Abstract
It has long been apparent that the three cytoskeletal systems of eukaryotic cells, microtubules, f-actin, and intermediate filaments, act in a cooperative fashion, both to achieve their characteristic distributions within cells (1–3), and to execute a variety of complex cellular phenomena (4). However, defining the nature of interactions between the different systems has proven difficult. These difficulties stem from several different features of the three cytoskeletal systems. The filamentous nature of the systems makes it difficult to apply standard biochemical approaches, since most such approaches cannot distinguish between specific interactions and those that result from fortuitous entanglement. Similarly, because all three systems are very abundant in the typical cell, and because all three systems are to greater or lesser degrees dynamic, simultaneous imaging of the different systems in living cells may be less than informative. Further, in most cell types, it is difficult, if not impossible to adequately preserve all three systems in the same preparation using standard immunofluorescence approaches. For example, in Xenopus oocytes and eggs, entirely different fixation protocols are required for visualization of microtubules (5), f-actin (6), and intermediate filaments (7).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Gurland G. and Gundersen G. G. (1995) Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts. J. Cell Biol. 131, 1275–1290.
Waterman-Storer C. M. and Salmon E. D. (1997) Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influence micro-tubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling. J. Cell Biol. 139, 417–434.
Yoon M., Moir R. D., Prahlad V., and Goldman R. D. (1998) Motile properties of vimentin intermediate filament networks in living cells. J. Cell. Biol. 143, 147–157.
Waterman-Storer C. M. and Salmon E. D. (1999) Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr. Opin. Cell Biol. 11, 61–67.
Gard D. L. (1991) Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy. Dev. Biol. 143, 346–362.
Roeder A. D. and Gard D. L. (1994) Confocal microscopy of F-actin distribution in Xenopus oocytes. Zygote 2, 111–124.
Gard D. L. (1997) The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon f-actin and microtubules. Dev. Biol. 184, 95–114.
Canman J. C. and Bement W. M. (1997) Microtubules suppress actomyosin-based cortical flow in Xenopus oocytes. J. Cell Sci. 110, 1907–1917.
Bement W. M., Mandato C. A., and Kirsch M. N. (1999) Wound-induced assembly and closure of an actomyosin purse string in Xenopus oocytes. In Press. Curr. Biol.
Sider J. R., Mandato C. A., Weber K. L., Zandy A. J., Beach D., Finst R. J., Skobel J., and Bement W. M. (1999) Direct observation of microtubule-F-actin interaction in cell free lysates. J. Cell Sci. 112, 1947–1956.
Murray A. W. (1991) Cell cycle extracts. Methods Cell Biol. 36, 581–605.
Leno G. H. and Laskey R. A. (1991) DNA replication in cell-free extracts from Xenopus laevis. Methods Cell Biol. 36, 561–79.
Weber K. L. and Bement W. M. (1998) Investigation of the interaction between cytoskeletal elements in cell-free Xenopus lysates. Mol. Biol. Cell. 9, 39a.
Stearns T. and Kirschner M. (1994) In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 76, 623–637.
Belmont L. D., Hyman A. A., Sawin K. E., and Mitchison T. J. (1990) Real time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62, 579–589.
Theriot J. A., Rosenblatt J., Portnoy D. A., Goldschmidt-Clermont P. J., and Mitchison T. J. (1994) Involvement of profilin in the actin-based motility of L. monocytogenes in cells and cell free extracts. Cell 76, 505–517.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Mandato, C.A., Weber, *.K.L., Zandy, *.A.J., Keating, T.J., Bement, W.M. (2001). XenopusEgg Extracts as a Model System for Analysis of Microtubule, Actin Filament, and Intermediate Filament Interactions. In: Gavin, R.H. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology™, vol 161. Humana Press. https://doi.org/10.1385/1-59259-051-9:229
Download citation
DOI: https://doi.org/10.1385/1-59259-051-9:229
Publisher Name: Humana Press
Print ISBN: 978-0-89603-771-7
Online ISBN: 978-1-59259-051-3
eBook Packages: Springer Protocols