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Title
Membrane lipid domains and dynamics as detected by Laurdan fluorescence.
Permalink
https://escholarship.org/uc/item/54x5q1gs
Journal
Journal of fluorescence, 5(1)
ISSN
1053-0509
Authors
Parasassi, T
Gratton, E
Publication Date
1995-03-01
DOI
10.1007/bf00718783
Copyright Information
This work is made available under the terms of a Creative Commons Attribution License,
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Journal of Fluorescence, Vol. 5, No. 1, 1995
Membrane Lipid Domains and Dynamics as Detected by
Laurdan Fluorescence
Tiziana Parasassi 1 and Enrico Gratton ~,z
Received February 16, 1994; revised September 27, 1994; accepted September 27, 1994
2-Dimethylamino-6-1auroylnaphthalene (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 emis-
sion 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 re-
stricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influ-
ence 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.
KEY WORDS: Cholesterol; domains; Laurdan; generalized polarization; membrane; phospholipids.
THE FLUID MOSAIC MODEL IN 1972
It is well established that phospholipids in the bi-
layer aggregation can be found in two main phase states,
l Istituto di Medicina Sperimentale, CNR, Viale Marx 15, 00137
Rome, Italy.
2 Laboratory for Fluorescence Dynamics, University of Illinois at Ur-
bana-Champaign, Urbana, Illinois 61801.
59
the gel and the liquid-crystalline [1]. Instead, lipids in
biological membranes were proposed, in 1972 [2], to be
in a homogeneously fluid phase state. This
fluid
state
was identified with the liquid-crystalline phase, in which
molecules can freely diffuse in the plane of the mem-
brane. Natural membranes show a complex lipid com-
position, each component having specific phase
properties. For phospholipids, the lipid class present at
the highest concentration in membranes, the differences
1053~)509/95/0300-0059507.50/0 9 1995 Plenum Publishing Corporation
60 Parasassi and Gratton
in the nature of their polar heads, in the length and un-
saturation of their acyl residues, give rise to very differ-
ent temperatures of transition from the gel to the
liquid-crystalline phase [3,4]. This leads to the hypoth-
esis of a possible coexistence of domains of different
phases in the plane of the membrane at the physiological
temperature: gel-like domains composed of phospholip-
ids with higher transition temperature and liquid-crys-
talline-like domains composed of phospholipids with
lower transition temperature. These coexisting domains
will possess different molecular dynamics and different
kinetics of in-plane diffusion. A corollary of this hy-
pothesis was the possibility of modulating cell functions
by a preferential partition of selected enzymes between
the two types of domains [5,6]. In general, phase do-
mains could create separate compartments for the dif-
ferent membrane activities, each requiring peculiar local
dynamical properties.
THE SEARCH FOR COEXISTING
PHASE
DOMAINS IN VESICLES AND IN MEMBRANES
Several spectroscopic techniques have been used to
study the bilayer phase state [3,4,7-9]. Fluorescence
spectroscopy offers several advantages for both the in-
trinsic time scale of the fluorescence, allowing obser-
vations on events occurring in the nanosecond time
scale, which is typical of several biochemical events
[10], and for the advantages offered by this technique of
measurement. Fluorescence measurements are fast and
require a small amount of sample, and the low concen-
tration of the fluorophore makes it possible to exclude
any significant perturbing effect. These are important
considerations when working with "living" biological
material, such as cells in culture. For studies on the lipid
components of membrane, the more interesting fluores-
cent probes are those with a similar chemical structure
so that the lipid organization is not disturbed. The probe
should have a high quantum yield in hydrophobic en-
vironment and virtually nil in water, negligible affinity
with other components of natural membranes, such as
proteins, and high sensitivity to the membrane phase
state. 1,6-Diphenyl- 1,3,5-hexatriene (DPH) is one of the
most popular membrane probes, widely utilized for
measurements of the average fluidity by its fluorescence
polarization [11-13]. DPH fluorescence decay is sensi-
tive to the polarity of its surroundings. The fluorescence
lifetime decreases with the increase of polarity [14]. In
phospholipids, this sensitivity to polarity results in
higher average lifetime values in the gel with respect to
the liquid-crystalline phase [ 15,16]. In vesicles of known
composition, the DPH decay has been resolved into two
components, corresponding to the lifetime values meas-
ured in each pure phase, and the associated relative frac-
tions were in a good agreement with the reported phase
diagrams [16]. Nevertheless, the characteristic lifetime
values are also dependent on the temperature at which
measurements are performed, so that in samples of un-
known composition the quantitative resolution of two
coexisting phases cannot be obtained in a simple way
[14]. Additional experimental problems prevented the
use of DPH for the detection and the resolution of co-
existing domains in membranes: (i) The difference in the
average lifetime values measured in each pure phase is
small. (ii) DPH decay in phospholipid vesicles as a func-
tion of temperature can be equally described by a linear
superposition of the properties arising from the two
phases or by a continuous variation of the properties of
the probe along the phase transition. During the phase
transition the intermediate lifetime value can originate
from the contribution of two coexisting phases or from
a homogeneous phase with intermediate properties. (iii)
DPH decay is better described by a continuous distri-
bution of lifetime values [17,18], the largest difference
between the two phospholipid phases residing in the
value of its width, narrow in the gel and broader in the
liquid-crystalline phase. The width of DPH lifetime dis-
tribution reflects the microheterogeneity of its environ-
ment, reflecting water concentration differences along
the membrane normal [14] (see also C. Stubbs in this
issue). The measurement of the width of DPH lifetime
distribution can be used to monitor membrane alterations
that affect the water gradient, such as oxidative damage
[19,20], but does not help for the quantitation of lipid
phases.
The fluorescence properties of the two isomers of
parinaric acid have also been studied for the detection
and quantitation of coexisting domains in membranes
[21 ]. Due to the different configuration of their unsatur-
ations, the two isomers show a different preferential par-
titioning between the two phases. When measured in the
same sample, the polarization of the
cis
isomer is gen-
erally lower than that of the
trans
isomer [21]. Never-
theless, these two probes show quite a complex decay
of fluorescence. In isotropic solvents their emission de-
cay has been described by three discrete exponential
components [22] or by a two-component Lorentzian dis-
tribution [23,24]. Even in vesicles composed of synthetic
phospholipids, the resolution of coexisting domains re-
quires long and delicate measurements [23,24].
To ascertain the coexistence of lipid-phase domains
in membranes, the ideal probe should possess all the
properties of the membrane probes described above,
The Fluid Mosaic by Laurdan Fluorescence 61
Excitation
Emission
t'~ i /ii~//~:4~" ~( ""'".
//[v.._\ I
\ ..' ".,,
N \
":"
~ ~ iil
~I/-~/
i,.,../~\\,
.. \
\,
. " 1 ~ \\ ",.,
~.~
\
"..
",.. \ ".,,
0.0
- '
""
300 400 500 600
Wavelength (nm)
Fig.
1. Normalized Laurdan excitation and emission spectra in phos-
pholipid multilamellar vesicles composed of gel (continuous line), liq-
uid-crystalline (dotted line), and an equimolar mixture of the two
phases (dashed line). Phospholipid composition of the vesicles and the
temperature of measurements are dilauroylphosphatidylcholine at 40~
(dotted line), dipalmitoylphosphatidylcholine at 5~ (continuous line),
and an equimolar mix~res of the two phospholipids at 20~
coupled with a high sensitivity to the phospholipid-phase
state. It should display a limited set of parameters, typ-
ical for each phase, that could be easily resolved, i.e., it
should display a steady-state, spectral sensitivity to the
lipid phase.
LAURDAN FLUORESCENCE CAN RESOLVE
COEXISTING DOMAINS IN VESICLES
Laurdan has all the properties needed for the de-
tection and quantitation of coexisting phase domains in
membranes. This probe was synthesized by G. Weber
for the study of the effect of solvent polarity on the
fluorescence emission [25,26]. In solvents of higher po-
larity, Laurdan displays a red shift of its emission spec-
trum [27], due to dipolar relaxation. The dipole moment
of the fluorescent moiety of the Laurdan molecule in-
creases several debyes upon absorption. If the molecular
dynamics of solvent molecules is of the same time scale
as that of the Laurdan fluorescence lifetime, part of the
energy of the probe in the excited state can be spent for
the reorientation of the solvent dipoles in the close vi-
cinity. Lanrdan emission is thus red-shifted [28]. When
the solvent is constituted of phospholipids, Laurdan
emission strongly depends on their phase state, being
blue, nonrelaxed, in the gel phase, and red, relaxed, in
the liquid-crystalline phase (Fig. 1) [29,30]. From the
gel to the liquid-crystalline phase, the Laurdan emission
maximum shifts by about 50 nm, from a maximum at
440 nm in the gel to a maximum at 490 nm in the liquid-
crystalline phase. This behavior indicates that in the gel
phase the molecular dynamics of the dipoles surrounding
Laurdan is slower than the probe lifetime. During the
phospholipid transition, and in vesicles composed of a
mixture of phospholipids in the two phases, Laurdan
emission spectra with intermediate maximum wave-
length and center of mass are observed [29,30]. Also the
excitation spectrum of Laurdan is modified by the po-
larity of solvents and by the phase state of phospholipids
[27,30]. In isotropic nonpolar solvents, the Laurdan ex-
citation spectrum shows a single blue band, with the
maximum at about 340 nm. In polar solvents, the Laur-
dan excitation spectrum is red-shifted, with a maximum
at about 370 nm, and displays a second excitation band
with a maximum at about 390 urn. This second, red,
excitation band increases its intensity with the increase
of the polarity of the solvents and has been attributed to
the stabilization of the probe ground-state Lc~ confor-
mation, due to polar solvent molecules oriented around
the probe dipole, i.e., already relaxed [27]. When Law'-
dan is inserted in phospholipid bilayers, the red excita-
tion band is present and its intensity depends upon the
phase state of phospholipids. In the gel phase, this ex-
citation band is particularly intense, constituting the
maximum excitation (Fig. 1), while in the liquid-crys-
talline phase, the red band is still present, but the max-
imum excitation is at 355 nm, corresponding to the blue
excitation band [30]. Thus the intensity of the red ex-
citation band depends both on the polarity of the Laur-
dan environment and, when inserted in phospholipid
vesicles, also on their phase state. Laurdan molecules
that populate this red band are those molecules sur-
rounded by oriented dipoles and, if present, by phos-
pholipids in the gel phase.
LAURDAN GENERALIZED POLARIZATION
A method for the treatment of the differences ob-
served in both the excitation and emission spectra of
Laurdan has been developed. The generalized polariza-
tion (GP) is defined as GP = (Ig -/lo ) / (Ig +/~o), where
Ig and/~o are the intensities observed at the wavelengths
typical of the maximum excitation or, alternatively, of
the maximum emission in the gel and in the liquid-crys-
talline phase, respectively [30]. The choice of the precise
excitation and emission wavelengths for the calculation
of the GP value is dictated by the wavelengths of the
maximum intensity in the two phases. In our calculation
of excitation GP, the intensities of the emission at wave-
lengths of 440 and of 490 nm have been chosen. In our
calculation of emission GP, the intensities of the exci-
62 Parasassi and Gratton
Excitation
Emission
~q~'-"solid"\
0.4 -~--
n 0.1
coexisting domains
d"_____q~ ......
i
-0"51 360 41 0 460 51 0
Wavelength (nrn)
Fig. 2. Laurdan excitation and emission
GP
spectra of phospholipids
in different phases, as reported in Fig. 1. Excitation
GP
spectra were
calculated by
GP = (I44o - I49o)/(I44o +
/490), using excitation wave-
lengths from 320 to 420 nm. Emission
GP
spectra were calculated by
GP
= (I4~o -
I340)/(I4~o +
I34o), using emission wavelengths from 420
to
550 ran.
tation at 410 and 340 nm have been chosen, at the edges
of the two excitation bands. The two GP values were
then calculated by excitation GP
= (I440 - 1490)
/ (/44O +
1490) and emission GP = (I4~o -/34o) / (I410 +/340). With
the above definitions, high GP values are measured in
the gel, while low GP values are measured in the liquid-
crystalline phase [30]. The use of the GP offers several
advantages: (i) once characteristic values for the GP in
the gel and in the liquid-crystalline phase are deter-
mined, the property of additivity of fluorescence polar-
ization can be used to quantitate the domains [27]; (ii)
all properties of fluorescence polarization can be used,
among others the possibility of determining the kinetics
of dipolar relaxation by an equation equivalent to the
Perrin equation [27,30,31]; (iii) in contrast to common
ratiometric measurements used for probes displaying
spectral sensitivity to the properties of the environment,
such as pH and calcium concentration probes, the GP
measurement does not require calibration; and (iv) the
measurement is fast and easy.
GP values typical of the gel and of the liquid-crys-
talline phase have been determined, using vesicles com-
posed of phospholipids differing in their acyl and in their
polar head residues, at pH between 4 and 10 [27]. The
determined excitation GP values are 0.6 and -0.2 for
the gel and liquid-crystalline phases, respectively, using
an excitation wavelength of 340 nm and emission wave-
lengths of 440 and 490 nm, while the emission GP val-
ues are 0.4 and -0.5 for the gel and liquid-crystalline
phases, respectively, using an emission wavelength of
440 nm and excitation wavelengths of 410 and 340 nm
[27]. The GP value has been found to depend only on
the phospholipid-phase state, and not on the type and
charge of their polar residue. Following these observa-
tions, the process of dipolar relaxation which determines
the spectral properties must then be attributed to a few
water molecules, present at the hydrophobic-hydrophilic
interface of the bilayer, where the fluorescent moiety of
Laurdan is located [27]. Rotational rean'angement of the
Laurdan molecule itself during its excited state has been
excluded as the origin of the dipolar relaxation, from the
measurement of Laurdan "classical" polarization along
its emission spectrum [30] and from experiments using
various Laurdan derivatives (unpublished observations).
With respect to the "bulk" water, whose rotational ki-
netics is known to be on the picosecond time scale, those
water molecules in the bilayer involved in the relaxation
should rotate more slowly. Moreover, the dynamics of
these water molecules is of the order of nanoseconds
only when phospholipids are in the liquid-crystalline
state. The reason for the absence of dipolar relaxation in
the gel state can be an even slower motion of water
molecules.
WAVELENGTH DEPENDENCE OF THE
GENERALIZED POLARIZATION TO
ASCERTAIN THE COEXISTENCE OF PHASE
DOMAINS
Once a GP value intermediate between the high
value of the gel and the low value of the liquid-crystal-
line phase is observed, the property of additivity can be
utilized to quantitate each domain [27]. An intermediate
value per se is not a proof of the coexistence of different
domains, since it can originate from a homogeneous en-
vironment with intermediate properties. The behavior of
the GP value as a function of excitation and of emission
wavelength can be used to distinguish between these two
cases. Both excitation and emission GP spectra are flat
in the gel phase (Fig. 2). In the liquid-crystalline phase,
the excitation GP spectrum shows decreasing values
with increasing excitation wavelength, while the emis-
sion GP spectrum shows increasing values with increas-
ing emission wavelength. When domains of different
phases coexist in the membrane, the behavior of the GP
value is opposite to that observed in the liquid-crystal-
line phase. The excitation GP increases and the emission
GP decreases with increasing excitation and emission
wavelength, respectively (Fig. 2) [27]. For the favorable
spectroscopic properties of Laurdan, we can easily dis-
tinguish between a homogeneous liquid-crystalline
phase and a mixed phase of coexisting domains. The red
band of the excitation spectrum is populated by Laurdan