Abstract
Fluorescence recovery after photobleaching (FRAP) is a microscopy-based technique that can be used to ask how lipid rafts impact protein and lipid diffusion in cells. This chapter, describes how to perform FRAP measurements of putative raft and nonraft proteins and lipids using a confocal microscope. Methods have been outlined for (1) transfecting cells with plasmids encoding for the expression of green fluorescent protein-tagged proteins, (2) labeling cells with fluorescent lipid analogs or with the lipid-binding toxin cholera toxin B-subunit, (3) depleting and loading cholesterol into cell membranes using methyl-β-cyclodextrin, and (4) performing and analyzing confocal FRAP measurements.
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
Jacobson, K., Sheets, E. D., and Simson, R. (1995) Revisiting the fluid mosaic model of membranes. Science 268, 1441–1442.
Edidin, M. (1992) Patches, posts and fences: proteins and plasma membrane domains. Trends Cell Biol. 2, 376–380.
Kenworthy, A. K. (2005) Fleeting glimpses of lipid rafts: how biophysics is being used to track them. J. Invest. Med. 53, 312–317.
Lommerse, P. H., Spaink, H. P., and Schmidt, T. (2004) In vivo plasma membrane organization: results of biophysical approaches. Biochim. Biophys. Acta 1664, 119–131.
Lagerholm, B. C., Weinreb, G. E., Jacobson, K., and Thompson, N. L. (2005) Detecting Microdomains In Intact Cell Membranes. Annu. Rev. Phys. Chem. 56, 309–336.
Kenworthy, A. K., Nichols, B. J., Remmert, C. L., et al. (2004) Dynamics of putative raft-associated proteins at the cell surface. J. Cell Biol. 165, 735–746.
Goodwin, J. S., Drake, K. R., Remmert, C. L., and Kenworthy, A. K. (2005) Ras diffusion is sensitive to plasma membrane viscosity. Biophys. J. 89, 1398–1410.
Niv, H., Gutman, O., Henis, Y. I., and Kloog, Y. (1999) Membrane interactions of a constitutively active GFP-Ki-Ras 4B and their role in signaling. Evidence from lateral mobility studies. J. Biol. Chem. 274, 1606–1613.
Rotblat, B., Prior, I. A., Muncke, C., et al. (2004) Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane. Mol. Cell Biol. 24, 6799–6810.
Niv, H., Gutman, O., Kloog, Y., and Henis, Y. I. (2002) Activated K-Ras and H-Ras display different interactions with saturable nonraft sites at the surface of live cells. J. Cell Biol. 157, 865–872.
Roy, S., Plowman, S., Rotblat, B., et al. (2005) Individual palmitoyl residues serve distinct roles in H-ras trafficking, microlocalization, and signaling. Mol. Cell Biol. 25, 6722–6733.
Hao, M., Mukherjee, S., and Maxfield, F. R. (2001) Cholesterol depletion induces large scale domain segregation in living cell membranes. Proc. Natl. Acad. Sci. USA 98, 13,072–13,077.
Meder, D., Moreno, M. J., Verkade, P., Vaz, W. L., and Simons, K. (2006) Phase coexistence and connectivity in the apical membrane of polarized epithelial cells. Proc. Natl. Acad. Sci. USA 103, 329–334.
Shvartsman, D. E., Gutman, O., Tietz, A., and Henis, Y. I. (2006) Cyclodextrins but not compactin inhibit the lateral diffusion of membrane proteins independent of cholesterol. Traffic 7, 917–926.
Nicolau, D. V., Jr., Burrage, K., Parton, R. G., and Hancock, J. F. (2006) Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane. Mol. Cell Biol. 26, 313–323.
Lippincott-Schwartz, J., Snapp, E., and Kenworthy, A. K. (2001) Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444–456.
Lippincott-Schwartz, J., Altan-Bonnet, N., and Patterson, G. H. (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. (Suppl) S7–S14.
Goodwin, J. S. and Kenworthy, A. K. (2005) Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells. Methods 37, 154–164.
Snapp, E. L., Altan, N., and Lippincott-Schwartz, J. (2003) Current Protocols in Cell Biology, vol. 3, (Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J., and Yamada, K., eds.), pp. 21.21–21.24.
Feder, T. J., Brust-Mascher, I., Slattery, J. P., Baird, B., and Webb, W. W. (1996) Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys. J. 70, 2767–2773.
Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W. (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069.
Soumpasis, D. M. (1983) Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys. J. 41, 95–97.
Adams, C. L., Chen, Y.-T., Smith, S. J., and Nelson, W. J. (1998) Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein. J. Cell Biol. 142, 1105–1119.
Schmidt, K. and Nichols, B. J. (2004) A barrier to lateral diffusion in the cleavage furrow of dividing mammalian cells. Curr. Biol. 14, 1002–1006.
Siggia, E. D., Lippincott-Schwartz, J., and Bekiranov, S. (2000) Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery. Biophys. J. 79, 1761–1770.
Ellenberg, J., Siggia, E. D., Moreira, J. E., et al. (1997) Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell Biol. 138, 1193–1206.
Subtil, A., Gaidarov, I., Kobylarz, K., Lampson, M. A., Keen, J. H., and McGraw, T. E. (1999) Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc. Natl. Acad. Sci. USA 96, 6775–6780.
Sharma, D. K., Brown, J. C., Choudhury, A., et al. (2004) Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol. Mol. Biol. Cell 15, 3114–3122.
Weiss, M. (2004) Challenges and artifacts in quantitative photobleaching experiments. Traffic 5, 662–671.
Sinnecker, D., Voigt, P., Hellwig, N., and Schaefer, M. (2005) Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44, 7085–7094.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Kenworthy, A.K. (2007). Fluorescence Recovery After Photobleaching Studies of Lipid Rafts. In: McIntosh, T.J. (eds) Lipid Rafts. Methods in Molecular Biology, vol 398. Humana Press. https://doi.org/10.1007/978-1-59745-513-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-59745-513-8_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-729-7
Online ISBN: 978-1-59745-513-8
eBook Packages: Springer Protocols