt, J. K., and Simons, K. (1994) Involvement of microtubule motors in basolateral and apical transport in kidney cells. Nature 372 801–803.
Pimplikar, S. W., Ikonen, E., and Simons, K. (1994) Basolateral protein transport in streptolysin O-permeabilized MDCK cells. J. Cell Biol. 125, 1025–1035.
Wendland, M. and Subramani, S. (1993) Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J. Cell Biol. 120, 675–685.
Murray, A. W. and Kirschner, M. W. (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280.
Chen, R. H. and Murray, A. W. (1997) Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzvmol. 283, 572–584.
Shorter, J. and Warren, G. (2002) Golgi architecture and inheritance. Anna. Rev. Cell Dev. Biol. 18, 379–420.
Rossanese, O. W. and Glick, B. S. (2001) Deconstructing Golgi inheritance. Traffic 2, 589–596.
Schnaar, R. L., Weigel, P. H., Kuhlenschmidt, M. S., Lee, Y. C, and Roseman, S. (1978) Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine. J. Biol. Chem. 253, 7940–7951.
Ahnert-Hilger, G., Bhakdi, S., and Gratzl, M. (1985) Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin. J. Biol. Chem. 260, 12,730–12,734.
Ahnert-Hilger, G., Bader, M. F., Bhakdi, S., and Gratzl, M. (1989) Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: inhibitory effect of tetanus toxin on catecholamine secretion. J. Neurochem. 52, 1751–1758.
Walev, I., Bhakdi, S. C, Hofmann, F., et al. (2001) Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proc. Natl. Acad. Sci. USA 98, 3185–3190.
Hakelien, A. M., Landsverk, H. B., Robl, J. M., Skalhegg, B. S., and Collas, P. (2002) Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat. Biotechnol. 20, 460–466.
Gaustad, K. G., Boquest, A. C, Anderson, B. E., Gerdes, A. M., and Collas, P. (2004) Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes. Biochem. Biophxs. Res. Commun. 314, 420–427.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Kano, F., Takenaka, K., Murata, M. (2006). Reconstitution of Golgi Disassembly by Mitotic Xenopus Egg Extract in Semi-Intact MDCK Cells. In: Liu, X.J. (eds) Xenopus Protocols. Methods in Molecular Biology™, vol 322. Humana Press. https://doi.org/10.1007/978-1-59745-000-3_25
Download citation
DOI: https://doi.org/10.1007/978-1-59745-000-3_25
Publisher Name: Humana Press
Print ISBN: 978-1-58829-362-6
Online ISBN: 978-1-59745-000-3
eBook Packages: Springer Protocols