Modulation and Dynamics of Phase Properties in Phospholipid Mixtures Detected by Laurdan Fluorescence*

Parasassi, Tiziana; Ravagnan, Giampietro; Rusch, Ruth M.; Gratton, Enrico

doi:10.1111/j.1751-1097.1993.tb02309.x

Cited by 107 publications

(132 citation statements)

References 23 publications

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“…Based on these previous studies and our observations, we propose that the stripe domains are in the gel phase. This assignment agrees with a model proposed by Parasassi et al that the network shaped domains are like gel filaments into a liquid crystalline phase (36).…”

Section: Discussionsupporting

confidence: 92%

Coexisting Stripe- and Patch-Shaped Domains in Giant Unilamellar Vesicles

Cheng

2006

Biochemistry

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We report a new type of gel-liquid phase segregation in giant unilamellar vesicles (GUVs) of mixed lipids. Coexisting patch and stripe shaped gel domains in GUV bilayers composed of DOPC/DPPC or DLPC/DPPC are observed by confocal fluorescence microscopy. The lipids in stripe domains are shown to be tilted according to the DiIC18 fluorescence intensity dependence on the excitation polarization. The patch domains are found to be mainly composed of d62-DPPC according to the coherent anti-Stokes Raman scattering (CARS) images of DOPC/d62-DPPC bilayers. When cooling GUVs from above the miscibility temperature, the patch domains start to appear between the chain melting and the pretransition temperature of DPPC. In GUVs containing high molar percentage of DPPC, the stripe domains form below the pretransition temperature. Our observations suggest that the patch and stripe domains are in the P β′ and L β′ gel phases, respectively. According to the thermoelastic properties of GUVs described by Needham and Evans (Biochemistry,27,[8261][8262][8263][8264][8265][8266][8267][8268][8269], the P β′ and L β′ phases are formed at relatively low and high membrane tensions respectively. GUVs with high DPPC percentage have high membrane surface tension and thus mainly exhibit L β′ domains, while GUVs with low DPPC percentage have low membrane surface tension and form P β′ domains accordingly. Adding negatively charged lipid to the lipid mixtures or applying an osmotic pressure to GUVs using sucrose solutions releases the surface tension and leads to the disappearance of L β′ gel phase. The relationship between the observed domains in free-standing GUV bilayers in and those in supported bilayers is discussed.Phase segregation plays important roles in membrane organization and intracellular trafficking (1-5). Phase behavior of lipid mixtures has been extensively studied using different welldefined model membrane systems. GUVs which mimic the plasma membrane with controlled lipid compositions and similar size as cells have been widely used to study the mechanisms of membrane phase segregation, fusion, and fission (6-10). In GUVs composed of a saturated lipid, an unsaturated lipid, and cholesterol, the observation of coexisting ordered and disordered liquid phases below miscibility transition temperature has provided supports for the existence of lipid rafts in cell membranes (11). Despite these advances, the lipid phase behavior in GUVs is far away from being well understood, even for binary lipid mixtures. Patch and stripe shaped domains in GUVs composed of DPPC and another phospholipid have been reported in different papers (7,8,12,13). However, the composition and lipid organization properties of these domains have not been well characterized. The relations between these two types of domains and the different gel phases (P β′ and L β′ ) observed in pure DPPC bilayers are elusive. In addition, the forces that govern the formation of these two types of domains are poorly investigated.To whom correspondence should be addressed....

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Section: Discussionsupporting

confidence: 92%

Coexisting Stripe- and Patch-Shaped Domains in Giant Unilamellar Vesicles

Cheng

2006

Biochemistry

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“…Thus, by moving the excitation toward the red, in the homogeneous liquidcrystalline phase the GP value decreases, while in bilayers composed of coexisting phases the GP value increases. Similar reasoning can be made for the GP values obtained at different emission wavelengths, as extensively reported and discussed elsewhere [32]. Note that, as reported above, the emission GP is calculated by the difference between the intensity of the red excitation band and that of the blue one, while the excitation GP is calculated by the difference between the intensity at the blue and that at the red part of the emission spectrum.…”

Section: Wavelength Dependence Of the Generalized Polarization To Ascsupporting

confidence: 64%

“…Time-resolved emission spectra of Laurdan in vesicles composed of phospholipids in the liquid-crystalline phase, i.e., in a relaxing environment, show a progressive red shift of the maximum and an increase of the center of mass of the emission with time. In vesicles composed of gelphase phospholipids, i.e., in a nonrelaxing environment, Laurdan emission does not change appreciably with the time after excitation [32]. When domains of the two phases coexist, the Laurdan maximum and center of mass increase up to about 20 ns after excitation; then the spectrum slightly shifts back toward the blue (Fig.…”

Section: Spectramentioning

confidence: 95%

“…When domains of the two phases coexist, the Laurdan maximum and center of mass increase up to about 20 ns after excitation; then the spectrum slightly shifts back toward the blue (Fig. 3) [30,32]. Such an inversion of the spectral shift has been interpreted as due to fluctuation between the different phases [30], with kinetics of about 25 ns, and imply per se the coexistence of separate phase domains.…”

Section: Spectramentioning

confidence: 99%

“…If phospholipids in the gel phase are not present, the red band is only populated by relaxed Laurdan molecules, with a red-shifted emission spectrum and a low GP value. If phospholipids in the gel phase are present, they mainly populate the red band of excitation, emitting with a blue spectrum and with a high GP value [32]. Thus, by moving the excitation toward the red, in the homogeneous liquidcrystalline phase the GP value decreases, while in bilayers composed of coexisting phases the GP value increases.…”

Section: Wavelength Dependence Of the Generalized Polarization To Ascmentioning

confidence: 99%

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Membrane lipid domains and dynamics as detected by Laurdan fluorescence

1995

Self Cite

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(Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation, ha bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). The GP value can be used for the quantitation of each phase. The wavelength dependence of the GP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined. GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in na~u'al membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 A.

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Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes

Cebecauer

Šachl

2016

Fluorescent Analogs of Biomolecular Building Blocks

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Modulation and Dynamics of Phase Properties in Phospholipid Mixtures Detected by Laurdan Fluorescence*

Cited by 107 publications

References 23 publications

Coexisting Stripe- and Patch-Shaped Domains in Giant Unilamellar Vesicles

Coexisting Stripe- and Patch-Shaped Domains in Giant Unilamellar Vesicles

Membrane lipid domains and dynamics as detected by Laurdan fluorescence

Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes

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