Abstract
Cell-free systems can be used to reconstitute complex actin or microtubule-based phenomena. For example, a number of extracts support actin-dependent propulsion of Listeria monocytogenes, whereas Xenopus laevis extracts support formation of a microtubule-based meiotic spindle. Working in vitro opens these complex processes to biochemical dissection. Here, we describe methods to view these in vitro preparations by thin-section electron microscopy.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Evans, L., Mitchison, T., and Kirschner, M. (1985) Influence of the centrosome on the structure of nucleated microtubules. J. Cell Biol. 100, 1185–1191.
Brieher, W. M., Coughlin, M., and Mitchison, T. J. (2004) Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex. J. Cell Biol. 165, 233–242.
Hyman, A., Drechsel, D., Kellogg, D., et al. (1991) Preparation of modified tubulins. Methods Enzymol. 196, 478–485.
Welch, M. D. and Mitchison, T. J. (1998) Purification and assay of the platelet Arp2/3 complex. Methods Enzymol. 298, 52–61.
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 in cell-free extracts. Cell 76, 505–517.
David, V., Gouin, E., Troys, M. V., et al. (1998) Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity approach. J. Cell Sci. 111, 2877–2884.
Noireaux, V., Golsteyn, R. M., Friederich, E., et al. (2000) Growing an actin gel on spherical surfaces. Biophys. J. 78, 1643–1654.
Loisel, T. P., Boujemaa, R., Pantaloni, D., and Carlier, M. F. (1999) Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature (London) 401, 613–616.
Boyles, J., Anderson, L., and Hutcherson, P. (1985) A new fixative for the preservation of actin filaments: fixation of pure actin filament pellets. J. Histochem. Cytochem. 33, 1116–1128.
McDonald, K. (1984) Osmium ferricyanide fixation improves microfilament preservation and membrane visualization in a variety of animal cell types. J. Ultrastruct. Res. 86, 107–118.
Carlemalm, E., Villiger, W., Actaruab, J.-D., and Kellenberger, E. (1986) Low temperature embedding, in Science of Biological Specimen Preparation (Mueller, M., Becker, R. P., Boyde, A., and Wolsewick, J. J., eds.), SEM Inc., AMF O’Hare, Chicago, pp. 24–59.
Murray, A. W. (1991) Cell cycle extracts, Methods Cell Biol. 36, 581–605.
Heidemann, S. R. and McIntosh, J. R. (1980) Visualization of the structural polarity of microtubules. Nature (London) 286, 517–519.
Euteneuer, U., Jackson, W. T., and McIntosh, J. R. (1982) Polarity of spindle microtubules in Haemanthus endosperm. J. Cell Biol. 94, 644–653.
Euteneuer, U. and McIntosh, J. R. (1981) Structural polarity of kinetochore microtubules in PtK1 cells. J. Cell Biol. 89, 338–345.
Heald, R., Tournebize, R., Habermann, A., Karsenti, E., and Hyman, A. (1997) Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J. Cell Biol.138, 615–628.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Coughlin, M., Brieher, W.M., Ohi, R. (2007). Cell-Free Extract Systems and the Cytoskeleton. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology™, vol 369. Humana Press. https://doi.org/10.1007/978-1-59745-294-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-294-6_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-573-6
Online ISBN: 978-1-59745-294-6
eBook Packages: Springer Protocols