A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study.

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).

[1]  E Gratton,et al.  Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach. , 1996, Biophysical journal.

[2]  D. S. Dimitrov,et al.  A mechanism of liposome electroformation , 1988 .

[3]  A. Lee,et al.  Fluorescence studies of chlorophyll a incorporated into lipid mixtures, and the interpretation of "phase" diagrams. , 1975, Biochimica et biophysica acta.

[4]  D. Mitov,et al.  Bending elasticities of model membranes: influences of temperature and sterol content. , 1997, Biophysical journal.

[5]  R. Weis,et al.  Cholesterol stabilizes the crystal-liquid interface in phospholipid monolayers , 1985 .

[6]  M. D. Mitov,et al.  Mechanical properties of model membranes studied from shape transformations of giant vesicles. , 1998, Biochimie.

[7]  L. Yang,et al.  Membrane domains containing phosphatidylserine and substrate can be important for the activation of protein kinase C. , 1995, Biochemistry.

[8]  F. Menger,et al.  Chemistry and physics of giant vesicles as biomembrane models. , 1998, Current opinion in chemical biology.

[9]  E. Gratton,et al.  Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. , 1997, Biophysical journal.

[10]  P. W. V. van Dijck,et al.  Miscibility properties of binary phosphatidylcholine mixtures. A calorimetric study. , 1977, Biochimica et biophysica acta.

[11]  M. Thoma,et al.  Domain formation in monolayers. , 1995, Molecular membrane biology.

[12]  R. Griffin,et al.  Phase equilibria, molecular conformation, and dynamics in phosphatidylcholine/phosphatidylethanolamine bilayers. , 1982, Biochemistry.

[13]  A. Raudino.  Lateral inhomogeneous lipid membranes: theoretical aspects. , 1995, Advances in colloid and interface science.

[14]  H. Clausen‐Schaumann,et al.  Direct detection of domains in phospholipid bilayers by grazing incidence diffraction of neutrons and atomic force microscopy. , 1998, Biophysical journal.

[15]  M. Sperotto,et al.  Phase equilibria and local structure in binary lipid bilayers. , 1993, Biochimica et biophysica acta.

[16]  E. Evans,et al.  Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. , 1988, Biochemistry.

[17]  E Gratton,et al.  Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence. , 1991, Biophysical journal.

[18]  D. Haverstick,et al.  Visualization of Ca2+-induced phospholipid domains. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Enrico Gratton,et al.  MODULATION AND DYNAMICS OF PHASE PROPERTIES IN PHOSPHOLIPID MIXTURES DETECTED BY LAURDAN FLUORESCENCE * , 1993, Photochemistry and photobiology.

[20]  P. Devaux,et al.  Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. , 1996, Biophysical journal.

[21]  M. Angelova,et al.  Preparation of giant vesicles by external AC electric fields. Kinetics and applications , 1992 .

[22]  J. Boggs,et al.  Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. , 1987, Biochimica et biophysica acta.

[23]  E. Evans,et al.  Mechanical calorimetry of large dimyristoylphosphatidylcholine vesicles in the phase transition region. , 1982, Biochemistry.

[24]  E Gratton,et al.  Giant phospholipid vesicles: comparison among the whole lipid sample characteristics using different preparation methods: a two photon fluorescence microscopy study. , 2000, Chemistry and physics of lipids.

[25]  E Gratton,et al.  Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. , 1990, Biophysical journal.

[26]  E. Sackmann,et al.  Membrane bending energy concept of vesicle‐ and cell‐shapes and shape‐transitions , 1994, FEBS letters.

[27]  J. Korlach,et al.  Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  E. Evans,et al.  Molecular friction and epitactic coupling between monolayers in supported bilayers , 1989 .

[29]  H. Ringsdorf,et al.  Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy. , 1990, Biochimica et biophysica acta.

[30]  E. Gratton,et al.  Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence. , 1998, Biophysical journal.

[31]  E Gratton,et al.  Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles. , 1999, Biophysical journal.

[32]  J. Sturtevant,et al.  Investigation of phase transitions of lipids and lipid mixtures by high sensitivity differential scanning calorimetry ( lipid bilayers / membranes / phase diagrams / transition heat capacity curves ) , 2022 .

[33]  O. G. Mouritsen,et al.  Phase separation dynamics and lateral organization of two-component lipid membranes. , 1995, Biophysical journal.

[34]  J M Sturtevant,et al.  Investigation of phase transitions of lipids and lipid mixtures by sensitivity differential scanning calorimetry. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[35]  E. Sackmann.  Dynamic Molecular Organization in Vesicles and Membranes , 1978 .

[36]  M. Caffrey,et al.  A temperature gradient method for lipid phase diagram construction using time-resolved x-ray diffraction. , 1987, Biophysical journal.

[37]  M. Angelova,et al.  Lipid swelling and liposome formation on solid surfaces in external electric fields , 1987 .

[38]  D. Haverstick,et al.  Influence of proteins on the reorganization of phospholipid bilayers into large domains. , 1989, Biophysical journal.

[39]  T. E. Thompson,et al.  Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 2 Two-component phosphatidylcholine liposomes. , 1976, Biochemistry.

[40]  H. Mcconnell,et al.  Lateral phase separation in phospholipid membranes. , 1973, Biochemistry.

[41]  E. Sackmann,et al.  Budding, fission and domain formation in mixed lipid vesicles induced by lateral phase separation and macromolecular condensation. , 1995, Molecular membrane biology.

[42]  E. Evans,et al.  Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C. , 1988, Biochemistry.

[43]  M. Angelova,et al.  DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles , 1999, European Biophysics Journal.

[44]  P. Kinnunen,et al.  Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. , 2000, Biophysical journal.

[45]  Enrico Gratton,et al.  Time-resolved fluorescence microscopy using two-photon excitation , 1995 .

[46]  P. Luisi,et al.  Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations. , 1996, Chemistry & biology.

[47]  E Gratton,et al.  Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. , 2000, Biophysical journal.

[48]  J. Ipsen,et al.  Modelling the phase equilibria in two-component membranes of phospholipids with different acyl-chain lengths. , 1988, Biochimica et biophysica acta.

[49]  D. Hammer,et al.  Area Expansion and Permeation of Phospholipid Membrane Bilayers by Influenza Fusion Peptides and Melittin , 1998 .

[50]  K. Arnold,et al.  31p-NMR investigations of phase separation in phosphatidylcholine/phosphatidylethanolamine mixtures. , 1981, Biochimica et biophysica acta.