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Quantitative imaging of membrane lipid order in cells and organisms

Nature Protocols volume 7, pages 24–35 (2012)Cite this article

Abstract

It is now recognized that lipids and proteins in cellular membranes are not homogenously distributed. A high degree of membrane order is the biophysical hallmark of cholesterol-enriched lipid rafts, which may induce the lateral sorting of proteins within the membrane. Here we describe a quantitative fluorescence microscopy technique for imaging localized lipid environments and measuring membrane lipid order in live and fixed cells, as well as in intact tissues. The method is based on the spectral ratiometric imaging of the polarity-sensitive membrane dyes Laurdan and di-4-ANEPPDHQ. Laurdan typically requires multiphoton excitation, making it suitable for the imaging of tissues such as whole, living zebrafish embryos, whereas di-4-ANEPPDHQ imaging can be achieved with standard confocal microscopes. This approach, which takes around 4 h, directly examines the organization of cellular membranes and is distinct from alternative approaches that infer membrane order by measuring probe partitioning or dynamics.

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Figure 1: Schematic of the probes and their emission spectra in different lipid environments.
Figure 2: Initialization of lasers for Laurdan microscopy in the Leica LAS software.
Figure 3: Activation of lasers for Laurdan microscopy in the Leica LAS software.
Figure 4: Detection settings for Laurdan microscopy in the Leica LAS software.
Figure 5: Scan settings for Laurdan microscopy in the Leica LAS software.
Figure 6: Generating GP images and pseudocolored GP-intensity-merged images.
Figure 7: Laurdan or di-4-ANEPPDHQ GP image processing.
Figure 8: Laurdan imaging of live zebrafish embryos.
Figure 9: Laurdan imaging of live HeLa cells.
Figure 10: Di-4-ANEPPDHQ imaging of live HeLa cells.

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References

  1. Simons, K. et al. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    Article  CAS  Google Scholar 

  2. Pike, L.J. Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J. Lipid Res. 47, 1597–1598 (2006).

    Article  CAS  Google Scholar 

  3. Tanimura, N. et al. Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation. J. Cell Biol. 160, 125–135 (2003).

    Article  CAS  Google Scholar 

  4. Owen, D.M. et al. Quantitative microscopy: protein dynamics and membrane organisation. Traffic 10, 962–971 (2009).

    Article  CAS  Google Scholar 

  5. Gaus, K. et al. Condensation of the plasma membrane at the site of T lymphocyte activation. J. Cell Biol. 171, 121–131 (2005).

    Article  CAS  Google Scholar 

  6. Owen, D.M. et al. High plasma membrane lipid order imaged at the immunological synapse periphery in live T cells. Mol. Membr. Biol. 27, 178–189 (2010).

    Article  CAS  Google Scholar 

  7. Rentero, C. et al. Functional implications of plasma membrane condensation for T cell activation. PLoS ONE 3, e2262 (2008).

    Article  Google Scholar 

  8. Gupta, N. et al. Lipid rafts and B cell signaling. Semin. Cell Dev. Biol. 18, 616–626 (2007).

    Article  CAS  Google Scholar 

  9. Gaus, K. et al. Integrin-mediated adhesion regulates membrane order. J. Cell Biol. 174, 725–734 (2006).

    Article  CAS  Google Scholar 

  10. Barman, S. et al. Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells. J. Virol. 81, 12169–12178 (2007).

    Article  CAS  Google Scholar 

  11. del Real, G. et al. Blocking of HIV-1 infection by targeting CD4 to nonraft membrane domains. J. Exp. Med. 196, 293–301 (2002).

    Article  CAS  Google Scholar 

  12. Scheiffele, P. et al. Influenza viruses select ordered lipid domains during budding from the plasma membrane. J. Biol. Chem. 274, 2038–2044 (1999).

    Article  CAS  Google Scholar 

  13. Ikonen, E. Roles of lipid rafts in membrane transport. Curr. Opin. Cell Biol. 13, 470–477 (2001).

    Article  CAS  Google Scholar 

  14. Hanzal-Bayer, M.F. et al. Lipid rafts and membrane traffic. FEBS Lett. 581, 2098–2104 (2007).

    Article  CAS  Google Scholar 

  15. Weber, G. & Farris, F.J. Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene. Biochemistry 18, 3075–3078 (1979).

    Article  CAS  Google Scholar 

  16. Parasassi, T. et al. Laurdan and Prodan as polarity-sensitive fluorescent membrane probes. J. Fluoresc. 8, 365–737 (1998).

    Article  CAS  Google Scholar 

  17. Viard, M. et al. Laurdan solvatochromism: solvent dielectric relaxation and intramolecular excited-state reaction. Biophys. J. 73, 2221–2234 (1997).

    Article  CAS  Google Scholar 

  18. Vincent, M. et al. Nanosecond dynamics of a mimicked membrane-water interface observed by time-resolved stokes shift of LAURDAN. Biophys. J. 88, 4337–4350 (2005).

    Article  CAS  Google Scholar 

  19. Parasassi, T. et al. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys. J. 57, 1179–1186 (1990).

    Article  CAS  Google Scholar 

  20. Parasassi, T. et al. Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. Biophys. J. 72, 2413–2429 (1997).

    Article  CAS  Google Scholar 

  21. Owen, D.M. et al. Imaging membrane lipid order in whole, living vertebrate organisms. Biophys. J. 99, L7–L9 (2010).

    Article  Google Scholar 

  22. Yu, W. et al. Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach. Biophys. J. 70, 626–636 (1996).

    Article  CAS  Google Scholar 

  23. Gaus, K. et al. Visualizing lipid structure and raft domains in living cells with two-photon microscopy. Proc. Natl. Acad. Sci. USA 100, 15554–15559 (2003).

    Article  CAS  Google Scholar 

  24. Gaus, K. et al. Condensation of the plasma membrane at the site of T lymphocyte activation. J. Cell Biol. 171, 121–131 (2005).

    Article  CAS  Google Scholar 

  25. Kaiser, H. et al. Order of lipid phases in model and plasma membranes. Proc. Natl. Acad. Sci. USA 106, 16645–16650 (2009).

    Article  CAS  Google Scholar 

  26. Römer, W. et al. Shiga toxin induces tubular membrane invaginations for its uptake into cells. Nature 450, 670–675 (2007).

    Article  Google Scholar 

  27. Römer, W. et al. Actin dynamics drive membrane reorganization and scission in clathrin-independent endocytosis. Cell 140, 540–553 (2010).

    Article  Google Scholar 

  28. Obaid, A.L. et al. Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis. J. Neurosci. Meth. 134, 179–190 (2004).

    Article  CAS  Google Scholar 

  29. Jin, L. et al. Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear and nonlinear optics. Biophys. J. 89, L04–L06 (2005).

    Article  CAS  Google Scholar 

  30. Jin, L. et al. Characterization and application of a new optical probe for membrane lipid domains. Biophys. J. 90, 2563–2575 (2006).

    Article  CAS  Google Scholar 

  31. Dinic, J. et al. Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing. Biochim. Biophys. Acta 1808, 298–306 (2011).

    Article  CAS  Google Scholar 

  32. Janes, P.W. et al. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J. Cell Biol. 147, 447–461 (1999).

    Article  CAS  Google Scholar 

  33. Zhang, R.-G. et al. The three-dimensional crystal structure of cholera toxin. J. Mol. Biol. 251, 563–573 (1995).

    Article  CAS  Google Scholar 

  34. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1163 (2009).

    Article  CAS  Google Scholar 

  35. Wawrezinieck, L. et al. Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization. Biophys. J. 89, 4029–4042 (2005).

    Article  CAS  Google Scholar 

  36. Bacia, K. et al. Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys. J. 87, 1034–1043 (2004).

    Article  CAS  Google Scholar 

  37. Kahya, N. et al. Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy. J. Biol. Chem. 278, 28109–28115 (2003).

    Article  CAS  Google Scholar 

  38. Lommerse, P.H.M. et al. Single-molecule diffusion reveals similar mobility for the Lck, H-Ras, and K-Ras membrane anchors. Biophys. J. 91, 1090–1097 (2006).

    Article  CAS  Google Scholar 

  39. Schneckenburger, H. et al. Time-gated total internal reflection fluorescence spectroscopy (TG-TIRFS): application to the membrane marker laurdan. J. Microsc. 211, 30–36 (2003).

    Article  CAS  Google Scholar 

  40. Grant, D.M. et al. High speed optically sectioned fluorescence lifetime imaging permits study of live cell signaling events. Opt. Express 15, 15656–15673 (2007).

    Article  CAS  Google Scholar 

  41. Miguel, L. et al. Primary human CD4+ T cells have diverse levels of membrane lipid order that correlate with their function. J. Immunol. 186, 3505–3516 (2011).

    Article  CAS  Google Scholar 

  42. Kim, H.M. et al. A two-photon fluorescent probe for lipid raft imaging: C-Laurdan. Chembiochem 8, 553–559 (2007).

    Article  CAS  Google Scholar 

  43. Kim, H.M. et al. Two-photon fluorescent turn-on probe for lipid rafts in live cell and tissue. J. Am. Chem. Soc. 130, 4246–4247 (2008).

    Article  CAS  Google Scholar 

  44. Veatch, S.L. et al. Fluorescent probes alter miscibility phase boundaries in ternary vesicles. J. Phys. Chem. 111, 502–504 (2007).

    Article  CAS  Google Scholar 

  45. Gaus, K. et al. Visualizing membrane microdomains by Laurdan 2-photon microscopy. Mol. Membr. Biol. 23, 41–48 (2006).

    Article  CAS  Google Scholar 

  46. Westerfield, M. The Zebrafish Book: A Guide For the Laboratory Use of Zebrafish (Danio rerio) 4th edn. (University of Oregon Press, 2000).

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Acknowledgements

D.M.O. and K.G. acknowledge funding from the Australian Research Council; K.G. also receives funding from the National Health and Medical Research Council of Australia and the Human Frontier Science Program. C.R. is grateful to the CONSOLIDER-INGENIO fellowship (CSD2009-00016, Ministerio de Innovación, Ciencia y Tecnología).

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Authors and Affiliations

  1. Centre for Vascular Research, University of New South Wales, Sydney, Australia

    Dylan M Owen, Astrid Magenau, Ahmed Abu-Siniyeh & Katharina Gaus

  2. Facultat de Medicina, Departament de Biologia Cel.lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain

    Carles Rentero

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Contributions

D.M.O. developed the di-4-ANEPPDHQ protocol and wrote the manuscript. C.R. wrote the GP analysis macro. D.M.O. and A.M. conducted the experiments presented here. D.M.O., A.M. and A.A-S. developed the zebrafish protocol. K.G. developed the Laurdan protocol and wrote the manuscript.

Corresponding authors

Correspondence to Dylan M Owen or Katharina Gaus.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

ImageJ macro for processing two channel Laurdan or di-4-ANEPPDHQ into GP images. The macro imports TIFF images from two different spectral windows. Using the data flow shown in Figure 6, it generates GP images and pseudo-coloured GP-intensity merged images. (TXT 16 kb)

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Owen, D., Rentero, C., Magenau, A. et al. Quantitative imaging of membrane lipid order in cells and organisms. Nat Protoc 7, 24–35 (2012). https://doi.org/10.1038/nprot.2011.419

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