Lipid packing is a crucial feature of cellular membranes. Quantitative analysis of membrane lipid packing can be achieved using polarity sensitive probes whose emission spectrum depends on the lipid packing. However, detailed insights into the exact mechanisms that cause the changes in the spectra are necessary to interpret experimental fluorescence emission data correctly. Here, we analysed frequently used polarity sensitive probes, Laurdan and di-4-ANEPPDHQ, to test whether the underlying physical mechanisms of their spectral changes are the same and, thus, whether they report on the same physico-chemical properties of the cell membrane. Steady-state spectra as well as time-resolved emission spectra of the probes in solvents and model membranes revealed that they probe different properties of the lipid membrane. Our findings are important for the application of these dyes in cell biology.

Department of Biophysical Chemistry J Heyrovský Institute of Physical Chemistry of the C A S v v i Dolejskova 3 182 23 Prague Czechia

MRC Human Immunology Unit OX39DS University of Oxford Oxford United Kingdom

Zobrazit více v PubMed

Singer S J, Nicolson G L. The fluid mosaic model of the structure of cell membranes. Science. 1972;175:720. doi: 10.1126/science.175.4023.720. PubMed DOI

Yeagle P L, Albert A D, Boeszebattaglia K, Young J, Frye J. Cholesterol dynamics in membranes. Biophys. J. 1990;57:413–24. doi: 10.1016/S0006-3495(90)82558-3. PubMed DOI PMC

Needham D, McIntosh T J, Evans E. Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. Biochemistry. 1988;27:4668–73. doi: 10.1021/bi00413a013. PubMed DOI

Parasassi T, Distefano M, Loiero M, Ravagnan G, Gratton E. Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe. Biophys. J. 1994;66:763–8. doi: 10.1016/S0006-3495(94)80852-5. PubMed DOI PMC

Veya L, Piguet J, Vogel H. Single molecule imaging deciphers the relation between mobility and signaling of a prototypical g protein-coupled receptor in living cells. J. Biol. Chem. 2015;290:27723–35. doi: 10.1074/jbc.m115.666677. PubMed DOI PMC

Blouin C M, et al. Glycosylation-dependent IFN-gammaR partitioning in lipid and actin nanodomains is critical for JAK activation. Cell. 2016;166:920–34. doi: 10.1016/j.cell.2016.07.003. PubMed DOI

Edgcomb M R, Sirimanne S, Wilkinson B J, Drouin P, Morse R D. Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes. Biochim. Biophys. Acta. 2000;1463:31–42. doi: 10.1016/S0005-2736(99)00179-0. PubMed DOI

Stewart G S, Eaton M W, Johnstone K, Barrett M D, Ellar D J. An investigation of membrane fluidity changes during sporulation and germination of Bacillus megaterium K.M. measured by electron spin and nuclear magnetic resonance spectroscopy. Biochim. Biophys. Acta. 1980;600:270–90. doi: 10.1016/0005-2736(80)90432-0. PubMed DOI

Klein C, Pillot T, Chambaz J, Drouet B. Determination of plasma membrane fluidity with a fluorescent analogue of sphingomyelin by FRAP measurement using a standard confocal microscope. Brain Res. Brain Res. Protocols. 2003;11:46–51. doi: 10.1016/S1385-299X(03)00016-3. PubMed DOI

Marczak A. Fluorescence anisotropy of membrane fluidity probes in human erythrocytes incubated with anthracyclines and glutaraldehyde. Bioelectrochemistry. 2009;74:236–9. doi: 10.1016/j.bioelechem.2008.11.004. PubMed DOI

Kuhry J G, Duportail G, Bronner C, Laustriat G. Plasma membrane fluidity measurements on whole living cells by fluorescence anisotropy of trimethylammoniumdiphenylhexatriene. Biochim. Biophys. Acta. 1985;845:60–7. doi: 10.1016/0167-4889(85)90055-2. PubMed DOI

Parasassi T, Krasnowska E K, Bagatolli L, Gratton E. Laurdan and Prodan as polarity-sensitive fluorescent membrane probes. J. Fluoresc. 1998;8:365–73. doi: 10.1023/A:1020528716621. DOI

Sanchez S A, Tricerri M A, Gunther G, Gratton E. Laurdan generalized polarization: from cuvette to microscope. In: Méndez-Vilas A, Díaz J, editors. Modern Research and Educational Topics in Microscopy. Badajoz: Formatex Research Center; 2007.

Sachl R, Stepanek M, Prochazka K, Humpolickova J, Hof M. Fluorescence study of the solvation of fluorescent probes prodan and laurdan in poly(epsilon-caprolactone)-block-poly(ethylene oxide) vesicles in aqueous solutions with tetrahydrofurane. Langmuir. 2008;24:288–95. doi: 10.1021/la702277t. PubMed DOI

Lucio A D, Vequi-Suplicy C C, Fernandez R M, Lamy M T. Laurdan spectrum decomposition as a tool for the analysis of surface bilayer structure and polarity: a study with DMPG, peptides and cholesterol. J. Fluoresc. 2010;20:473–82. doi: 10.1007/s10895-009-0569-5. PubMed DOI

Barucha-Kraszewska J, Kraszewski S, Ramseyer C. Will C-Laurdan dethrone Laurdan in fluorescent solvent relaxation techniques for lipid membrane studies? Langmuir. 2013;29:1174–82. doi: 10.1021/la304235r. PubMed DOI

Bacalum M, Zorila B, Radu M. Fluorescence spectra decomposition by asymmetric functions: Laurdan spectrum revisited. Anal. Biochem. 2013;440:123–9. doi: 10.1016/j.ab.2013.05.031. PubMed DOI

Golfetto O, Hinde E, Gratton E. The Laurdan spectral phasor method to explore membrane micro-heterogeneity and lipid domains in live cells. Methods Mol. Biol. 2014;1232:273–90. doi: 10.1007/978-1-4939-1752-5_19. PubMed DOI

Vequi-Suplicy C C, Coutinho K, Lamy M T. New insights on the fluorescent emission spectra of Prodan and Laurdan. J. Fluoresc. 2015;25:621–9. doi: 10.1007/s10895-015-1545-x. PubMed DOI

Malacrida L, Astrada S, Briva A, Bollati-Fogolin M, Gratton E, Bagatolli L A. Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures. Biochim. Biophys. Acta. 2016;1858:2625–35. doi: 10.1016/j.bbamem.2016.07.017. PubMed DOI PMC

Bagatolli L A, Sanchez S A, Hazlett T, Gratton E. Giant vesicles, Laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers. Methods Enzymol. 2003;360:481–500. doi: 10.1016/s0076-6879(03)60124-2. PubMed DOI

Dinic J, Biverstahl H, Maler L, Parmryd I. Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing. Biochim. Biophys. Acta. 2011;1808:298–306. doi: 10.1016/j.bbamem.2010.10.002. PubMed DOI

Zhao X, Li R, Lu C, Baluska F, Wan Y. Di-4-ANEPPDHQ, a fluorescent probe for the visualisation of membrane microdomains in living Arabidopsis thaliana cells. Plant Physiol. Biochem. 2015;87:53–60. doi: 10.1016/j.plaphy.2014.12.015. PubMed DOI

Sezgin E, Sadowski T, Simons K. Measuring lipid packing of model and cellular membranes with environment sensitive probes. Langmuir. 2014;30:8160–6. doi: 10.1021/la501226v. PubMed DOI

Jin L, Millard A C, Wuskell J P, Clark H A, Loew L M. Cholesterol-enriched lipid domains can be visualized by di-4-ANEPPDHQ with linear and nonlinear optics. Biophys. J. 2005;89:L4–6. doi: 10.1529/biophysj.105.064816. PubMed DOI PMC

Parasassi T, De Stasio G, Ravagnan G, Rusch R M, Gratton E. Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. Biophys. J. 1991;60:179–89. doi: 10.1016/S0006-3495(91)82041-0. PubMed DOI PMC

Sezgin E, Kaiser H-J, Baumgart T, Schwille P, Simons K, Levental I. Elucidating membrane structure and protein behavior using giant plasma membrane vesicles. Nat. Protocols. 2012;7:1042–51. doi: 10.1038/nprot.2012.059. PubMed DOI

Sezgin E, Waithe D, Bernardino de la Serna J, Eggeling C. Spectral imaging to measure heterogeneity in membrane lipid packing. ChemPhysChem. 2015;16:1387–94. doi: 10.1002/cphc.201402794. PubMed DOI PMC

Horng M L, Gardecki J A, Papazyan A, Maroncelli M. Subpicosecond measurements of polar solvation dynamics: coumarin 153 revisited. J. Phys. Chem. 1995;99:17311–37. doi: 10.1021/j100048a004. DOI

Owen D M, Rentero C, Magenau A, Abu-Siniyeh A, Gaus K. Quantitative imaging of membrane lipid order in cells and organisms. Nat. Protocols. 2012;7:24–35. doi: 10.1038/nprot.2011.419. PubMed DOI

Amaro M, Sachl R, Jurkiewicz P, Coutinho A, Prieto M, Hof M. Time-resolved fluorescence in lipid bilayers: selected applications and advantages over steady state. Biophys. J. 2014;107:2751–60. doi: 10.1016/j.bpj.2014.10.058. PubMed DOI PMC

Machan R, et al. Peripheral and integral membrane binding of peptides characterized by time-dependent fluorescence shifts: focus on antimicrobial peptide LAH(4) Langmuir. 2014;30:6171–9. doi: 10.1021/la5006314. PubMed DOI

Kulig W, et al. Experimental determination and computational interpretation of biophysical properties of lipid bilayers enriched by cholesteryl hemisuccinate. Biochim. Biophys. Acta. 2015;1848:422–32. doi: 10.1016/j.bbamem.2014.10.032. PubMed DOI

Jin L, et al. Characterization and application of a new optical probe for membrane lipid domains. Biophys. J. 2006;90:2563–75. doi: 10.1529/biophysj.105.072884. PubMed DOI PMC

de Almeida R F, Fedorov A, Prieto M. Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts. Biophys. J. 2003;85:2406–16. doi: 10.1016/S0006-3495(03)74664-5. PubMed DOI PMC

Oldfield E, Meadows M, Rice D, Jacobs R. Spectroscopic studies of specifically deuterium labeled membrane systems. Nuclear magnetic resonance investigation of the effects of cholesterol in model systems. Biochemistry. 1978;17:2727–40. doi: 10.1021/bi00607a006. PubMed DOI

Bloom M, Evans E, Mouritsen O G. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q. Rev. Biophys. 1991;24:293–397. doi: 10.1017/S0033583500003735. PubMed DOI

McMullen T P W, McElhaney R N. Physical studies of cholesterol-phospholipid interactions. Curr. Opin. Colloid Interface Sci. 1996;1:83–90. doi: 10.1016/S1359-0294(96)80048-3. DOI

Alwarawrah M, Dai J, Huang J. A molecular view of the cholesterol condensing effect in DOPC lipid bilayers. J. Phys. Chem. B. 2010;114:7516–23. doi: 10.1021/jp101415g. PubMed DOI PMC

Hofsass C, Lindahl E, Edholm O. Molecular dynamics simulations of phospholipid bilayers with cholesterol. Biophys. J. 2003;84:2192–206. doi: 10.1016/S0006-3495(03)75025-5. PubMed DOI PMC

Fee R S, Maroncelli M. Estimating the time-zero spectrum in time-resolved emmsion measurements of solvation dynamics. Chem. Phys. 1994;183:235–47. doi: 10.1016/0301-0104(94)00019-0. DOI

Richert R, Stickel F, Fee R S, Maroncelli M. Solvation dynamics and the dielectric response in a glass-forming solvent: from picoseconds to seconds. Chem. Phys. Lett. 1994;229:302–8. doi: 10.1016/0009-2614(94)01032-3. DOI

Hof M. Applied Fluorescence in Chemistry, Biology and Medicine. Berlin: Springer; 1999. Solvent relaxation in biomembranes; pp. pp 439–56. DOI

Sykora J, et al. ABA-C-15: a new dye for probing solvent relaxation in phospholipid bilayers. Langmuir. 2002;18:9276–82. doi: 10.1021/la026435c. DOI

Yang M, Richert R. Observation of heterogeneity in the nanosecond dynamics of a liquid. J. Chem. Phys. 2001;115:2676–80. doi: 10.1063/1.1380206. DOI

Richert R. Spectral diffusion in liquids with fluctuating solvent responses: dynamical heterogeneity and rate exchange. J. Chem. Phys. 2001;115:1429–34. doi: 10.1063/1.1380209. DOI

Hutterer R, Schneider F W, Hof M. Time-resolved emission spectra and anisotropy profiles for symmetric diacyl- and dietherphosphatidylcholines. J. Fluoresc. 1997;7:27–33. doi: 10.1007/BF02764574. DOI

Sykora J, Kapusta P, Fidler V, Hof M. On what time scale does solvent relaxation in phospholipid bilayers happen? Langmuir. 2002;18:571–4. doi: 10.1021/la011337x. DOI

Fluhler E, Burnham V G, Loew L M. Spectra, membrane binding, and potentiometric responses of new charge shift probes. Biochemistry. 1985;24:5749–55. doi: 10.1021/bi00342a010. PubMed DOI

Obaid A L, Loew L M, Wuskell J P, Salzberg B M. Novel naphthylstyryl-pyridinium potentiometric dyes offer advantages for neural network analysis. J. Neurosci. Methods. 2004;134:179–90. doi: 10.1016/j.jneumeth.2003.11.011. PubMed DOI

Kao W Y, Davis C E, Kim Y I, Beach J M. Fluorescence emission spectral shift measurements of membrane potential in single cells. Biophys. J. 2001;81:1163–70. doi: 10.1016/S0006-3495(01)75773-6. PubMed DOI PMC

Starke-Peterkovic T, Turner N, Vitha M F, Waller M P, Hibbs D E, Clarke R J. Cholesterol effect on the dipole potential of lipid membranes. Biophys. J. 2006;90:4060–70. doi: 10.1529/biophysj.105.074666. PubMed DOI PMC

Szabo G. Dual mechanism for the action of cholesterol on membrane permeability. Nature. 1974;252:47–9. doi: 10.1038/252047a0. PubMed DOI

McIntosh T J, Magid A D, Simon S A. Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes. Biochemistry. 1989;28:17–25. doi: 10.1021/bi00427a004. PubMed DOI

De Vequi-Suplicy C C, Benatti C R, Lamy M T. Laurdan in fluid bilayers: position and structural sensitivity. J. Fluoresc. 2006;16:431–9. doi: 10.1007/s10895-005-0059-3. PubMed DOI

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties

What Does Time-Dependent Fluorescence Shift (TDFS) in Biomembranes (and Proteins) Report on?