Summary
This chapter describes the use of laser-scanning confocal fluorescence microscopy for determining the localization of fluorescently tagged proteins within bacterial cells, discussing the problems caused by the limited resolution of an optical microscope. It also explains a relatively simple method for using fluorescence recovery after photobleaching (FRAP) to observe and quantify the diffusion of fluorescently tagged proteins in bacterial cells. The techniques are illustrated with reference to measurements on green fluorescent protein (GFP)-tagged proteins in Escherichia coli.
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
Reits, E. A. J. and Neefjes, J .J. (2001) From fixed to FRAP: measuring protein mobility and activity in living cells. Nature Cell Biol. 3,145–147.
Blonk, J. C. G., van Aalst, D. H., and Birmingham, J. J. (1993) Fluorescence photobleaching recovery in the confocal scanning light microscope. J. Microsc. 169, 363–374.
Kubitscheck, U., Wedekind, P., and Peters, R. (1994) Lateral diffusion measurements at high spatial resolution by scanning microphotolysis in a confocal microscope. Biophys. J. 67,948–956.
Mullineaux, C. W., Nenninger, A., Ray, N., and Robinson, C. (2006) Diffusion of green fluorescent protein in three cell environments in Escherichia coli. J. Bacteriol. 188,3442–3448.
Elowitz, M. B., Surette, M. G., Wolf, P.-E., Stock, J. B., and Leibler, S. (1999) Protein mobility in the cytoplasm of Escherichia coli. J. Bacteriol. 181,197–203.
Ray, N., Nenninger, A., Mullineaux, C. W., and Robinson, C. (2005) Location and mobility of twin-arginine translocase subunits in the Escherichia coli plasma membrane. J. Biol. Chem. 280,17961–17968.
Ma, X., Ehrhardt, D. W., and Margolin, W. (1996) Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli coli cells by using green fluorescent protein. Proc. Natl. Acad. Sci. USA 93,12998–123003.
Sourjik, V. and Berg, H. C. (2000) Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions. Mol. Microbiol. 37,740–751.
Mashanov, G. I., Tacon, D., Knight, A. E., Peckham, M., and Molloy, J. E. (2003) Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 29,142–152.
Brass, J. M., Higgins, C. F., Foley, M., Rugman, P. A, and Birmingham, J. (1986) Lateral diffusion of proteins in the periplasm of Escherichia coli. J. Bacteriol. 165,787–795 .
Mullineaux, C. W., Tobin, M. J. and Jones, G. R. (1997) Mobility of photosynthetic complexes in thylakoid membranes. Nature 390,421–424.
Cowan, A. E., Koppel, D. E., Setlow, B., and Setlow, P. (2003) A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy. Proc. Nat. Acad. Sci. USA 100,4209–4214.
Cormack, B. P., Valdivia, R. H. and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173,33–38.
Barrett, C. M. L., Ray, N., Thomas, J. D., Robinson, C., and Bolhuis, A. (2003) Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli. Biochem. Biophys. Res. Comm. 304,279–284.
Mullineaux, C. W. (2004) FRAP analysis of photosynthetic membranes. J. Exp. Bot. 55,1207–1211.
Acknowledgments
The E. coli transformants shown in Figs. 1-3 were produced by Colin Robinson (University of Warwick). The method used for estimating Z-resolution was developed in collaboration with Helmut Kirchhoff (University of Münster) during a stay in CWM’s lab funded by the Deutsche Forschungsgemeinschaft. Work in CWM’s laboratory is supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.
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
Mullineaux, C.W. (2007). Localization and Mobility of Bacterial Proteins by Confocal Microscopy and Fluorescence Recovery After Photobleaching. In: van der Giezen, M. (eds) Protein Targeting Protocols. Methods in Molecular Biology™, vol 390. Humana Press. https://doi.org/10.1007/978-1-59745-466-7_1
Download citation
DOI: https://doi.org/10.1007/978-1-59745-466-7_1
Publisher Name: Humana Press
Print ISBN: 978-1-58829-702-0
Online ISBN: 978-1-59745-466-7
eBook Packages: Springer Protocols