 |
INTRODUCTION |
Cholesterol is essential for optimal membrane transport,
receptor-effector coupling, cell recognition, and other eukaryotic cellular processes (reviewed in Ref. 1). Increasing evidence indicates
that plasma membrane cholesterol is organized into lateral and
transbilayer cholesterol-rich microdomains (reviewed in Ref. 2) such as
lipid rafts and caveolae (reviewed in Refs. 2-6). These domains
contain proteins involved in multiple cellular functions including
signaling and cholesterol transport. For example, overexpression of
caveolin-1 in mice stimulates high density lipoprotein uptake via
plasma membrane caveolae and markedly increases plasma high density
lipoprotein-cholesterol (8). In contrast, caveolin-1-deficient mice are
lean and resistant to diet-induced obesity but show
hypertriglyceridemia and exhibit vascular abnormalities (9-11). There
is a strong association of cholesterol abnormalities with cytotoxicity,
sickle cell acanthacytosis, Niemann-Pick C disease, Alzheimer's
disease, atherosclerosis, diabetes, and obesity (reviewed in Refs.
12-16). However, almost nothing is known regarding cholesterol
organization within cholesterol-rich microdomains.
The available evidence suggests that the cholesterol content of plasma
membrane microdomains is very high, possibly sufficient for cholesterol
phase separation into crystals. Plasma membranes exhibit the highest
cholesterol:phospholipid molar ratio (e.g. 0.5-1.0) for any
intracellular membrane, and this ratio appears to be closely regulated
(reviewed in Ref. 2). Excess membrane cholesterol phase separates into
crystalline cholesterol at cholesterol:phospholipid molar ratios higher
than 1.0 in model membrane bilayers (reviewed in Ref. 17), plasma
membranes (18), and the lysosomal matrix as well as cytosol of
macrophage foam cells (19). Cholesterol monohydrate crystals within
plasma membranes of smooth muscle cells (18, 20, 21), lysosomes and
cytoplasm of macrophage-derived foam cells (14, 19), and
atherosclerotic plaques (13) are cytotoxic. Plasma membranes of certain
types of cell derivation are composed of cholesterol-rich cytofacial
leaflets (reviewed in Ref. 22) and lateral lipid raft/caveolar
microdomains (reviewed in Refs. 2, 3, 5, 23, and 24), wherein the
cholesterol:phospholipid ratio is expected to be
1. However, if
cholesterol phase separates into crystalline cholesterol at such
ratios, it is difficult to conceive how these microdomains effectively
facilitate cholesterol transport, potocytosis, and signaling (2,
3, 5, 23, 24). Therefore, it is important to determine the structural form of cholesterol within the plasma membrane, especially in lipid
rafts/caveolae, as well as in other intracellular organelles and
membranes. Unfortunately, relatively few noninvasive, nonperturbing techniques exist for real time visualization of cholesterol structures in biological membranes or in living cells (18, 25).
The present investigation addresses these issues by taking advantage of
two major technological advances. First, the spectral properties of a
naturally occurring fluorescent sterol, dehydroergosterol (DHE),1 differ in monomeric
and crystalline forms (26-30). DHE consists of up to 20% membrane
sterol in yeast and sponge membranes, is taken up from the culture
medium of microorganisms and cultured fibroblasts, codistributes with
endogenous sterol among intracellular membranes (replaces nearly 90%
of endogenous membrane cholesterol), and does not alter cell growth,
membrane structure, or function of sterol-sensitive membrane proteins
(reviewed in Refs. 1, 2, 26, and 31). The structural, transfer, and
functional properties of DHE closely mimic those of cholesterol in
lipoproteins and membranes (1, 2, 22, 26). Second, the development of
new fluorescence imaging technologies, i.e. multiphoton
laser scanning microscopy (MPLSM), now makes it possible to resolve multiple membrane and cellular forms of cholesterol in living cells
(32, 33). Because DHE absorption occurs in the ultraviolet region,
single photon excitation (used in conventional, video, and confocal
imaging microscopy) results in significant photobleaching and
phototoxicity (32, 34). In contrast, MPLSM utilizes infrared radiation
to overcome these problems (32, 33) and, as shown herein, now provides
high resolution images of multiple structural forms of sterol within
living cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Sucrose was purchased from Sigma. Cholesterol and
ergosterol were obtained from Steraloids (Newport, RI).
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was from
Avanti Polar Lipids (Alabaster, AL). Stock cholesterol (10 mg/ml)
solutions were prepared in 95% ethanol with 1 mol % butylated
hydroxytoluene and stored at
70 °C. Human recombinant sterol
carrier protein-2 was prepared as described earlier (35). Anti-caveolin-1 and anti-flotillin-1 antisera were obtained from Affinity Bioreagents (Golden, CO) and Transduction Laboratories (San Jose, CA), respectively. 1,6-Diphenyl-1,3,5-hexatriene (DPH), LysoTracker Green, BODIPY FL C5-ceramide, and Nile Red were
obtained from Molecular Probes (Eugene, OR). Dehydroergosterol
(
5,7,9(11),22-ergostatetraen-3
-ol) was synthesized from
ergosterol (Steraloids, Newport, RI) (36) or purchased from Sigma and
further purified by high performance liquid chromatography (HPLC) (36).
DHE was prepared in 100% ethanol as a stock solution (5 mg/ml)
containing 1 mol % butylated hydroxytoluene and stored at
70 °C.
All solvents were HPLC grade or better, and aqueous buffers herein
described will be filtered 10 mM PIPES, pH 7.4.
Cell Culture, Cellular Subfractionation, and Membrane
Isolation--
L-cell fibroblasts were obtained and cultured as
described earlier (37). Cells were cultured for 2 days at 37 °C in a
CO2 incubator with Higuchi medium containing 10% fetal
bovine serum supplemented with DHE (20 µg/ml medium) (38). Cells were
subfractionated to obtain plasma membranes (38), endoplasmic reticulum
(2), lysosomes (39), mitochondria (40), and lipid droplets (41). Lysosomal membranes were resolved from DHE crystals present in the
lysosomal matrix by lysing purified lysosomes in hypotonic buffer (39)
followed by overlayering 1.2-ml aliquots (in 10 mM Tris, 1 mM EDTA, pH 7.4) on a discontinuous sucrose gradient composed of 0.2 ml of 30% (w/v) sucrose (d = 1.1328),
0.3 ml of 20% (w/v) sucrose (d = 1.0854), and 0.4 ml
of 14% (w/v) sucrose (d = 1.0586) in 10 mM
Tris, 1 mM EDTA, pH 7.4. After 18 h of centrifugation at 200,000 × g using a RP55S swinging bucket rotor and
RC M120 Micro-ultracentrifuge (Sorvall, Wilmington, DE), pure sterol
crystals appeared at d <1.054 (interface between 14%
sucrose layer and the sample) (13), whereas lysosomal membranes
appeared at the bottom of the gradient. Individual membrane fraction
purity was determined by quantitative Western blotting with antisera to
appropriate markers for each membrane fraction as described in the
cited papers.
Isolation of Lipid Rafts from the Plasma Membrane
Fraction--
Lipid rafts/caveolae-enriched fractions were isolated by
a nondetergent method (42, 43). The plasma membrane fraction was
layered on a discontinuous sucrose gradient (5 ml of 24% (w/v) sucrose
(d = 1.093), 5 ml of 36% (w/v) sucrose
(d = 1.139)) in 10 mM Tris, 1 mM EDTA, pH 7.8. After 90 min at 39,000 rpm on an SW40Ti
swinging bucket rotor and Beckman XL-90 ultracentrifuge (Beckman
Instruments, Fullerton, CA), pure sterol crystals appeared at
d <1.054 (13), whereas plasma membrane-enriched fractions appeared at the 36-24% (w/v) sucrose interface (43). The relative enrichment of the lipid raft/caveolar membrane fraction was determined by quantitative Western blotting using antisera to caveolin-1 and
flotillin-1, basically as described for other membrane markers (39).
Measurement of Force-area Isotherms of Pure and Mixed
Monolayers--
Pure cholesterol or dehydroergosterol monolayers or
mixed monolayers containing varying amounts of sterol together with
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine were
compressed on 140 mM NaCl in water at 22 °C (under an
argon atmosphere in the dark) with a KSV surface barostat (KSV
Instruments Ltd., Helsinki, Finland). The barrier speed during
compression did not exceed 3.4 Å2/molecule/min. Isotherms
at a surface pressure of 35 mN/m were recorded using
proprietary KSV software (44).
Model Membrane Vesicle Preparation--
Small unilamellar
vesicles (SUV) and large unilamellar vesicles (LUV) were prepared to
contain 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dehydroergosterol (DHE), and cholesterol in the following indicated proportions (45): 65:35:0 and 65:5:30 (SUV and LUV); 40:60:0,
33:67:0, and 25:75:0 (LUV). The median radii of SUV and LUV curvature
were 15 ± 3 and 53 ± 10 nm, respectively, determined by a
Coulter N4 Plus Photon Correlation Spectrometer (Beckman Instruments,
Miami, FL).
Spectral Properties of DHE in Solvents and
Membranes--
Absorbance spectra were obtained with a Lambda 2 Dual
Wavelength Spectrophotometer (PerkinElmer life Sciences). Steady-state fluorescence excitation and emission spectra were obtained with a PC1
Photon Counting Fluorometer (ISS Instruments, Champaign, IL). Spectra
were analyzed with Grams32 (Thermo Galactic, Salem, NH). Light scatter
was avoided by use of narrow monochromator slits, low concentrations,
and appropriate cut-off filters. Any Raman scattering was subtracted
from all excitation and emission spectral data. Artifacts because of
inner filter effects were avoided by keeping the absorbance of sample
solutions at the excitation wavelength (324 nm) below 0.15. All
absorbance measurements were performed in 1 ml- or 3.5-ml quartz
cuvettes. Fluorescence measurements of organelles and associated
membranes were performed with samples in 2 ml of filtered 10 mM PIPES buffer, pH 7.4, in a quartz cuvette with the
temperature regulated to 37 ± 0.3 °C through use of a water
heating bath (Fisher). Six integrated intensity measurements (335-600
nm) were performed on concentrations of DHE in 10 mM PIPES,
pH 7.4, and 100% ethanol, ranging from 2.5 to 10 µM.
Aqueous Solutions of DHE and Cholesterol for Micelle
Determination--
Solutions of 2.5 and 10 µM
cholesterol or DHE were prepared in 95% EtOH and evaporated onto
glassware under dry nitrogen. Each of the deposited sterols was then
redissolved in 10 mM PIPES buffer at 37 °C under heavy
vortexing. Filtration of portions of these solutions was made with a
single pass through either an Avanti Mini-Extruder with a 0.1-µ
polycarbonate membrane (Whatman) or a 0.5-µ filter (Millipore,
Bedford, MA). Similarly, DPH in tetrahydrofuran was evaporated onto
glassware followed by addition of the unfiltrated and filtrated sterol
solutions and intense vortexing. The sterol:DPH ratio was maintained at
250:1. Fluorescence emission spectra were obtained as described above.
Determination of DHE Steady-state Polarization during Exchange
Assays--
Steady-state fluorescence polarization measurements of DHE
in plasma membranes, endoplasmic reticulum, mitochondria, lysosomes, and lysosomal membranes at 37 °C were performed as described earlier (38-40). Residual light scatter (from both donor and acceptor
membranes) contribution to polarization data was corrected by
converting polarization to anisotropy according to the equation
r = 2P/(3
P), and
subtracting the residual fluorescence anisotropy of both donor and
acceptor membranes (i.e. not containing DHE) from all
experimental data. Absorbance (324 nm) of sample solutions in 10 mM PIPES buffer, pH 7.4, was kept below 0.15.
Lysosomal Sterol Transfer--
Sterol transfer between isolated
lysosomes was determined using a fluorescent sterol (DHE) exchange
assay as described previously (39, 46). The basis of the assay (release
from self-quenching of DHE in the donor lysosome and transfer to
acceptor lysosome lacking DHE), validation of DHE as a probe for
cholesterol transfer, in-depth descriptions of the assay, proper
controls, and justification for the lack of polarization change in the
absence of acceptor membranes were provided in the above cited
publications. Standard curves for DHE in lysosomal-lysosomal membrane
exchanges were determined earlier (39, 46).
Multiphoton Laser Scanning Microscopy (MPLSM) and Image
Analysis--
MPLSM of DHE, Nile Red, LysoTracker Green, and BODIPY FL
C5-ceramide was performed on intact L-cells (L
arpt
tk
) cultured on two-well Lab-Tek
chambered cover glasses (VWR, Sugarland, TX). Prior to imaging, cells
were supplemented for 2 days with the addition to the medium of 20 µg/ml DHE, either from a 5 mg/ml stock solution of DHE in anhydrous
ethanol or with 10 mM 65:35 large unilamellar vesicles of
POPC and DHE (prepared as described above). For colocalization
experiments of DHE with Nile Red, the cells cultured with DHE were
washed with Puck's buffer and then incubated with 100-400
nM Nile Red for ~30 min. The concentration was titered to
different levels in this range depending upon the amount of excitation
power used in the multiphoton imaging process described below. For
imaging with LysoTracker Green, L-cells were grown to confluency on a
chambered cover glass supplemented with 20 µg/ml medium of DHE for 2 days and then washed in Puck's buffer (1 mM
Na2HPO4, 0.9 mM
H2PO4, 5.0 mM KCl, 1.8 mM CaCl2, 0.6 mM MgSO4,
6 mM glucose, 138 mM NaCl, and 10 mM HEPES). Cells were then incubated with 50 nM
LysoTracker Green in Puck's buffer at 37 °C for 1 h; medium
was removed, and cells were washed several times with Puck's buffer.
To ascertain crystalline DHE and Golgi colocalization, BODIPY FL
C5-ceramide was purchased as already made complexes with
bovine serum albumin. Murine L-cells with DHE (20 µg/ml medium) were
grown on chambered cover glasses for 2 days and then washed with a
Hanks' buffered saline solution (HBSS) with 10 mM HEPES,
pH 7.4, subsequently termed HBSS/HEPES. After washing, the cells were
loaded for 30 min with 5 µM BODIPY FL
C5-ceramide-bovine serum albumin at 4 °C in HBSS/HEPES
medium. Afterward, the medium was removed, and the cells were washed
with HBSS/HEPES at 4 °C. After replacing the medium, the chambered cover glass were then placed back in the incubator for 30 min at
37 °C. The cells were once again washed with HBSS/HEPES medium and
immediately imaged under multiphoton excitation.
MPLSM fluorescence imaging was performed (33) using an MRC1024
Multiphoton Laser Scanning Microscope controlled by LaserSharp software
and equipped with an external descanned 3 detector unit (Bio-Rad) (47).
Briefly, the excitation source was a femtosecond Coherent Mira 900 mode-locked Ti:Sapphire laser with broadband optics pumped at 12 watts
with a Coherent Sabre argon ion laser (Coherent, Palo Alto, CA). The
excitation light (900-930 nm) was delivered to an Axiovert 135 (Zeiss
Inc., New York, NY) microscope stage via a modified epiluminescence
light path. A Zeiss 63× Plan-Apochromat (1.4 N.A.) or 100×
Fluar (1.3 N.A. with higher transmittance between 350 and 400 nm) oil immersion objective was used for all images. Selection of
900-925 nm as the multiphoton excitation wavelength range allowed
simultaneous excitation of crystalline DHE and monomeric DHE
(three-photon excitation), with either Nile Red, LysoTracker Green, or
BODIPY FL C5-ceramide (two-photon excitation). The
fluorescence emission of the respective probe molecules was collected
by the same objective and passed into the Bio-Rad external detector
unit where the emission was appropriately separated into different wavelength regions and collected by three photomultiplier tubes (PMT)
as follows: (i) multialkali PMT1 collected emission through a
D455:30-nm filter; (ii) multialkali PMT2 collected emission through a
HQ575:150-nm filter; (iii) bi-alkali PMT3 collected emission through a
D375:50-nm filter. All filters and dichroics used in detection were
from Chroma Technology (Brattleboro, VT). Images (Kalman filtered) were
analyzed and presented using a combination of software packages
including MetaMorph Image Analysis Software (Advanced Scientific
Imaging, Meraux, LA), Adobe Photoshop 5.0 (San Jose, CA), CorelDraw 9 (Ottawa, Ontario, Canada), and National Instruments LabView
6i equipped with IMAQ Vision 5.0 (Austin, TX). MPLSM of
autofluorescence in L-cells was obtained for each experiment at the
same excitation power and wavelength as used for collecting probe
fluorescence. The gain and black levels of each photomultiplier tube
were optimized to minimize the autofluorescence signal and to maximize
the fluorescence signal in the probe-supplemented cells. Any residual
autofluorescence signal at high detection sensitivity in the 375:50-nm
channel was subtracted.
In order to separate crystalline and monomeric DHE signals for
colocalization with other probes and thus minimize the spectral "bleed through" of each form into the other channel, software was
written using LabView 6i equipped with IMAQ Vision 5.0 utilizing the pixel color intensity representation of pixel
fluorograms. The program, called FLUOROGRAM_IM, provided the user with
a way to import an RGB image and form a pixel fluorogram from the red and green components by plotting the red intensities along the vertical
axis and the green intensities along the horizontal axis. Once a pixel
fluorogram was created, the program allowed the user to collectively
select pixels by drawing a region (specifiable in any shape or size)
around the pixels belonging to either form of DHE. Pixels belonging to
crystalline DHE were chosen based upon the ratio
I475nm:I375nm
1, whereas the remaining pixels with
I475nm:I375nm <1 were
deemed monomeric (intensity ratio = 1 along the diagonal of the
fluorogram). After the selection was made, the software program turned
off those pixels in the original image whose correlating intensities do
not fall in the selected region. The new gray scale images
corresponding to each form (crystalline, 455 channel; monomeric, 375 channel) is then saved for further colocalization analysis with the
second probe (third channel) used in the experiment. Areas of low
concentration and/or low sensitivity as well as saturable conditions
produce ambiguous correlations but mostly can be overcome by imaging
cells at different levels of sensitivities depending upon the focus of
the localizable product. Generally, the bulk of the intensity was
separable and highly indicative of the structural form of sterol.
After background correction, colocalization coefficients for each
pseudo-color (red or green channel) were calculated using Equation 1
(Bio-Rad Tech Note 8) (48),
|
(Eq. 1)
|
where Cred and
Cgreen are the colocalization coefficients;
I
and
I
represent the red
and green intensities of the ith pixel after background
subtraction, and
rg(i) and
gr(i) are Kronecker deltas that represent
intensity independent colocalization weights. For every ith
pixel,
rg(i) =
gr(i) = 0, if there is only a red intensity
or green intensity but not both.
rg(i) =
gr(i) = 1, only if there is both a red and
green intensity associated with the ith pixel. Therefore, a
coefficient represents the fraction of the total intensity of the
filtered fluorescence of the corresponding probe in an image that is
colocalizing with the filtered fluorescence of another probe.
 |
RESULTS |
Synthesis and Purification of DHE--
To determine structural
properties of sterol in membranes of living cells, it was essential
that DHE be free of contaminants. A commercially available DHE as well
as DHE freshly synthesized and purified herein (see "Experimental
Procedures") were examined. Absorbance spectra of the two
preparations differed only slightly in the region where DHE maximally
absorbs (peak maxima near 311, 324, and 340 nm) (Fig.
1A). However, below 275 nm the
absorbance of the two preparations deviated significantly, indicating
the presence of impurities. HPLCs of commercially available DHE (10 µg/10 µl solvent) revealed four peaks (Fig. 1B).
However, only peak 3 (representing 83% of total) exhibited absorption
characteristics of DHE. Peaks 1 and 2 were sample impurities, whereas
peak 4 was due to a small solvent impurity. In contrast, the DHE
synthesized and purified as indicated under "Experimental
Procedures" was 98% pure as indicated by HPLC (Fig. 1C).
When Peak 3 (pure DHE in Fig. 1C) was pooled from a number
of HPLC runs, dried under nitrogen, and reinjected on the HPLC column,
the resultant DHE was 99.7% pure (Fig. 1D, peak
3).

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Fig. 1.
Synthesis and purification of DHE.
A, absorbance spectra of DHE (19 µg/ml ethanol).
Curve 1 represents DHE purchased commercially as indicated
under "Experimental Procedures." Curve 2 represents
recrystallized DHE that was synthesized and freshly prepared as
indicated under "Experimental Procedures." B, HPLC of
commercially available DHE (10 µg/10 µl solvent). Peak
3, the only material absorbing at 325 nm (absorption maximum of
DHE) represents 83% of total. Peaks 1 and
2 represent impurities present in the sample, and peak
4 is due to a small solvent impurity. C, HPLC scan of
DHE (10 µg/10 µl solvent) synthesized and recrystallized as
indicated under "Experimental Procedures." DHE present in
peak 3 represents 98% of total. D, DHE was
collected from chromatograms (peak 3 in C), dried
under nitrogen, and reinjected (30 µg/10 µl solvent) on the HPLC
column. The final purified DHE (peak 3 in D)
represented 99.7% pure DHE. All HPLC eluants were monitored by
absorbance at 205 nm as indicated under "Experimental
Procedures."
|
|
Absorbance Spectral Properties of DHE in Ethanol and in Aqueous
Buffer--
Although DHE is monomeric at low concentrations in
ethanol, aggregation of DHE occurs in aqueous buffers due to the low
critical micellar concentration (20-30 nM for cholesterol
and DHE) of sterols (26-30). Absorbance spectral properties of DHE in
aqueous buffers (10 µM in 10 mM PIPES, pH
7.4) differed significantly from those of monomeric DHE (10 µM in ethanol). In ethanol DHE displayed maxima at 311, 324, and 340 nm (Fig. 2A). In
contrast, DHE in aqueous buffer (Fig. 2C) exhibited the
following differences. (i) The absorption spectrum in aqueous buffer
was slightly broader with maxima (314, 329, and 347 nm) that were
red-shifted by 3, 5, and 7 nm from those in ethanol. (ii) The
absorption peak profile of DHE in aqueous buffer was significantly
altered. The ratio of absorption maxima at 329:314 nm was 1.03, significantly lower than that for absorption maxima at 324:311 nm in
ethanol near 1.14. (iii) The molar extinction coefficient of DHE in 10 mM PIPES, pH 7.4 (EM
~5500 M
1 cm
1) was 2.4-fold
lower than for DHE in anhydrous ethanol
(EM ~13,000
M
1 cm
1), both measured at 324 nm.

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Fig. 2.
Spectral properties of DHE in ethanol and
aqueous buffer. A, absorbance spectrum of DHE (10 µM) in ethanol. B, fluorescence emission
spectrum of DHE (10 µM) in ethanol, upon excitation at
324 nm. C, absorbance spectrum of DHE (10 µM)
in 10 mM PIPES, pH 7.4. D, fluorescence emission
spectrum of DHE (10 µM) in 10 mM PIPES, pH
7.4, upon excitation at 324 nm. E, (fluorescence emission
spectrum of DHE (10 µM) in 10 mM PIPES, pH
7.4, in D) (fluorescence emission spectrum of DHE
(10 µM) in ethanol, B (normalized to the
emission at 375 nm in B)). In order to normalize the
fluorescence emission spectrum of DHE in ethanol (B) to that
in 10 mM PIPES, pH 7.4, the fluorescence emission spectrum
in B was slightly (5 nm) red-shifted to be superimposable on
that in D. The emission intensity at 375 nm was then
normalized to the fluorescence emission intensity at 375 nm in
D. This allowed subtraction of the two spectra to obtain the
fluorescence emission spectrum of crystalline DHE.
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|
Fluorescence Emission Characteristics of DHE in Ethanol and in 10 mM PIPES Buffer, pH 7.4--
The fluorescence emission
spectral properties of DHE in ethanol and aqueous buffer differed even
more than the absorption spectra. The fluorescence emission of DHE in
ethanol (Fig. 2B) was Stokes shifted ~46 nm as compared
with the absorption spectrum (Fig. 2A), essentially the same
as that obtained for DHE in other alcohols (1-butanol, 2-propanol, and
methanol) (28). The fluorescence emission spectrum of DHE in ethanol
(Fig. 2B) was a near mirror image of the absorption spectrum
(Fig. 2A) and exhibited vibrationally resolved fluorescence
emission maxima near 354, 370, and 390 nm (Fig. 2B). In
contrast, the aqueous fluorescence emission spectrum of DHE was
consistent not only with the presence of monomeric DHE spectral
features as observed for DHE in ethanol (Fig. 2B) but also
indicated the appearance of a completely new fluorescent entity
differing markedly from that in ethanol (Fig. 2B) as
follows. (i) DHE emission maxima (356 and 375 nm) were red-shifted as
compared with those in ethanol. (ii) New fluorescence emission maxima
appeared at 403 and 426 nm (Fig. 2C). (iii) The relative
emission intensity of the new emission maxima was severalfold greater
than those at 356 and 375 nm (Fig. 2D). This increased
intensity was at some expense of the DHE emission at 356 and 375 nm
because their intensities were 0.65 that of the emission maxima in
ethanol (Fig. 2B). However, the overall integrated intensity
of the emission from the DHE in 10 mM PIPES was 2.7 ± 0.2 greater than that in ethanol. (iv) The quantum yield (88) of DHE in
10 mM PIPES, pH 7.4, was increased 6-fold from 0.04 in
ethanol (56) to 0.25 ± 0.02, due to the overall increase in
integrated emission and decrease in absorption.
A fluorescence emission spectrum of the new DHE fluorescing species,
formed in 10 mM PIPES, pH 7.4 (Fig. 2E), was
resolved from that of the monomeric DHE by subtraction of the
normalized fluorescence emission spectrum of DHE in ethanol from that
in aqueous buffer. The new spectral profile of DHE exhibited
fluorescence emission maxima at 403 and 426 nm, with a slight shoulder
at 460 nm. The appearance of this new fluorescence could be due to an impurity in the DHE preparation. Although this may potentially be the
case in studies where partially pure DHE (e.g. Fig. 1, A and C) was used, in the present investigation
the DHE was highly purified (99.7% pure, Fig. 1D). In
contrast to the commercially available preparation, the highly purified
DHE did not contain significant impurities. These data indicated that
the DHE species emitting maximally at 426 nm in PIPES, pH 7.4, was not
due to a contaminant in the DHE preparation, but rather it was due to the presence of aggregating DHE in the form of microcrystals or micelles. That the DHE was present primarily as microcrystals, rather
than micelles, was resolved as follows.
First, aqueous solutions of DHE or cholesterol were examined under
polarized light microscopy using the Zeiss Axiovert Microscope with
63× oil objective. Clearly visible under cross-polarization of DHE
solutions were small birefringent crystals. For comparison, aqueous
solutions of cholesterol were also made and examined using the same
technique. Under these conditions, small birefringent crystals of
cholesterol monohydrate were detected, confirming earlier results of
others (49). Importantly, the DHE microcrystals were similar in size
and shape as that those of cholesterol.
Second, aliquots of the aqueous solutions of DHE (2.5 and 10 µM) were filtered through 0.1-µm filters, which would
allow passage of micelles (but not microcrystals) with sizes on the
order of 4 nm. Emission spectra over the region 350-600 nm of the
aqueous solutions of DHE were recorded before and after filtration
using an excitation wavelength of 324 nm. Before filtration, the
spectrum of DHE showed the new fluorescing species as shown in Fig.
2D. However, after filtration no spectral emission was
observed above the background noise. By increasing the slit sizes on
the monochromator, sensitivity was increased but without any detectable
DHE emission. In an effort to determine whether there are possible
contributions from larger aggregates of micelles, the filtration was
repeated using a 0.5-µm filter. Once again no DHE emission was
detected in the filtrate.
Third, the fluorescence probe DPH was used to detect the presence of
micelles. In order to excite DPH and not DHE, the excitation wavelength
was changed to 369 nm, and emission spectra were recorded over the
range 385-600 nm. DPH was added to the unfiltrated and filtrated
solutions of DHE as described under "Experimental Procedures." Initially, a spectrum was obtained on DPH in buffer without DHE to
determine any background level of DPH emission. No detectable emission
was observed. Next, the spectra of DPH added to the DHE solutions
before and after filtration were obtained, also showing no DPH
emission. As a control, similar concentrations of cholesterol in
aqueous buffer were prepared and filtered similarly to that of the DHE.
As was the case with DHE, no DPH emission was detected for cholesterol.
These experiments suggest that the spectral emission of the unfiltered
solutions of DHE is derived from microcrystals larger than 0.5 µm.
This is supported by comparison of spectral characteristics of aqueous
dispersions of DHE with those of DHE in the form of crystalline powder
(30). Although the purity of the latter preparation of DHE is unknown,
the fluorescence emission spectrum and lifetime components of this
crystalline powder were remarkably similar to that shown for the new
aqueous form (Fig. 2D). In summary, the fluorescence
emission spectrum of the new, microcrystalline form of DHE appearing in
10 mM PIPES, pH 7.4, was distinct from that of monomeric
DHE appearing exclusively in ethanol.
Stability of Crystalline DHE in Aqueous Media--
The stability
of DHE microcrystals in 10% fetal bovine serum-containing media (used
to supplement and incorporate DHE into cultured cells over a period of
2-3 days) was determined to show if serum constituents in the cell
culture medium could solubilize DHE microcrystals. Spectra of DHE in
media at 37 °C were collected for different time points over a
period of 70 h, followed by calculation of emission peak ratios
(426:355 and 426:373 nm) and comparison to those of DHE in ethanol and
10 mM PIPES, pH 7.4. At time t = 0 (Table
I) monomeric DHE emission peak ratios at
426:355 and 426:373 were very low, 0.42 ± 0.01 and 0.298 ± 0.004, respectively (n = 6). This was in contrast to
those of crystalline DHE in aqueous buffer, which were 10-fold higher,
4.8 ± 0.1 and 3.4 ± 0.2 (n = 6) (Table I).
In media containing 10% fetal bovine serum at time 0, the ratios of
the crystalline DHE peak at 426 nm to those of monomeric DHE (355 or
373 nm) were 2.91 ± 0.07 and 2.81 ± 0.05, respectively
(n = 6). These ratios were not significantly altered with increasing incubation time over 70 h (Table I). A similar finding has been reported for crystalline cholesterol in the presence of plasma (13).
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Table I
Relative proportions of crystalline/monomeric DHE in
solvents and membranes
DHE was 10 µM in solvents and model membranes. Where
indicated, values represent the mean ± S.E., n = 5-7. LUV refers to large unilamellar vesicle membranes. SUV refers to
small unilamellar vesicle membranes. All membrane spectra of DHE
emission were obtained in 10 mM PIPES, pH 7.4. Control
L-cell fibroblasts were cultured with 10% fetal bovine serum medium
containing 20 µg/ml DHE, and subcellular organelles and lysosomes
were isolated as described under "Experimental Procedures." ND, not
determined.
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Spectral Properties of DHE in Model Membranes, Effect of
Sterol:Phospholipid Ratio--
As indicated in the Introduction, it is
thought that sterol phase separates at molar ratios of
sterol:phospholipid >1.0. Yet relatively little is known regarding the
exact nature of phase-separated sterol (transbilayer dimers, pure
crystalline phase, and superlattices) in membranes. Furthermore, the
methods (NMR and x-ray crystallography) used to detect the phase
separation are not sensitive enough to detect the presence of low
quantities/clusters/microdomains of phase-separated sterol in
membranes. To resolve these issues, DHE was incorporated into model
membranes (large unilamellar vesicles (LUV)) composed of varying molar
ratios of DHE and
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).
The fluorescence emission spectrum of DHE in LUVs composed of POPC:DHE
at 65:35 (i.e. sterol:phospholipid molar ratio of 0.54) exhibited emission maxima near 355, 373, and 394 nm, as well as a small
shoulder near 426 nm (Fig.
3A). These maxima were shifted from those observed in ethanol but not quite to the extent of those
found in aqueous buffer. The ratios of DHE fluorescence emission peak
intensities at 426:355 and 426:373 were 0.54 and 0.33, respectively,
slightly higher than for monomeric DHE in ethanol and suggesting the
presence of a small amount of crystalline DHE (Table I). When the
emission spectrum of DHE in ethanol was normalized to that of DHE (at
373 nm) in the LUV and subtracted, the difference spectrum (Fig.
3B) exhibited emission maxima characteristic of crystalline
DHE in aqueous buffer (Fig. 2E). The spectra before and
after subtraction were integrated to give values for the fluorescence emission that were corrected for the increased quantum yield exhibited by the crystalline DHE. These corrected values were used to obtain the
approximate % of DHE in a crystalline arrangement which was very small
(~0.2%) for LUV composed of POPC:DHE at 65:35 (i.e. DHE:phospholipid molar ratio of 0.54) (Table I). It should be noted
that in membranes composed of POPC:DHE at 65:35, the DHE undergoes
significant self-quenching by Forster energy transfer (29). The
fluorescence emission intensity was 63% lower than expected, based on
comparison with that of DHE in membranes composed of
POPC:DHE:cholesterol of 65:5:30 (Fig. 3A) and corrected for dilution. However, as expected from Forster energy transfer, the shape
of the DHE emission spectrum was unaffected. Consequently, the
self-quenching did not account for the presence of the small percentage
of crystalline DHE in these membranes.

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Fig. 3.
Excitation, emission, and difference emission
spectra of DHE in model membrane bilayers. DHE was incorporated
into large unilamellar vesicles (LUV, A) or small
unilamellar vesicles (SUV, C) as indicated under
"Experimental Procedures." All fluorescence emission difference
spectra (B and D) were obtained by subtraction of
the emission spectrum of DHE in ethanol, normalized to the emission of
DHE at 373 nm in the respective membrane. The values indicated in the
figure represent the ratios of POPC:DHE:cholesterol in the respective
membrane preparations. Fluorescence (A) spectra of LUV are
shown where the proportions of these lipids are as follows: Curve
1, 25:75:0; curve 2, 65:5:35; curve 3,
33:66:0; curve 4, 40:60:0; and curve 5, 65:35:0.
Difference (B) spectra of LUV are shown with proportions
65:5:35 (curve 1), 65:35:0 (curve 2), 25:75:0
(curve 3), 33:66:0 (curve 4), and 40:60:0
(curve 5). The proportions of lipids for fluorescence
(C) and difference (D) spectra of SUV were
65:5:30 (curve 1) and 65:35:0 (curve 2).
Fluorescence excitation spectra (200-360 nm) were obtained with
emission detected at 375 nm. Fluorescence emission spectra (340-600
nm) were obtained with excitation at 324 nm.
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The fluorescence emission spectrum of DHE in LUV composed of POPC:DHE
at 40:60 (i.e. sterol:phospholipid molar ratio of 1.5) exhibited very similar emission maxima near 355, 373, and 395 nm, along
with a more distinct shoulder near 426 nm (Fig. 3A) as
compared with that at 0.54 DHE:phospholipid ratio. At 1.5 DHE:phospholipid molar ratio, the ratios of intensities of the DHE
fluorescence emission peaks at 426:355 and 426:373 nm were 0.84 and
0.47, respectively (Table I). These ratios were significantly higher
than those of monomeric DHE in ethanol, higher than for DHE in LUV with
0.54 molar ratio of sterol:phospholipid, and thereby suggested the presence of a small amount of crystalline DHE (Table I). Subtraction of
the normalized emission spectrum of DHE in ethanol (normalized to that
of 1.5 molar ratio DHE:phospholipid LUV at 373 nm) yielded a difference
spectrum, which again exhibited emission maxima (Fig. 3B)
characteristic of crystalline DHE in aqueous buffer (Fig. 2E). After correction for the higher quantum yield of
crystalline DHE, this indicated that increasing the molar ratio of
sterol:phospholipid by 3-fold (from 0.54 to 1.5) increased by 15-fold
the amount of crystalline sterol to 7.7% (Table I). Further increment
in DHE:phospholipid ratio to 2 and 3 increased the crystalline DHE
content to 9.5% (Table I). Thus, emission spectral properties of DHE
detected the presence of small amounts (1-10%) of crystalline sterol
in LUV membranes composed of a broad range of sterol:phospholipid molar ratios (i.e. 0.54-3.00).
Force-area Isotherms of Monolayers Formed from DHE or Cholesterol
or Mixtures of the Two Sterols with POPC--
In order to examine
whether the DHE separation into crystalline form in membrane lipids
reflects that of cholesterol, force-area isotherms of DHE and
cholesterol in POPC monolayers were compared (Table
II) at a surface compression of 35 mN/m, in the range of that typical of lipid bilayers (44,
50). Increasing the mol % of either DHE or cholesterol in the
monolayer reduced the mean molecular area (Å2), due to the
condensing effect of sterols on phospholipid membranes. At 35 mol %
sterol, where no crystalline cholesterol was detected by x-ray
crystallography and birefringence (51), the mean molecular area of the
monolayer composed of DHE and POPC, 43.6 Å2, was not
statistically different from that composed of cholesterol and POPC,
43.4 Å2 (Table II). Even at 70 mol % sterol, where
crystalline cholesterol was detected by x-ray crystallography and
birefringence (51), the mean molecular area of the monolayer composed
of DHE and POPC, 41.0 Å2, was not statistically different
from that composed of cholesterol and POPC, 39.9 Å2 (Table
II). Finally, there was no statistical difference in mean molecular
area occupied by pure DHE (40.40 Å2) versus
pure cholesterol (39.15 Å2) (Table II). Because there was
no difference in the condensing effects of DHE versus
cholesterol in the POPC monolayers, these data are consistent with DHE
behaving similarly to cholesterol with regard to formation of
crystalline forms in monolayers at high mol % of sterol.
Spectral Properties of DHE in Model Membranes, Effect of Membrane
Curvature--
Although the transbilayer distribution of sterol in LUV
is nearly equal, packing constraints significantly affect the
transbilayer distribution of sterol in small unilamellar model
membranes (SUV). In SUV with cholesterol:phospholipid ratios
0.3
(i.e.
23 mol % cholesterol), the molar ratios of
sterol:phospholipid in the inner leaflet and outer leaflet are about
1.3 and 0.9, respectively (52), and sterol in the inner leaflet is much
more tightly packed than in the outer leaflet (22). To examine if this
increase might induce the formation of crystalline phase sterol, SUVs
with limiting radii of curvature were prepared as described under
"Experimental Procedures." The radii of curvature in the SUV and
LUV, measured by photon correlations spectroscopy, were 15 ± 3 and 53 ± 10 nm, respectively. The fluorescence emission spectrum
of DHE in SUV (POPC:DHE of 65:35, i.e. sterol:phospholipid
molar ratio of 0.54) exhibited emission maxima (Fig. 3C)
indistinguishable from those in LUV with the same composition (Fig.
3A). The ratios of intensities of the DHE fluorescence
emission peaks in the SUV at 426:355 and 426:373 nm were 0.69 and 0.43, respectively, slightly higher than for monomeric DHE in ethanol,
thereby suggesting the presence of low levels of crystalline DHE in SUV
(Table I). When the emission spectrum of DHE in ethanol was normalized
to that of DHE (at 370 nm) in the SUV and subtracted, the difference
spectrum (Fig. 3D) exhibited emission maxima characteristic
of crystalline DHE in aqueous buffer (Fig. 2E).
Because any DHE expelled from the membrane would form microcrystals in
the buffer and give rise to spectra resembling Fig. 2E, the
small unilamellar vesicles were subjected to a single pass extrusion
using a 0.1-µm membrane as performed for the aqueous solutions of
DHE. The spectra obtained before and after the extrusion were exactly
the same, decreasing the likelihood that the spectra (Fig.
2E) originates from extraneous DHE in the buffer. Also, an
additional centrifugation run like the one used to sediment debris and
multilamellar vesicles (sediments >85% of the microcrystals) did not
produce any variability in the amount of the crystalline portion of the
SUV DHE spectrum. Model membrane data from Table I indicated that with
decreasing radius of curvature of the membrane the crystalline sterol
increased 30-fold from ~0.2% in LUV to ~6% in SUV.
Spectral Properties of DHE in Plasma Membranes Isolated from L-cell
Fibroblasts--
The plasma membrane exhibits the highest molar ratio
of sterol:phospholipid ratio in the cell, 0.5-1.0 (2). The fact that the inner (cytofacial) leaflet of the plasma membrane contains 80-90%
of plasma membrane sterol but only half of the plasma membrane phospholipid (2, 53 54) indicates molar ratios of
sterol:phospholipid in the cytofacial leaflet as high as 1.8. However, it is not known if this sterol in the plasma membrane may be
phase-separated into crystalline sterol. Therefore, L-cells were
cultured in the presence of DHE, and the DHE-enriched plasma membranes
were isolated as described under "Experimental Procedures."
Excitation and emission spectral intensities of DHE were higher in
plasma membranes than any other cellular membrane fractions (Fig.
4A). Whereas emission spectra
of DHE in plasma membranes (Fig. 4A) largely resembled those
of DHE in model membranes (Fig. 3), after subtraction of the monomeric
DHE (Fig. 4C), a small amount of crystalline DHE (about
5.4% of total) was detectable in the plasma membrane fraction (Table
I). This was confirmed by examination of emission spectral peak ratios,
all of which were greater than those of monomeric DHE (Table I).
Detection of a small amount of crystalline sterol in these membranes
was not due to adherence of DHE crystals to the membranes followed by
coisolation on sucrose gradients. On the contrary, plasma membrane vesicles appeared at much higher density (27-30% sucrose) than did
DHE crystals (14% sucrose). Furthermore, mixing of crystalline DHE
with membranes not containing DHE, followed by re-isolation on sucrose
gradients, showed that there was no cross-contamination of the purified
membranes with crystalline DHE.

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Fig. 4.
Spectral properties of DHE in subcellular
membrane fractions and organelles isolated from L-cell
fibroblasts. Excitation spectra were obtained at 37 °C while
monitoring emission at 375 nm. Emission spectra were obtained at
37 °C upon excitation at 324 nm. All fluorescence emission
difference spectra were obtained by subtraction of the emission
spectrum of DHE in ethanol, normalized to the emission of DHE at 373 nm
in the respective membrane. Protein concentration in all samples was 14 µg/ml in 10 mM PIPES, pH 7.4. A, excitation
(left side) and emission (right side) spectra of
DHE in isolated plasma membrane vesicles (PM), microsomal
membrane vesicles (ER), mitochondrial membrane vesicles
(MITO), and lipid droplets (LD). Lipid droplet
spectrum shown is the result of subtraction of a spectrum of lipid
droplets without DHE from a spectrum of lipid droplets with DHE.
B, emission spectra of DHE in lipid raft/caveolar membrane
subfractions. The fold enrichment of caveolin-1 and flotillin-1 in
these subfractions is shown as a subpanel. C, emission
difference spectra of DHE in plasma membrane vesicles (PM),
lysosomal membrane (LM), and caveolar membrane
(CAV)-enriched fractions. D, excitation
(left side) and emission (right side) spectra of
DHE in isolated lysosomes (LYSO). E, effect of
increasing ethanol on emission spectra of DHE in isolated lysosomes.
Curve 1 shows DHE emission upon addition in lysosomes (14 µg of protein/ml 10 mM PIPES, pH 7.4). Curve 2 shows the same sample as in curve 1 diluted with ethanol to
a final concentration 25%. Curve 3 shows the same sample as
in curve 1 diluted with ethanol to a final concentration
50%. Curve 4 shows the lysosomes (14 µg of protein) added
directly to ethanol (final ethanol concentration >99%).
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Spectral Properties of DHE in Plasma Membrane Microdomains, Lipid
Raft-enriched Subfractions from L-cell Fibroblasts--
Plasma
membrane sterol is laterally distributed into cholesterol-rich (lipid
rafts and caveolae) and cholesterol-poor regions. Lipid rafts/caveolae
account for about 10% of total plasma membrane sterol but consist of
only 1% of plasma membrane surface (reviewed in Ref. 23). Thus, the
small amount (i.e. 5.4%) of crystalline sterol in the
plasma membrane may be highly enriched in these microdomains. To
examine this possibility, DHE containing lipid raft/caveolae-enriched
subfractions were isolated from the plasma membrane fraction by a
nondetergent technique (see "Experimental Procedures"). The lipid
raft/caveolar subfraction was enriched nearly 3-fold in caveolin-1 and
flotillin-1 as compared with the parent plasma membrane fraction (Fig.
4B). The emission spectra of DHE in lipid raft/caveolar
membrane subfraction (Fig. 4B) were similar to those of
plasma membranes (Fig. 4A). DHE exhibited low intensities of
crystalline DHE emission peaks at 403 and 426 nm (Fig. 4B),
in the range of those observed for plasma membranes (Fig.
4A) and model membranes containing low molar ratios of
DHE:phospholipid (Fig. 3A). Close examination of the DHE
emission spectra in lipid raft/caveolar membrane subfraction after
subtraction of monomeric DHE (Fig. 4C) showed <0.5%
crystalline DHE, considerably less than in the plasma membrane. The
very small and almost negligible presence of the crystalline DHE in the
lipid raft/caveolar membrane subfraction was confirmed by examination
of emission spectral peak ratios, all of which were only slightly
greater than those of monomeric DHE (Table I).
Spectral Properties of DHE in Lysosomes--
In order to examine
the structural form of DHE in the intracellular organelle membrane
containing the second highest cellular sterol phospholipid ratio,
i.e. lysosomal membrane, it was necessary to first isolate
the lysosome organelle. Lysosomes contained high quantities of
crystalline sterol as follows. (i) The emission spectrum of DHE in
isolated lysosomes exhibited large peaks near 426 and 403 nm (Fig. 4,
D and E). (ii) The DHE emission spectral peak
ratios at 426:355 and 426:373 were 2.02 ± 0.15 and 1.42 ± 0.06, respectively (n = 3) (Table I). These ratios were
much higher than for monomeric DHE in ethanol (Table I) but were more similar to those of crystalline DHE in buffer or cell culture medium
(Table I). (iii) Quantitative evaluation of the emission spectra showed
that 75% of the fluorescence intensity was representative of the
crystalline form. (iv) Lysosomes had very high ratios of sterol:phospholipid, consistent with the separation of DHE into a
crystalline phase, crystallization of DHE in the lysosomal matrix, and/or incomplete solubilization/efflux of DHE crystals from the lysosomal matrix into the cell interior. The total DHE:phospholipid molar ratios in the lipids extracted from the lysosomes, determined from the extinction coefficient of DHE and by HPLC analysis, were 1.6 and 1.5, respectively.
Spectral Properties of DHE in Lysosomal Membranes--
To
determine whether the crystalline DHE detected in the lysosome (see
above) was largely confined to the lysosomal matrix or actually a part
of the lysosomal membrane, lysosomes were subjected to hypotonic lysis
followed by an additional sucrose gradient purification to resolve DHE
crystals (matrix-derived) from lysosomal membranes (see "Experimental
Procedures"). The lysosomal membranes (density >30% sucrose) were
clearly resolved from lysosomal matrix-derived crystals of DHE (density
>16% sucrose). The molar ratio of total sterol:phospholipid
(determined by HPLC of lipids extracted from purified lysosomal
membranes) was 0.38, consistent with earlier reports (39). This molar
ratio was more than 4-fold lower than that of intact lysosomes,
confirming the presence of a large amount of crystalline DHE in the
lysosomal matrix.
The emission spectral intensity of DHE in the lysosomal membrane
fraction (not shown) was lower than that of DHE in plasma membranes but
higher than that of DHE in endoplasmic reticulum and mitochondrial
membranes (Fig. 4A). This reflected the relative sterol
content of the lysosomal membranes versus plasma membranes, microsomes (endoplasmic reticulum), and mitochondrial membranes. Close
examination of the DHE emission spectra in lysosomal membranes showed
(after subtraction of monomeric DHE) a clearly resolvable crystalline
DHE emission spectrum (Fig. 4C). The crystalline DHE present
in the purified lysosomal membrane fraction represented about 6.9% of
total sterol. The presence of a small amount of crystalline DHE in the
lysosomal membranes was also substantiated by examination of emission
spectral peak ratios, both of which were greater than those of
monomeric DHE (Table I).
The possibility that the crystalline DHE in the lysosomal membrane was
the result of DHE crystals (from the lysosomal matrix) sticking to the
lysosomal membrane and cosedimenting at the same density as lysosomal
membranes was considered as follows. (i) Crystalline DHE was mixed with
isolated lysosomal membranes that did not contain DHE. (ii) The mixture
was placed on the same sucrose gradient and the two fractions were
clearly separated. Examination of emission spectra of the two fractions
revealed that all the crystalline DHE appeared much higher in the
gradient (density <14% sucrose) and was clearly separated from the
much denser lysosomal membrane fraction (density >30% sucrose), which
did not contain any DHE. Finally, it is important to note that the
small amount of lysosomal membrane DHE found in the crystalline form
was in the same range as that observed for DHE in model membranes
wherein no crystalline DHE was present during the membrane preparation (Table I).
Spectral Properties of DHE in Microsomal Membrane and Mitochondrial
Membrane Fractions Isolated from L-cell Fibroblasts--
The ratio of
sterol:phospholipid in microsomes (endoplasmic reticulum) and
mitochondrial membranes, near 0.2 and <0.1, respectively, was much
lower than that in plasma membranes or lysosomal membranes (reviewed in
Refs. 32 and 39). The excitation and emission spectral intensities of
DHE in the microsomal and mitochondrial membranes were both lower than
in plasma membranes (Fig. 4A) or lysosomal membranes (not
shown). The low intensities of DHE emission spectra in these membrane
fractions (Fig. 4A) produced, upon subtraction of monomeric
DHE, spectra with large signal to noise but indicated only small
amounts of crystalline DHE, <1% of the total.
Disruption of Crystalline DHE in Lysosomes, Potential Role of
Sterol Carrier Protein-2--
Whereas crystalline DHE clearly
accumulated in lysosomes of L-cells supplemented with crystalline DHE
in the medium (Fig. 4D), crystalline DHE can be converted to
monomeric form both in vitro and in intact cells. Addition
of ethanol converted the emission spectrum of DHE in the lysosomes from
primarily crystalline to essentially monomeric (Fig. 4E).
This was confirmed upon examination of the DHE emission peak ratios at
426:355 and 426:373 nm (Table I).
The spontaneous transfer of DHE from lysosomal donors (enriched in
crystalline DHE) to lysosomal acceptors (containing no DHE) was
determined as the release from self-quenching and detected as increased
DHE polarization (see "Experimental Procedures"). Spontaneous DHE
transfer from lysosomes was relatively slow (Fig. 5A, solid circles)
with an initial rate of molecular sterol transfer of 0.048 ± 0.020 pmol/min. However, upon addition of 1.5 µM sterol carrier protein-2, the DHE fluorescence polarization and anisotropy increased rapidly, consistent with transfer of DHE from donor lysosomes
to acceptor lysosomes (Fig. 5A, open circles).
The initial rate of DHE transfer was enhanced nearly 13-fold, from
0.048 ± 0.020 to 0.601 ± 0.096 pmol/min. To determine
whether SCP-2-mediated DHE transfer from the lysosomes disrupted the
crystalline DHE or simply transferred DHE from the lysosomal membranes,
emission spectra of DHE were obtained for lysosomes (isolated from
L-cells supplemented with DHE) at the beginning (t = 0 min, spectrum 1, Fig. 5B) and at the end
(t = 270 min, spectrum 2, Fig.
5B) of SCP-2-mediated DHE transfer from lysosomal donors
(enriched in crystalline DHE) to lysosomal acceptors (containing no
DHE). DHE emission spectral peak ratios (426:355 and 426:373 nm)
decreased 3-4-fold to 0.54 and 0.36, respectively, after 270 min of
SCP-2-mediated sterol exchange (Table I). These ratios resembled those
observed for DHE in purified lysosomal membranes, 0.66 and 0.48, respectively (Table I). It should be noted that simply adding SCP-2 to
DHE crystals in the intact lysosomes did not alter the DHE emission spectral properties. These data suggest that SCP-2 converted the crystalline DHE to the monomeric spectral form by quickly transferring the DHE from the crystalline DHE in the donor lysosomes to acceptor lysosomes.

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Fig. 5.
SCP-2-mediated DHE transfer from intact
lysosomes disrupts crystalline DHE in vitro.
A, the transfer of DHE from lysosomal donors (enriched in
crystalline DHE) to lysosomal acceptors (contain no DHE) was determined
as described under "Experimental Procedures." In the absence of
sterol carrier protein-2, the spontaneous transfer of DHE was indicated
by slight change in the anisotropy over the time period of the
experiment. However, upon addition of 1.5 µM SCP-2, the
DHE fluorescence anisotropy increased rapidly, consistent with transfer
of DHE from donor lysosomes to acceptor lysosomes. B,
emission spectra of DHE in lysosomal membranes (isolated from L-cells
supplemented with DHE) at the beginning (curve 1,
t = 0 min, spectrum 1) and at the end (curve
2, t = 270 min, spectrum 2) of sterol carrier
protein-2-mediated DHE transfer from lysosomal donors (enriched in
crystalline DHE) to lysosomal acceptors (contain no DHE). C,
the bottom curve represents the anisotropy of 1.25 µg/ml
DHE in 10 mM PIPES buffer over 3 h. With the addition
of lysosomal membrane acceptors, a near-linear increase in anisotropy
occurs over the same period, indicating spontaneous exchange of DHE
with cholesterol. Upon addition of 1.5 µM SCP-2, the
protein-mediated transfer of DHE as measured by a change in anisotropy
follows an exponential rise over time.
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In order to confirm further that sterol exchange from DHE crystals was
facilitated by SCP-2, the anisotropy of the transfer of sterol between
DHE crystals and lysosomal membrane acceptors was monitored. The
experiment consisted of three steps involving donors of DHE crystals
created by adding 2.5 µg of DHE (similar to that seen in lysosomes)
to 2 ml of 10 mM PIPES buffer. (i) Anisotropy of donor
crystals was monitored for 3 h (the flat curve in Fig. 5C). (ii) A
spontaneous transfer was performed between the donor crystals and
lysosomal membrane acceptors (middle curve of Fig.
5C). (iii) 1.5 µM SCP-2 was included and once
again the anisotropy of donor crystal and lysosomal membrane acceptors
was monitored for 3 h (the top curve of Fig.
5C). Initial rates were calculated by using a standard curve
based upon previously published lysosomal membranes exchange data.
Exchanges between crystals and lysosomal membrane acceptors showed that
1.5 µM SCP-2 increased the initial rate of molecular
sterol transfer nearly 7-fold, from 4.67 ± 0.65 (spontaneous) to
30.73 ± 5.2 pmol/min (with 1.5 µM SCP-2). Clearly,
anisotropy increased for spontaneous sterol transfer from both
lysosomal and pure crystalline DHE donors to acceptor lysosomal
membranes, an effect 13- and 7-fold larger, respectively, in the
presence of SCP-2.

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Fig. 6.
Multiphoton laser scanning microscopy
of DHE crystals. DHE crystallized on coverslips by evaporation of
carrier ethanol solvent under a stream of nitrogen. DHE was excited at
900 nm, and fluorescence emission was monitored simultaneously through
three separate dichroic filters: A, green
(455:30-nm dichroic filter) preferentially detected
crystalline DHE; B, blue (375:50-nm dichroic
filter) preferentially detected monomeric DHE; C,
red (575:150-nm dichroic filter), detected neither monomeric
nor crystalline DHE at very low sensitivity levels. D
represents the merged images of A-C.
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Multiphoton Laser Scanning Microscopy of Crystalline DHE--
The
above differences in DHE fluorescence emission suggested that it might
be possible to visualize simultaneously monomeric and crystalline DHE
in living cells in real time through fluorescence microscopy.
Unfortunately, the fact that DHE absorbs in the ultraviolet region over
the range 275-345 nm (excitation maxima near 311, 324, and 340 nm,
Fig. 1A) makes visualization by conventional and confocal
fluorescence microscopy difficult because this requires the use of
quartz optics, results in significant photobleaching and
photodestruction, and yields images with limited resolution and
excessive photobleaching over time (32, 34). These difficulties are
largely avoided by the use of multiphoton excitation with infrared
light at 900-925 nm resulting in simultaneous absorption of three
infrared photons equivalent to single photon excitation in the
ultraviolet range (i.e. 300-308 nm). Furthermore, in
multiphoton excitation only those DHE molecules within the objective
focal volume (about 0.01 cu µ) are excited, thereby allowing
emission to be measured only from the DHE molecules excited in the
focal volume. Through use of scanning optics and external detectors, images similar to those obtained by confocal microscopy are obtained but lacking in the drawbacks of the latter and exhibiting dramatically reduced photobleaching. These features of multiphoton excitation were
therefore used to determine whether crystalline and monomeric forms of
DHE could be discriminated by multiphoton laser scanning microscopy.
DHE was crystallized on coverslips by evaporation of carrier ethanol
solvent under a stream of nitrogen followed by three-photon excitation
at 900 nm (Fig. 6). To resolve crystalline and monomeric DHE,
fluorescence emission was monitored simultaneously through separate
dichroic filters as described under "Experimental Procedures": crystalline DHE, distinctly visible as needle-like structures (up to 50 µm long and usually 1-10 µm wide), was preferentially detected by
monitoring emission with a 455:30-nm dichroic filter (Fig.
6A, green channel); monomeric DHE was
preferentially detected with a 375:50-nm dichroic filter (Fig.
6B, blue channel). For the sake of imaging the
crystals without saturation, the gain was decreased in the 455:30-nm
channel to prevent saturation, whereas the gain in the 375:50-nm
channel was maximal. The red channel (Fig. 6C, 575:150-nm
dichroic filter) detected neither monomeric nor crystalline DHE and was
used to establish whether DHE fluorescence emission spilled over into
this wavelength range. Analysis of the merged image (comprised of
A-C) revealed primarily green DHE crystals (Fig.
6D). Thus, multiphoton excitation at 900 nm was useful for
determining the presence of crystalline DHE by MPLSM utilizing
basically the same spectroscopic techniques as for the in
vitro studies except that emission was collected simultaneously in
the 455:30- and 375:50-nm channels.
Multiphoton Laser Scanning Microscopy of Crystalline DHE in
Lysosomes of Living L-cell Fibroblasts--
As indicated above, DHE
crystals were stable over many days in 10% fetal bovine cell culture
medium incubated at 37 °C. Because crystalline DHE was detected in
lysosomes isolated by subfractionation of L-cells cultured with DHE
crystals, consistent with the observation that L-cells actively
phagocytose particles as large as several microns (55), the possibility
that these crystals could be visualized in lysosomes by MPLSM was
examined. L-cell lysosomes, ranging from 0.4 to 3.5 µm in diameter,
were readily visualized by staining intact cells with LysoTracker and
monitoring LysoTracker emission with a 575:150-nm dichroic filter (Fig.
7, red pixels).
Microcrystalline DHE was simultaneously visualized by monitoring
emission through a 455:30-nm dichroic filter (blue pixels).
Finally, some of the crystalline DHE taken up by the L-cells was
solubilized/metabolized as indicated by the presence of monomeric DHE
detected by monitoring emission with a 375:50-nm dichroic filter (Fig.
7, green pixels). The merged image of all three
photomultiplier tubes (Fig. 7) revealed the following.

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Fig. 7.
Multiphoton laser scanning microscopy of DHE
and LysoTracker Green, a lysosomal marker, in L-cell fibroblasts.
L-cell fibroblasts were cultured on chambered cover glass for 2-days in
medium containing 10% fetal bovine serum and DHE (20 µg/ml), washed
3-4 times with Puck's buffer, and incubated with LysoTracker Green as
described under "Experimental Procedures." Excitation was at 925 nm. Red represents LysoTracker detected through a dichroic
filter (575:150 nm); green represents DHE detected through a
dichroic filter (375:50 nm), and blue represents DHE
detected through a dichroic filter (455:30 nm). Arrow 1 points to a representative white region where
red, green, and blue colocalize
(i.e. LysoTracker, monomeric DHE, crystalline DHE);
arrow 2 points to a representative magenta region
where red and blue colocalize (LysoTracker and
crystalline DHE); arrow 3 points to a representative
yellow pixilated area where green and
red colocalize (monomeric DHE and LysoTracker); arrow
4 points to a representative red region where
LysoTracker stains lysosomes containing neither monomeric nor
crystalline cholesterol; arrow 5 points to a representative
green region showing primarily monomeric DHE; arrow
6 points to a representative cyan region where
green and blue colocalize (monomeric DHE and
crystalline DHE).
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First, distinct white pixilated areas (indicating
colocalized crystalline DHE (blue), monomeric DHE
(green), and LysoTracker (red)) were present in
some lysosomes (Fig. 7, arrow 1). Second, distinct
magenta pixilated areas (indicating colocalized crystalline DHE (blue), but not monomeric DHE, and LysoTracker
(red)) were present in some lysosomes (Fig. 7, arrow
2). Third, distinct yellow/orange pixilated areas
(indicated colocalization of monomeric DHE (green), but
little crystalline DHE, and LysoTracker (red)) were present in some lysosomes (Fig. 7, arrow 3). Fourth, distinct
red pixilated areas (indicating LysoTracker staining regions
without either monomeric or crystalline cholesterol) showed that L-cell
fibroblasts had a substantial population of lysosomes that did not
appear to contain significant amounts of either monomeric or
crystalline DHE (Fig. 7, arrow 4). Fifth, distinct
green (Fig. 7, arrow 5) and cyan (Fig.
7, arrow 6) pixels showed that some monomeric and crystalline/monomeric DHEs, respectively, were not present in the lysosomes.
Resolution of the Two Forms of DHE in Living L-cell
Fibroblasts--
Due to the complexity of visually distinguishing the
different color tones necessary for deciphering the spatial
colocalization of crystalline and monomeric forms, a procedure for
separating the two forms utilizing the differences in spectral ratios
was developed (see "Experimental Procedures").
First, the 455:30-nm channel and the 375:50-nm channel were
pseudo-colored red and green, respectively, and merged (Fig.
8A). This was imported into
the software program that plotted the fluorogram (Fig. 8B)
as described under "Experimental Procedures." This fluorogram showed that there was a strong correlation in the more red
(crystalline) pixels, which clearly have a higher ratio over the more
green (monomeric) pixels and appeared in somewhat linear fashion along the ordinate axis. The more green pixels appeared in a more complex pattern due to the saturation of monomeric regions of localization, with a population aligned linearly along the horizontal axis and another population saturating along the right vertical axis. Some saturation was allowed in order to detect lower concentrations. As will
be shown later, at least some of those saturated monomeric DHE regions
in the pixelgram represented monomeric DHE in lipid droplets.

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Fig. 8.
Colocalization of LysoTracker Green with the
monomeric and crystalline forms of dehydroergosterol L-cell
fibroblasts. A, green and red
represents DHE detected through dichroic filters (375:50 and 455:30 nm,
respectively). B, the fluorogram produced from image in
A. C (showing the crystalline form) and
D (showing the monomeric form) are gray scale images derived
by selecting the pixels with intensities above and below the diagonal
in the fluorogram (B) as described under "Experimental
Procedures." E is a colocalization of crystalline DHE
(C, green) with LysoTracker (not shown,
red) with F as the corresponding fluorogram.
G is a colocalization of monomeric DHE (D,
green) with LysoTracker (not shown, red).
H is the corresponding fluorogram.
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Second, the more red pixels (pixels with
Ired:Igreen
1) were
selected by drawing a line along the diagonal and then enclosing a
triangle around the upper left scattering of more red pixels. The
resulting gray scale image was created (Fig. 8C) and then merged with the third or 575:150-nm channel (LysoTracker emission, not
shown). This merged image (Fig. 8E) showed the crystalline (green) colocalizing with LysoTracker Green (red)
displayed as a fluorogram (Fig. 8F) and colocalization
coefficients; Cgreen = 0.97 (crystalline DHE)
exhibited a strong correlation with the LysoTracker Green, whereas
Cred = 0.32 (LysoTracker Green) did not. This
was corroborated by the image (Fig. 7) that showed many lysosomes
colored in bright red and thereby evidencing the fact that they did not
have any crystalline DHE.
Third, the more green pixels (pixels with
Ired:Igreen <1) were
selected by drawing a triangle around the lower right scattering of
more green pixels. This resulting image was created (Fig.
8D) and then merged with the 575:150-nm channel (LysoTracker
emission, not shown). The resulting new merged image showed monomeric
DHE pseudo-colored green and LysoTracker pseudo-colored red (Fig. 8G). The fluorogram of the merged image (Fig. 8H)
indicated that the LysoTracker, Cred = 0.54, exhibited a slightly larger amount of colocalization with the monomeric
DHE, Cgreen = 0.46. Both coefficients indicated
additional separate localization of each probe in other cellular sites.
In summary, MPLSM allowed simultaneous three-photon excitation
(crystalline and monomeric DHE) and two-photon excitation (LysoTracker Green) and separate, simultaneous detection of each in living cells in
real time. This noninvasive technology took advantage of the enhanced
emission properties of crystalline DHE to image multiple structural
forms of sterol in living cells both within lysosomes and outside of
the lysosomal compartment. It should be noted that the intensities of
the crystalline DHE in L-cells were so bright that, to avoid saturating
the photomultiplier tubes, it was not possible in these images to
visualize simultaneously the DHE enriched in the plasma membrane.
However, the latter was clearly imaged under other conditions (see below).
Multiphoton Laser Scanning Microscopy of DHE and BODIPY FL
C5-ceramide, a Golgi Marker, in L-cell Fibroblasts--
To
determine whether crystalline DHE entered the Golgi compartment of
L-cell fibroblasts, cells were incubated with BODIPY FL
C5-ceramide (see "Experimental Procedures"). MPLSM at
900 nm excitation resulted in simultaneous three-photon excitation of DHE (monomeric and crystalline) and emission detected as above. Concomitantly, MPLSM at 900 nm resulted in simultaneous two-photon excitation of BODIPY FL C5-ceramide whose emission
was detected through a dichroic filter (575:150 nm). When the
images were treated as described above (not shown), the fluorogram of
the Golgi marker versus crystalline DHE (not shown)
indicated that only 9% of DHE in Golgi was crystalline. Thus, once the
DHE left the lysosomal compartment it did not significantly accumulate
in the Golgi in a crystalline form.
Multiphoton Laser Scanning Microscopy of DHE and Nile Red, a Lipid
Droplet Marker, in L-cell Fibroblasts--
To determine whether DHE
was translocated for storage in intracellular lipid droplets of L-cell
fibroblasts, the cells, as described under "Experimental
Procedures," were cultured on chambered cover glass and incubated
with Nile Red, a probe that accumulates primarily in neutral
lipid droplets. Tuning the Ti: Sapphire laser to 925 nm resulted in
simultaneous three-photon excitation of DHE (monomeric and crystalline)
and two-photon excitation of Nile Red. The fluorescence emission of all
fluorophores was simultaneously detected as follows: Nile Red with a
575:150-nm dichroic filter; crystalline and monomeric with a 455:35-
and a 375:50-nm dichroic filter, respectively. Two levels of
sensitivity and objectives were used to collect images as follows: (i)
no attenuation of excitation power and a Zeiss 100× Fluar oil
immersion lens (high transmission characteristics for 350-400 nm but
decreased flatness of field); (ii) 80% attenuation of excitation power
and a Zeiss 63× Plan Apochromat oil immersion lens. To determine the
degree of colocalization of the monomeric DHE with Nile Red in L-cell lipid droplets, a region of cells was selected showing little crystalline DHE; sensitivity was increased, and the images were merged
(Fig. 9A). The
blue/cyan regions showed the presence of very low
crystalline DHE (Fig. 9A, arrow 1). Importantly,
monomeric DHE was clearly visualized in plasma membranes and other
membranes (Fig. 9A, arrow 2). The bright
saturated yellow regions (Fig. 9A, arrow 3;
upper right corner of fluorogram, Fig. 9B) were lipid droplets wherein monomeric DHE colocalized with the lipid droplet probe, Nile Red. The colocalization coefficient,
Cred = 0.98 showed that almost all of the Nile
Red colocalized with monomeric DHE, whereas
Cgreen = 0.42 showed that just under half of the
monomeric DHE intensity was colocalized with Nile Red in the lipid
droplets (Fig. 9B). This implied that >50% of monomeric
DHE intensity was in membranes, vesicles, and in diffuse distributions
in the cytoplasm.

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Fig. 9.
Multiphoton laser scanning microscopy of DHE
and Nile Red, a lipid droplet marker, in L-cell fibroblasts.
L-cell fibroblasts were cultured on cover glass for 2-days in medium
containing 10% fetal bovine serum and DHE (20 µg/ml) as described
under "Experimental Procedures." The cells were washed 3-4 times
with Puck's buffer, followed by incubation with Nile Red as indicated
under "Experimental Procedures." Excitation at 925 nm elicited
simultaneous three-photon excitation of DHE (monomeric and crystalline)
and Nile Red. Nile Red emission (red pixels in A
and C) was detected through a 575:150-nm dichroic filter;
crystalline DHE (blue pixels in A and
C) was detected through a 455:30-nm dichroic
filter; monomeric DHE (green pixels in A and
C) was detected through a 375:50-nm dichroic filter.
A shows the merged image obtained from high power excitation
on a region of cells with small amounts crystals (arrow 1),
plasma membrane (arrow 2) as well as other intracellular
membranes, and lipid droplets (arrow 3). B is the
fluorogram resulting from colocalization of the monomeric DHE
(green) that was obtained using our software as described
under "Experimental Procedures" and Nile Red (red).
C is a merged image obtained from slightly lower power
excitation of a region of cells showing larger amounts of crystals
(arrow 1), organelles with DHE but with no Nile Red
(arrow 2), and lipid droplets (arrow 3).
D is the fluorogram of the merged images detected through
the dichroic filters 455:30 (red) and 375:50 nm
(green). The crystalline (upper reddish correlated
pixels) and the monomeric forms (lower greenish
correlated pixels) are clearly separated across the diagonal. As
described under "Experimental Procedures," each clustering of
pixels was selected to produce individual gray scale images
representative of crystalline and monomeric DHE. E is the
fluorogram determined by merging the crystalline portion with Nile Red,
and F is the fluorogram obtained from merging the monomeric
portion with Nile Red. Clearly, the correlation coefficients confirm
that the majority of Nile Red emission from lipid droplets colocalizes
with monomeric DHE.
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Some of the bright pixilated areas in the merged image were
cyan (Fig. 9A, arrow 1), representing
crystalline DHE colocalized with monomeric DHE. To obtain a more
quantitative estimation of the distinction between crystalline and
monomeric DHE with Nile Red, another image was taken at lower laser
excitation power and with the 63× objective centered upon cells
containing more phagocytosed DHE crystals. Once again the images were
merged under the same pseudo-colors as previously (Fig. 9C),
where the blue regions represented crystalline DHE (Fig.
9C, arrow 1), green regions showed
monomeric DHE (Fig. 9C, arrow 2), and the
yellow regions represented monomeric DHE colocalized with
Nile Red in lipid droplets (Fig. 9C, arrow 3).
Once again, an image was formed (not shown) composed of DHE alone:
crystalline (red) and monomeric (green). Two
distinct and well separated populations of pixels were obtained in this
pixel fluorogram (Fig. 9D). The high slope, upper pixel population was composed of crystalline DHE, whereas the lower pixel
population was composed of monomeric DHE. Each population was
encircled, and a corresponding gray scale image was created as
described under "Experimental Procedures." Each of these images was
merged with the 575:150-nm channel (Nile Red in lipid droplets, not
shown). The fluorograms resulting from the merged images of crystalline
DHE (green) with Nile Red (red) and monomeric DHE (green) with Nile Red (red) were shown in Fig. 9,
E and F, respectively. The coefficients of
colocalization with respect to the crystalline form were quite small
for both DHE and Nile Red: Cred = 0.01, Cgreen = 0.03 (Fig. 9E). However, the
monomeric form colocalized quite well with Nile Red in lipid
droplets. With this concentration of Nile Red, most of the
Nile Red was observed in the bright lipid droplets, confirmed in
the image (yellow round regions and no visible red
areas), by the fluorogram, and the coefficient,
Cred = 0.99 (Fig. 9F). The monomeric
DHE, on the other hand, clearly resided in other areas illustrated by
regions of green. At this excitation level, 74% of the
375:50 nm intensity integrated over the whole image colocalized to some
extent with the Nile Red in lipid droplets, as seen by a coefficient of
Cgreen = 0.74, whereas the other 25% was in
other cellular structures (Fig. 9F). Thus, monomeric DHE was
significantly colocalized with lipid droplets as well as other
intracellular structures and the plasma membrane. Crystalline DHE was
primarily associated with lysosomes.
Multiphoton Laser Scanning Microscopy of L-cell Fibroblasts
Cultured with Large Unilamellar Membrane Vesicles Containing
DHE--
To determine whether L-cells cultured with noncrystalline DHE
might exhibit the absence of crystalline DHE and/or a different intracellular DHE distribution, L-cell fibroblasts were cultured for 2 days with medium supplemented with DHE (20 µg/ml) in the form of LUV
composed of POPC:DHE (65:35) as described under "Experimental Procedures." As shown in Fig. 3, DHE in these LUVs was primarily in
monomeric form with <0.2% in crystalline form. After the cells were
washed 3-4 times with Puck's buffer and incubated with Nile Red (see
"Experimental Procedures"), all fluorophores were excited at 920 nm
by MPLSM. Fluorescence emission of all fluorophores was simultaneously
collected by the Zeiss 63× oil immersion objective and directed to
three separate external photomultiplier tubes: Nile Red (red pixels)
with a 575:150-nm dichroic filter; crystalline DHE (blue pixels), with
a 455:35-nm dichroic filter; and monomeric DHE (green pixels) with a
375:50-nm dichroic filter. To show the intracellular distribution of
DHE (in cells labeled with LUV DHE and with Nile Red), the
simultaneously acquired images from the three photomultiplier tubes
were merged (Fig. 10A). It
is clear from this merged image that supplementing the L-cells with DHE in the form of LUV (primarily noncrystalline DHE) resulted in a
somewhat different intracellular distribution of DHE as compared with
that detected in cells supplemented with crystalline DHE (Fig. 9).

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Fig. 10.
Multiphoton laser scanning microscopy of DHE
and Nile Red, a lipid droplet marker, in L-cell fibroblasts
supplemented with DHE in large unilamellar vesicles. L-cell
fibroblasts were cultured on cover glass for 2-days in medium
containing 10% fetal bovine serum and DHE (20 µg/ml) added in the
form of large unilamellar vesicles. The large unilamellar vesicles were
composed of POPC:DHE (65:35). The cells were washed 3-4 times with
Puck's buffer. Filters were as described in the legend to Fig. 9.
A shows the merged image. Arrow 1 points to the
plasma membrane region showing only monomeric DHE; arrow 2 points to a representative white pixilated area wherein
mostly crystalline DHE was localized with Nile Red; arrow 3 points to a representative yellow/orange area
where monomeric DHE colocalized with Nile Red. B is a pixel
fluorogram of only the 455:30-nm emission (red) and
375:50-nm emission (green). The crystalline and monomeric
portions were separated out as discussed. C shows the
monomeric portion merged with Nile Red emission. D is a
pixel fluorogram corresponding to C.
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The plasma membrane was prominent in the merged image (Fig.
10A, arrow 1) and contained primarily monomeric
DHE (green pixels, detected through a 375:50-nm dichroic
filter). An examination of the intracellular regions in the merged
image (Fig. 10A) showed that, within the perinuclear region
of some cells, a small amount of crystalline DHE
(blue/magenta, arrow 2) colocalizing with Nile Red was visible. As seen previously, monomeric DHE colocalized with
Nile Red (yellow/orange, arrow 3) in lipid droplets.
Once again, to help separate the forms of DHE, a merged image (not
shown) composed of the 375:50- (green) and 455:30-nm (red) channel was
created and imported into the newly developed software program. The
resultant DHE fluorogram is shown in Fig. 10B. The pixels
having a Ired:Igreen <1
and Ired:Igreen
1 were
selected to produce gray scale images. Both of these images were merged with the Nile Red gray scale image. The merged image of monomeric DHE
with Nile Red was shown in Fig. 10C with its corresponding fluorogram (Fig. 10D). With a Cred = 0.96, most of the Nile Red colocalized with monomeric DHE even though
it was not all in lipid droplets in this figure (Fig. 10D).
A higher concentration of the Nile Red probe was used in order to
delineate other nonpolar lipid regions within the cell. Again, the DHE
in monomeric form was distributed among cellular components, but >50%
of the total intensity of monomeric DHE colocalized to some degree with
Nile Red staining regions (including lipid droplets) as indicated by
Cgreen = 0.64 (Fig. 10D). Whereas the
brightest regions were the lipid droplets, the perinuclear regions also
appeared to accumulate significant colocalized Nile Red and monomeric cholesterol.
As indicated above, supplementation of L-cells with DHE in liposomal
form resulted in the near absence of crystalline DHE, with only a few
crystalline pixels detected colocalizing with Nile Red in the
perinuclear region (not shown). With a Cred = 0.04, almost none of the Nile Red colocalized with crystalline DHE.
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DISCUSSION |
Because of its influence on membrane fluidity, permeability,
cell-cell recognition, transport, receptor-effector coupling, and
microdomain (e.g. caveolae and rafts) function, the
cholesterol content of cellular membranes must be tightly regulated
(reviewed in Refs. 2 and 53). This is especially important in view of
the fact that abnormal regulation of membrane cholesterol content and/or distribution impairs membrane function (reviewed in Refs. 2, 32,
53, and 56-60) and thereby cell survival as indicated by cytotoxicity,
sickle cell acanthacytosis, Niemann-Pick C disease, Alzheimer's
disease, and atherosclerosis (reviewed in Refs. 12-16). Despite these
findings relatively little is known regarding the organization of
membrane cholesterol, especially when cholesterol is present in molar
excess over membrane phospholipid. Likewise, almost nothing is known
regarding the real time, direct visualization of different structural
forms of cholesterol in membranes or cells (reviewed in Refs. 18 and
25). The data presented here address these issues and provide the
following new insights.
First, crystalline and monomeric forms of sterol were differentiated
spectroscopically. Monomeric DHE exhibited emission maxima near 354, 370, and 394 nm in anhydrous ethanol, whereas those of crystalline DHE
were significantly red-shifted to 356, 403, and 426 nm. These emission
characteristics of crystalline DHE were not due to the presence of
impurities. The onset of the red-shift was concomitant with the
formation of DHE microcrystals in aqueous buffers (27, 30) at
concentrations similar to that where cholesterol has been shown to form
microcrystals in aqueous buffers (49, 61-63). Several techniques were
applied to determine whether the change in spectral characteristics
from ethanol to water was due to formation of microcrystals or of
micelles. Observation by polarizing light microscopy revealed
birefringent structures resembling those reported to be cholesterol
monohydrate microcrystals (60-63) and are therefore presumed to be
dehydroergosterol monohydrate crystals. Filtration up to 0.5 µm
eliminated all detectable emission, indicating that the fluorescence is
derived from an aggregate with a lower size limit of >0.5 µm, most
likely the observed microcrystals. In corroboration, the addition of
DPH to µM concentrations of DHE in aqueous buffer did not
reveal any incorporation into micelles of DHE, regardless of the
possible occurrence of micellar agglomeration. These results compared
favorably with the same experiments applied to cholesterol at similar
concentrations and conditions.
Although determination of the exact photophysical nature of crystalline
DHE involves a complex discussion of exciton theory (64), one can
consider a simple interaction mechanism with a nearest neighbor in the
form of a physical sandwich dimer, because steroid fused rings form
-face-to-
-face dimers as well as
-face-to-
-face dimers in
crystals (65). For example, the crystalline absorption spectrum
exhibited characteristics such as a bathochromatic shift accompanied by
spectral broadening, some increase in intensity of higher order
vibrational bands, and overall hypochromism. The crystalline spectral
emission exhibited a red shift in the emission maxima (354 to 356 nm
and 370 to 375 nm) whose intensities were decreased from those in the
monomer, whereas the lower energy transitions (exhibiting maxima at 403 and 426 nm) were increased dramatically in intensity. This was
indicative of enhanced Franck-Condon factors and fast relaxation times
into the lowest energy level where there must be less nonradiative
processes and stronger wave function overlap with higher vibrational
levels in the ground state. A larger lifetime component consistent with
a smaller nonradiative transfer rate was observed in the crystalline
form (30). The possibility that the enhanced emission could also be due
to the formation of excited state dimers (excimers) was considered
based on the fact that the conjugated triene double bonds in DHE are part of a planar, ring structure. Such structures, when stacked face-to-face, allow the overlap of the electron clouds of the two
molecules and thereby facilitate excimeric interaction. Typically, excimer formation is concentrationdependent and fluoresces at longer wavelengths than monomers as well as exhibits higher quantum yield than the monomer. However, excimeric emission is usually broad
and structureless because the excimer dissociates due to the strong
repulsive force between the molecules in the ground state. As can be
seen in Fig. 1D, vibrational structural features were
present in the subtracted spectrum. Regardless of whether the DHE
formed physical dimers and/or excimers, these differences in the
spectral emissive characteristics of the monomeric and crystalline DHE
allowed the determination of their relative proportions in solvents,
model membranes, biological membranes, and organelles (e.g. lysosomes).
Second, these differences in the spectral emission characteristics of
the monomeric and crystalline DHE permitted determination of their
relative proportions in model membranes. The structural form of DHE in
model membranes was primarily monomeric even at molar ratios of
sterol:phospholipid substantially >1. This observation was surprising
in view of previous studies on sterol packing in model membranes. While
at low mol % the sterol is uniformly dispersed/solubilized in the
membrane, and increasing the sterol:phospholipid molar ratio from 0.25 to 1 results in formation of interdigitated, transbilayer sterol dimers
(66). Over the same concentration, DHE self-quenches in model membranes
by Forster energy transfer (29, 30). The Forster energy transfer
distance for this homotransfer between face-to-face oriented DHE
molecules is 13.3 Å (30). Because sterol and phosphatidylcholine are 6 (13) and 8 Å (30) thick, respectively, Forster homotransfer of energy
is efficient even if the DHE molecules are separated by one
phospholipid molecule. In contrast, at sterol:phospholipid molar ratios
>1 an immiscible sterol phase forms in model membranes (reviewed in
Refs. 13, 67, and 68). Based on x-ray and NMR data, it had previously been assumed that this immiscible sterol phase is identical to pure
crystalline sterol monohydrate (reviewed in Refs. 13, 67, and 68). If
massive formation of pure crystalline DHE had occurred in membranes
with DHE:phospholipid molar ratios >1, the concomitant formation of
face-to-face DHE dimers would have resulted in preponderant emission at
longer wavelength. On the contrary, the data presented here with DHE
clearly showed that even at sterol:phospholipid molar ratios >1 there
was only a small amount of phase-separated DHE with properties
identical to that in pure DHE crystals. The molecular basis for this
apparent contradiction between results obtained by the various
techniques (x-ray and NMR versus fluorescence) may be
explained as follows.
One possibility is that the different techniques detecting
phase-separated immiscible sterol may not necessarily report on the
same aspects of sterol structural packing in membranes. Although the
x-ray and NMR techniques largely report on transbilayer
thickness/distance between sterol molecules, it is unclear whether
these techniques differentiate the multiple potential forms of lateral
phase-separated sterol in membranes: pure cholesterol patches, ribbons
of sterol, and/or sterol-sterol with different orientations
(edge-to-edge,
-face-to-
-face,
-face-to-
-face, and
-face-to-
-face), etc. (reviewed in Refs. 65, 69, and 70). In
contrast, DHE emission characteristics detect the formation of
face-to-face lateral interactions between sterols, which emit with
greater quantum yield and at higher wavelength. As shown here over the
DHE:phospholipid molar ratio ranging from 0.5 to 3, DHE detected only
small amounts (1-10%) of immiscible DHE whose structure (identified
by longer wavelength emission maxima) appeared identical to pure
crystalline DHE or other face-to-face sterol packings.
An alternative explanation of the different results obtained by the
x-ray and NMR of cholesterol versus fluorescence of DHE is
that the phase behavior of DHE may differ markedly from that of
cholesterol in model membranes. Although it is important to recognize
that DHE and cholesterol are not identical, this latter possibility was
considered unlikely based on the very similar behavior of these sterols
in a variety of model membrane systems (reviewed in Refs. 26, 30, 71,
and 72) summarized briefly as follows. (i) In pure and mixed monolayers
at pressures similar to bilayer systems, cholesterol and DHE showed
similar packing arrangements even at mol % where cholesterol has been
shown by x-ray crystallography and birefringence to separate into
crystalline structures (51). (ii) The dissociation constants of
spontaneous desorption of cholesterol and DHE from membranes were 0.044 and 0.031 mol/mol, respectively (30). In contrast, due to the apparent higher affinity of cyclodextrin for DHE, the cyclodextrin-catalyzed extraction of cholesterol from model membranes was severalfold slower
than that of cyclodextrin-catalyzed extraction of DHE (73). (iii) The
rate constants of spontaneous transfer of cholesterol and DHE between
POPC containing model membranes did not significantly differ (71). (iv)
The fractional distribution of cholesterol into kinetically resolvable
sterol domains was not significantly different from that of DHE in POPC
containing model membranes (71). (v) Cholesterol and DHE similarly
shifted the midpoint of the phase transition temperature of POPC
similarly to higher temperature (reviewed in Ref. 26). (vi) Cholesterol
and DHE similarly abolished the phase transition of POPC at high
mol % sterol, although slightly higher DHE was required (reviewed in Ref. 26). (vii) DHE (fluorescence, lifetime, and polarization) detected
the same phase transition temperatures as detected using other methods
for a variety of phospholipids (reviewed in Ref. 22). (viii) DHE
codistributed with cholesterol (i.e. did not phase separate
from cholesterol) in membranes and formed superlattices similarly to
those of cholesterol in model membranes (reviewed in Refs. 30 and 72).
These observations were in dramatic contrast to those obtained with
nitroxide-labeled sterols or other types of fluorescent-labeled
cholesterol whose properties in model membranes differed much more
markedly (4-10-fold) from those of cholesterol (reviewed in Refs. 26
and 34). Thus, the phase properties of DHE in model membrane bilayers
reflect those containing cholesterol.
Third, it was demonstrated that crystalline and monomeric membrane
sterol could be differentiated spectroscopically in vitro in
subcellular membrane fractions isolated from L-cell fibroblasts. Plasma
membranes, lysosomal membranes, microsomes (endoplasmic reticulum), and
mitochondrial membranes differ markedly in sterol:phospholipid ratio
ranging from near 1.0 in plasma membranes to as low as 0.04 in
mitochondria (reviewed in Refs. 2 and 53). When these membrane
fractions were isolated from L-cell fibroblasts supplemented with DHE,
only a small proportion (1-7%) of crystalline DHE was detected. This
was confirmed by MPLSM of L-cells supplemented with DHE in monomeric
form (i.e. LUV) and represents the first real time imaging
of a fluorescent sterol in the plasma membrane of a living cell as well
as the first real time resolution of multiple structural forms of DHE
in the plasma membrane of a living cell. Consistent with the findings
of only a small amount of crystalline sterol in the plasma membrane,
other investigators (18) using x-ray crystallography also showed that
plasma membranes isolated from control smooth muscle cells did not
contain significant levels of crystalline sterol.
The lack of substantial quantities of crystalline sterol in plasma
membranes, the most highly sterol-enriched membrane in the cell, was
especially interesting because the transbilayer distribution of sterol
in the plasma membrane is asymmetric, i.e. 4-fold higher in
the cytofacial leaflet (reviewed in Refs. 22 and 58). Because the
L-cell plasma membranes in this study exhibited a sterol:phospholipid
molar ratio of 1.2 (38), this would imply that the sterol:phospholipid
molar ratio in the cytofacial leaflet was almost 2.0. Nevertheless,
neither DHE in isolated plasma membranes in vitro, x-ray
crystallography in vitro, nor DHE in real time multiphoton
images of living cells detected very much crystalline sterol in plasma
membranes. It should be noted that these observations for the
structure of DHE in biological membranes were not due to DHE perturbing
biological membrane structure or function because of the following: DHE
is a natural product found in high percentage in the membranes of
eukaryotes such as sponge and yeast; DHE can be supplemented to
microorganisms and cultured L-cells to replace nearly 90% of
endogenous sterol; DHE has no adverse effect on sterol:phospholipid
ratio or phospholipid composition, fatty acid composition, cell growth,
or cholesterol-sensitive enzymes; DHE codistributes with endogenous
sterol among intracellular membranes; and DHE does not alter the
structure of the plasma membrane or the function of sterol-sensitive
membrane proteins (reviewed in Refs. 26 and 31).
Fourth, the detection of crystalline DHE in the isolated plasma
membrane fraction suggested that this small amount of crystalline sterol might represent microdomains present in small amounts in the
plasma membrane. It is known that the lateral distribution of sterol in
the plane of the bilayer is not uniform because cholesterol-rich lipid
rafts/caveolae have been detected in the cell surface and isolated by
several methods (reviewed in Ref. 23). Caveolae represent only 1-2%
of the cell surface membrane area, but lipid raft/caveolar
membrane-enriched fractions exhibit severalfold higher sterol content
than the plasma membrane (reviewed in Ref. 23). If the small amount of
crystalline DHE detected in L-cell plasma membranes was due to its
presence in lipid raft/caveolar membranes, then an enrichment of
severalfold might be predicted therein with DHE:phospholipid ratios
near 2-3. However, model membranes with such DHE:phospholipid ratios
contained 7.1-9.5% crystalline DHE. Surprisingly, the level of
crystalline DHE in isolated lipid raft/caveolar membranes was <0.3%,
18- and up to 32-fold lower than in the bulk plasma membrane and model
membranes, respectively (Table I). These observations, showing that
lipid rafts/caveolar membranes contain very little crystalline sterol despite their high sterol content, may explain for the first time why
so many processes (signaling, transport, etc.) important to cell
viability readily function in these domains. The future challenge will
be to determine what features of lipid raft/caveolar proteins and/or
lipids prevent crystalline sterol formation.
Fifth, MPLSM for the first time resolved the uptake and intracellular
distribution of crystalline sterol in real time in living cells. L-cell
fibroblasts ingested large amounts of crystalline DHE that accumulated
in lysosomes as evidenced by colocalization with LysoTracker Green, a
lysosomal stain, in living cells. The lysosomal localization of
substantial amounts of crystalline DHE was confirmed by examination of
the spectral properties of DHE in lysosomes isolated from L-cells
cultured with crystalline DHE. In addition, some crystalline DHE was
also detected outside the lysosomal compartment. However, very little
extralysosomal crystalline DHE colocalized with BODIPY FL
C5-ceramide (Golgi marker) or Nile Red (lipid droplet
marker) in living cells. Consistent with this observation, crystalline
cholesterol has been detected in macrophage foam cells, both within
lysosomes and outside (as crystals surrounded by a membrane) the
lysosomal compartment (13, 14, 19). The phagocytosis of DHE crystals
was consistent with the known ability of L-cells to phagocytose
particles as large as several microns and by the fact that L-cells
endocytose the equivalent of their entire cell surface membrane within
about 2 h (55, 74), similar to the activity of macrophages (14,
19).
Sixth, MPLSM for the first time resolved the uptake and intracellular
distribution of monomeric sterol in real time in living cells. The
pattern of monomeric sterol distribution depended on the mode of DHE
supplementation to the cells. (i) Although most cells incubated with
crystalline DHE exhibited high amounts of crystalline form in
lysosomes, those cells, which had digested/solubilized the ingested
crystals, distributed monomeric DHE throughout the cell. The highest
concentrations of monomers were in lipid droplets, followed by
perinuclear regions, the plasma membrane, and punctate areas resembling
vesicles in the cytoplasm (Fig. 10). (ii) In contrast, when cells were
incubated with DHE in monomeric form (i.e. vesicles composed
of POPC:DHE, 65:35) almost no crystalline form was detected in the
cells. Instead highest concentrations of monomeric DHE were found in
the plasma membrane and lipid droplets, followed by lower levels in the
perinuclear region. Much less monomeric DHE was present in a punctate
vesicular pattern throughout the cytoplasm. Thus, the use of
multiphoton laser scanning microscopy together with the spectral
differences in monomeric versus crystalline DHE allowed for
the first time the noninvasive, real time imaging of multiple forms of
sterol in living cells.
In summary, the data presented herein demonstrate that the spectral
properties of DHE, together with multiphoton laser scanning microscopy,
form a powerful tool to noninvasively resolve and visualize the
individual dynamics of multiple structural forms of sterol in real time
in living cells. Crystalline DHE was enriched within lysosomes but not
plasma membranes or lipid raft/caveolar membrane-enriched subfractions.
Interestingly, the presence of sterol carrier protein-2 in
vitro significantly enhanced the sterol transfer of crystalline
DHE (in donor lysosomes) to acceptor lysosomes (containing no DHE) as
evidenced by the change in spectral characteristics of the DHE emission
from crystalline to monomeric. These data for the first time showed a
potential protective role for sterol carrier protein-2 in mitigating
the deleterious effects of crystalline DHE in the cell. Consistent with
this possibility, the cellular level of sterol carrier protein-2 was
up-regulated as much as 3-fold in macrophage foam cells (7, 75) wherein
crystalline cholesterol is known to accumulate and become cytotoxic
(13, 14, 19). These noninvasive, nonperturbing methods also
demonstrated for the first time that multiple structural forms of
sterol normally occurred within cell membranes and could, for the first
time, be visualized real time in membranes and intracellular organelles of living cells. These findings are especially important because relatively few noninvasive, nonperturbing techniques exist for real
time visualization of cholesterol structures in biological membranes or
in living cells (reviewed in Refs. 18 and 25).