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The Journal of Neuroscience, January 15, 2001, 21(2):504-512
Membrane Lipid Rafts Are Necessary for the Maintenance of the
 7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary
Neurons
Juan L.
Brusés,
Norbert
Chauvet, and
Urs
Rutishauser
Cellular Biochemistry and Biophysics Program, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
Calcium-permeable neurotransmitter receptors are concentrated into
structurally and biochemically isolated cellular compartments to
localize calcium-mediated events during neurotransmission. The
cytoplasmic membrane contains lipid microdomains called lipid rafts,
which can gather into microscopically visible clusters, and thus the
association of a particular protein with lipid rafts can result in its
redistribution on the cell surface. The present study asks whether
lipid rafts participate in the formation and maintenance of the
calcium-permeable  7-subunit nicotinic acetylcholine receptor
(7nAChR) clusters found in somatic spines of ciliary neurons. Lipid
rafts and 7nAChR become progressively colocalized within somatic
spines during synaptogenesis. To determine whether these rafts are
required for the maintenance of 7nAChR aggregates, cholesterol was
extracted from dissociated ciliary neurons by treatment with
methyl- -cyclodextrin. This treatment caused the dispersion of lipid
rafts and the redistribution of 7nAChR into small clusters over the
cell surface, suggesting that the integrity of lipid rafts is required
to maintain the receptor clustering. However, lipid raft dispersion
also caused the depolymerization of the F-actin cytoskeleton, which can
also tether the receptor at specific sites. To assess whether
interaction between rafts and 7nAChR is independent of F-actin
filaments, the lipid raft patches were stabilized with a combination of
the cholera toxin B subunit (CTX), which specifically binds to the raft
component ganglioside GM1, and an antibody against CTX. The
stabilized rafts were then treated with latrunculin-A to depolymerize
F-actin. Under these conditions, large patches of CTX persisted and
were colocalized with 7nAChR, indicating that the aggregates of
receptors can be maintained independently of the underlying F-actin
cytoskeleton. Moreover, it was found that the 7nAChR is resistant to
detergent extraction at 4°C and floats with the caveolin-containing
lipid-rich fraction during density gradient centrifugation, properties
that are consistent with a direct association between the receptor and
the membrane microdomains.
Key words:
ciliary ganglion; somatic spines; lipid rafts; 7
nicotinic acetylcholine receptor; receptor clusters; actin
cytoskeleton; synapse formation
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INTRODUCTION |
Neurotransmitter receptors are
highly concentrated in the area of functional contact between excitable
cells. The localization and clustering of the relevant receptor on the
cell surface at and within the appropriate contact site are critical
for the triggering and integration of neurotransmission.
The plasma membrane is mainly composed of a fluid bilayer of
phospholipids within which integral and peripheral membrane proteins diffuse. Therefore diffusion forces must be neutralized to maintain a
high receptor density in the area of synaptic contact. To this end,
there are two basic mechanisms by which lateral dispersion of receptors
can be influenced: the assembly of molecular scaffolds via
protein-protein interactions that link the receptor to the cytoskeleton and the existence of specific domains of the membrane to
which receptors are either inserted or recruited.
Several proteins have been identified that interact with
neurotransmitter receptors, including rapsyn (Froehner, 1991 ; Phillips et al., 1991; Gautam et al., 1995; Ramarao and Cohen, 1998), gephyrin (Prior et al., 1992; Meyer et al., 1995), GABA-R-associated
protein (Wang et al., 1999), postsynaptic density 95 (PSD95)
(Kornau et al., 1995), and PKC-interacting protein 1 (PICK1)
(Xia et al., 1999). Rapsyn and gephyrin have been shown to be required
for clustering of the muscular nicotinic acetylcholine receptor (nAChR) and neuronal glycine receptors, respectively (Gautam et al., 1995; Feng
et al., 1998b), whereas PSD95 and PICK1 use PDZ domains to couple the glutamatergic NMDA and AMPA receptor to other cell components or functions (Kornau et al., 1997; Craven and Bredt, 1998).
Additional mechanisms must also exist because, for example, the C
terminal of a subfamily of ligand-activated ion channel subunits (ACh,
GABA, serotonin, and glycine receptors) cannot bind to PDZ proteins
(Barnard, 1992), and although the clustering of nAChR in muscle (Gautam
et al., 1995) requires rapsyn, its close homolog in autonomic neurons
does not (Burns et al., 1997; Feng et al., 1998a; Conroy and Berg,
1999).
In addition to the fluid bilayer, the cytoplasmic membrane contains
small lipid membrane microdomains called lipid rafts, which are
enriched in sphingolipids and cholesterol and are resistant to nonionic
detergent extraction at 4°C (Simons and Ikonen, 1997; Brown and
London, 1998). Membrane proteins partition differentially into these
lipid microdomains. For example,
glycosylphos-phatidylinositol-linked proteins, some transmembrane
proteins, and tyrosine kinases of the src family are
enriched in lipid rafts (Simons and Ikonen, 1997; Jacobson and
Dietrich, 1999). Because lipid rafts can move laterally and cluster
into larger patches (Harder et al., 1998), the association of a
particular protein with rafts can result in its redistribution, and
therefore they have been proposed to function as membrane platforms for
the assembly of signaling complexes and for the sorting of surface
molecules to particular cellular structures (Simons and Ikonen, 1997;
Stauffer and Meyer, 1997; Viola et al., 1999).
On this basis it is reasonable to ask whether lipid rafts might be a
factor in the redistribution of some neurotransmitter receptors, as
well as other synaptic components. The present study was designed to
determine whether cholesterol-rich lipid microdomains influence the
clustering of acetylcholine receptor within somatic spines in neurons.
To this end, the ciliary neurons of the chick ciliary ganglion (CG)
have been used as an experimental model. These cholinergic neurons
express high levels of the homomeric bungarotoxin
(BTX)-sensitive 7nAChR that is highly concentrated in
somatic spine aggregates localized over discrete areas of the ciliary
neuron soma (Shoop et al., 1999). 7nAChRs are permeable to calcium,
and their clustering in somatic spines is required for its proper
function (Liu and Berg, 1999).
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MATERIALS AND METHODS |
Animals. Fertilized White Leghorn chicken eggs from
SPAFAS (Roanoke, IL) were incubated in a forced-draft incubator at
39°C under a humidified atmosphere until the desired embryonic stage (St) was reached (Hamburger and Hamilton, 1951).
Labeling of intact CG. CGs from varying embryonic stages
were dissected and treated with collagenase A (Boehringer
Mannheim, Indianapolis, IN; 2 mg/ml in PBS for 20 min at 37°C
in a water bath) to facilitate penetration of markers. Ganglia were
then incubated in tetramethylrhodamine-conjugated BTX (RITC-BTX; 2 µg/ml; Molecular Probes, Eugene, OR) and biotin-conjugated cholera toxin B subunit (biotin-CTX; 20 µg/ml; Sigma, St. Louis, MO) for 2 hr
at 16°C. Thereafter, the ganglia were washed, fixed in 4% paraformaldehyde in phosphate buffer (0.1 M), pH 7.4, for
30 min at room temperature (RT), incubated in Cy2-conjugated
streptavidin (2 µg/ml; Jackson ImmunoResearch, West Grove, PA)
for 2 hr at RT, mounted in Mowiol, and observed by confocal microscopy
with an LSM510 Zeiss Axiovert microscope.
Labeling of cultured ciliary neurons. St 40 CGs were
dissected and collected in
Ca2+-Mg2+-free
Tyrode's solution on ice. The ganglia were then incubated in
collagenase A (2 mg/ml in PBS) for 45 min at 37°C, mechanically dissociated with fire-polished Pasteur pipettes, and plated on plastic
tissue culture dishes precoated with poly-D,L-ornithine or
poly-D,L-lysine (0.2 mg/ml) and laminin (1 µg/cm2). Cells were then cultured in
DMEM and F12 plus insulin (5 µg/ml) for 1 hr in a tissue culture
incubator at 37°C. Labeling of 7nAChR was performed by incubating
the cultured cells with RITC-BTX (2 µg/ml) in Dulbecco's PBS
(D-PBS; Life Technologies, Gaithersburg, MD), pH 7.4, for 1 hr on ice.
In some cases, Alexa488-conjugated BTX (Alexa488-BTX; Jackson
ImmunoResearch) was used at a concentration of 10 µg/ml to label
7nAChR.
CTX was used as a marker for cholesterol-rich lipid
microdomains. CTX binds to the ganglioside GM1 (Schon and Freire,
1989), which is an abundant lipid raft component and thus has been
extensively used as a lipid raft marker in a variety of cells (Harder
et al., 1998; Viola et al., 1999) including neurons (Ledesma et al.,
1998). The ability of CTX to label cholesterol-rich lipid microdomains in ciliary neurons was tested by removing cholesterol from the cultured
cells. As has been observed in other cell types (Janes et al., 1999),
cholesterol removal causes the dispersion of lipid raft patches in
ciliary neurons (see Fig. 4). Cultured ciliary neurons were
incubated with fluorescein-conjugated CTX (FITC-CTX; Sigma) at a
concentration of 10 µg/ml in D-PBS for 1 hr on ice. Cells were then
washed with D-PBS and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min at RT. The neural cell adhesion molecule (NCAM) was immunodetected with a
monoclonal antibody (5e) that recognizes all NCAM isoforms (Watanabe et
al., 1986) followed by Cy2-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch) at 7.5 µg/ml for 1 hr. Polymerized
actin filaments (F-actin) were detected with
tetramethylrhodamine-conjugated phalloidin (RITC-phalloidin; Molecular
Probes), a fungus venom that specifically binds to F-actin
(Vandekerckhove et al., 1985). Cultured cells were fixed as described
above, permeabilized in D-PBS with 0.05% Triton X-100 for 20 min, and
then incubated for 1 hr in D-PBS containing RITC-phalloidin.
Electron microscopy. Dissociated ciliary neurons were plated
on Aclar and cultured for 1 hr in a tissue culture incubator at 37°C.
Cells were then incubated with peroxidase-conjugated CTX (10 µg/ml;
Sigma) for 1 hr on ice, washed, and incubated in a 1:50 dilution of
anti-peroxidase antibody conjugated to 6 nm immunogold particles
(Jackson ImmunoResearch) in D-PBS with 2% BSA for 1 hr on ice. Cells
were then fixed with 4% paraformaldehyde and 2.5% glutaraldehyde for
1 hr, washed, postfixed in 1% osmium tetroxide, dehydrated, and
infiltrated by Epon resin. Ultrathin sections were cut and observed
using a JEOL electron microscope. The number of immunogold
particles per unit membrane length was estimated from electron
microphotographs of ciliary neurons following the procedure of Shoop et
al. (1999). Briefly, neuronal membrane was divided into two classes:
"spiny" and "nonspiny" membrane. A spine was defined as a
protrusion from the cell body of at least 0.2 µm. A nonspiny or
smooth cell surface was defined as a flat cell membrane with no
depressions or bumps that could indicate the origin of a spine. The
total number of gold particles along each type of membrane was summed
and divided by the total length of membrane observed from ~20 cells.
Membrane length was measured using NIH Image software.
Quantification of fluorescence intensities and the fluorescence
intensity ratio. NIH Image software was used to determine the
average fluorescence intensity of various markers independently over
distinct areas of the cell surface from mid-cell section confocal
images (1 µm thick). Midcell sections were selected because they help
to determine whether a marker is localized at the cell surface. To
analyze the differential distribution of molecules over discrete
regions of the cell surface, ciliary neurons were double-stained with
RITC-BTX or Alexa488-BTX and with one of the following markers:
FITC-CTX, RITC-phalloidin, or Cy2-conjugated anti-mouse IgG to label
anti-NCAM antibody. Fluorescently labeled BTX was used to identify
7nAChR clusters in somatic spines. The average fluorescence
intensity of a predetermined area (18 × 18 pixel square
corresponding to 2.94 µm2) was measured
within the BTX-positive region of the cell and divided by the
average fluorescence intensity of an area of the same size positioned
over an BTX-negative region of the membrane, which corresponded to
the smooth surface of the cell. The ratio between these two
fluorescence intensity values was calculated for ~20 cells from each
experimental condition, with a ratio of 1 indicating a homogeneous
distribution of the marker. A similar procedure for quantifying the
synaptic clustering of molecules has been reported (Craven et al.,
1999).
Line profiles. To determine the line profiles of the
membrane fluorescence intensity for a given marker, a line was drawn over the cell circumference from a confocal section, and the
fluorescence intensity was determined independently for each marker
over the same area and plotted as relative intensity versus relative distance.
Assessment of the number and size of molecular clusters. To
estimate the magnitude of the effect of drug treatments on the 7nAChR clustering, two parameters were selected: the average number
of clusters of a particular molecular marker per cell section and the
average size of the clusters (total cluster area divided by the number
of clusters in the cell section). An arbitrary fix threshold for the
average fluorescent intensity of a minimum cell area was set to detect
molecular clusters automatically. A cluster was defined as an area of
20 or more adjacent pixels (0.18 µm2)
with an average fluorescence intensity above threshold.
Triton X-100 flotation gradients. To analyze
detergent-insoluble components of the cell membrane, the method of
Bruckner et al. (1999) was followed with small modifications. Briefly,
50-60 St 40-42 CGs were collected on ice and homogenized by 20 strokes in a Dounce with 300 µl of ice-cold homogenization buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 250 mM sucrose, and 1 mM DTT) and protease
inhibitor cocktail (Complete, Mini, EDTA-free; Boehringer-Mannheim; one
tablet in 10 ml). The extract was then passed five times through a 22 gauge needle and centrifuged at 735 × g (3000 RPM) for 10 min in an Eppendorf microcentrifuge at 4°C. The
supernatant was saved, and the pellet was reextracted with 100 µl of
homogenization buffer by passing five times through a 22 gauge needle
and spun as described above. Both supernatants were combined in an
ultracentrifuge tube, brought to 35% Optiprep (Nycomed Pharma, Oslo,
Norway) by adding 550 µl of 60% Optiprep, and overlayered
successively with 1 ml of 30, 20, and 5% Optiprep in homogenization
buffer. The sample was centrifuged in a SW55Ti rotor at 285,000 × g (49,000 RPM) for 3 hr at 4°C, and 12 fractions were
collected. The third and fourth fractions from the top, containing the
membrane, were combined and adjusted to 0.1% Triton X-100 (Calbiochem,
La Jolla, CA; protein grade) and incubated on wet ice for 30 min.
Thereafter, the sample was adjusted to 35% Optiprep, overlayered with
2.5 ml of 30% Optiprep plus 0.1% Triton X-100 and 0.5 ml of
homogenization buffer plus 0.1% Triton X-100, and spun as described
above for 4 hr. After centrifugation, six equal fractions were
collected. A 60 µl aliquot from each sample was put aside, and 500 µl of solubilization solution was added to the remaining sample and incubated with 75 µl of BTX conjugated-Actigel overnight at 4°C with rotation. The samples were then washed three times with
solubilization solution, twice with of 0.1 M
phosphate buffer, pH 7.3, 0.5% Triton X-100, and 1 M NaCl, and twice with 0.1 M phosphate buffer, pH 7.3, and 0.5% Triton
X-100 and resuspended in 50 µl of Laemmli sample buffer. Both sets of
samples were analyzed by 10% SDS-PAGE and Western blotting with the
rat monoclonal antibody 319 against 7nAChR (kindly provided by Drs.
W. Conroy and D. Berg of the University of California at San Diego) and
a rabbit polyclonal against caveolin (Transduction Laboratories,
Lexington, KY; #13630).
Drugs and treatments. Methyl--cyclodextrin (MBC; Sigma)
was used to extract cholesterol from the cell membrane. MBC contains a
hydrophobic cavity that specifically binds cholesterol, rendering it
soluble in aqueous solutions, and thus reduces the cholesterol content
of the cell (Klein et al., 1996). Cultured cells were incubated for 1 hr at 37°C in a tissue culture incubator in culture medium containing
8 mM MBC. This procedure was found to extract ~70% of
cellular cholesterol (Harder et al., 1998; Keller and Simons, 1998). To
destabilize the F-actin cortical cytoskeleton of cultured ciliary
neurons, cells were incubated for 2 hr at 37°C with latrunculin-A
(LAT-A; Molecular Probes) at a concentration of 20 µg/ml. LAT-A is a
membrane-permeant toxin isolated from the Red Sea sponge Negombata
(Spector et al., 1983) that specifically binds to monomeric actin and
therefore sequesters G-actin. This results in a depolymerization of
actin filaments and as a consequence causes the collapse of spines
(Allison et al., 1998; Shoop et al., 2000). Two hours of LAT-A
treatment did not induce a complete disappearance of F-actin filaments
but caused a substantial reduction of the marker. Longer treatments
were not performed because somatic spines of cultured ciliary neurons
tend to collapse spontaneously after 4-6 hr (Shoop et al., 2000).
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RESULTS |
Membrane lipid rafts become progressively colocalized with the
7nAChR during development of a ciliary neuron
The chick CG is part of the autonomic system that brings the
parasympathetic innervation to the internal muscles of the eye. Synapse
formation on ciliary neurons can be divided into two major periods.
Soon after the preganglionic fibers arrive at the ganglion (St 25), the
presynaptic terminals form active synaptic contacts with ciliary
neurons. This first phase extends up to St 33-34, and by this time
100% of the neurons in the ganglion respond to preganglionic
stimulation (Landmesser and Pilar, 1972). Although these bouton-like
contacts are active, they are morphologically immature, and the
resulting EPSPs are of small amplitude. After a brief period of
multiple innervation, the bouton-like contacts are progressively
replaced by a single synaptic contact. This second phase in formation
of the calyciform synapse (from St 34 to 42), which includes the events
studied here, involves the growth of the presynaptic calyx, the
formation and maturation of synaptic complexes, and the development of
somatic spines. By the end of this phase (St 42), the cell body is
primarily covered by a single presynaptic calyx and is wrapped by
Schwann cells (Landmesser and Pilar, 1972, 1976, 1978).
To determine whether changes in the distribution of organized lipid
membrane components correlate with the formation of 7nAChR-rich somatic spines in ciliary neurons, the developmental pattern of distribution of cholesterol-containing membrane microdomains and 7nAChR was analyzed from the beginning (St 33) to the end (St 42) of
the second phase of synapse formation. Intact CGs from St 33, 37, and
42 embryos were stained with RITC-BTX and biotin-CTX followed by
FITC-streptavidin and observed by confocal microscopy. Because the B
subunit of CTX specifically binds to GM1, which is enriched in
cholesterol-rich membrane microdomains, CTX is commonly used as a raft
marker (Harder et al., 1998; Viola et al., 1999).
In accordance with previous reports (Corriveau and Berg, 1993;
Blumenthal et al., 1999), it was found that 7nAChR expression at St
33 is low (Fig. 1a). At this
time, CTX labeling is distributed over the entire surface of ciliary
neurons (Fig. 1d). As development progresses (St 37), the
expression of 7nAChR increases and becomes restricted to distinct
regions of the cell surface (Fig. 1b). This process is
accompanied by changes in the surface distribution of CTX, which
becomes more concentrated, although not exclusively, into the same
BTX-positive regions (Fig. 1e,h, arrowheads).
By the end of synapse formation (St 42), ciliary neurons have adopted a
mature cellular architecture with clumps of somatic appendages distributed on discrete regions of the cell. These appendages that are
~0.5 µm wide by 2 µm long resemble dendritic spines (De Stefano
et al., 1993) and by St 40-42 have formed tightly folded aggregates or mats that contain high concentrations of 7nAChR (Shoop
et al., 1999). Accordingly, BTX labeling reveals a few large, well
defined patches over the cell surface (Fig. 1c,
arrowheads), and CTX labeling is almost exclusively
restricted to these same cellular structures (Fig. 1f,i,
arrowheads). Thus, the similarity in the developmental
pattern of 7nAChR expression and lipid raft location suggests a
possible association between this lipid membrane compartment and the
neurotransmitter receptor.
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Figure 1.
Colocalization of 7nAChR and membrane lipid
rafts during ciliary neuron development. Intact CGs from St 33, 37, and
42 chick embryos were double-labeled with RITC-BTX
(red) and FITC-CTX (green) to
analyze the developmental distribution of 7nAChR and membrane lipid
rafts, respectively. The samples were observed by confocal microscopy,
and the same cell sections are presented for both markers.
a, At St 33, BTX is expressed over the surface of the
ciliary neurons in poorly defined patches. d, g, CTX
(d) is detected over the entire cell body and
only partially colocalizes with BTX (g;
yellow). b, By St 37, BTX labeling is
more intense and concentrated in defined patches
(arrowheads). e, h, CTX
(e) also labels the BTX-positive areas
indicated by the arrowheads
(yellow, arrowheads in
h represent the areas where both markers overlap);
however CTX is also localized to areas not labeled by BTX
(h; green). c, f, At St
42, when ciliary neurons have matured and formed distinct mats of
somatic spines along the cell surface, both BTX and CTX,
respectively, are only detected on well defined patches
(arrowheads). i, At this stage both
markers are almost completely colocalized as indicated by the overlay
(arrowheads). Scale bar, 10 µm.
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Cholesterol-rich lipid microdomains colocalize with 7nAChR in
somatic spines of dissociated cells
At St 40-42, ciliary neurons can be isolated from CG by
collagenase treatment and mechanical dissociation. This procedure preserves much of the surface composition and cellular architecture of
the cells, including the 7nAChR and somatic spines, the latter remaining intact for ~6 hr after plating (Shoop et al., 2000). To
determine whether lipid rafts are differentially distributed on the
surface of cultured ciliary neurons, freshly dissociated neurons were
double-stained with RITC-BTX and antibodies against NCAM or with
RITC-BTX and FITC-CTX. NCAM has been found previously to be evenly
distributed on the surface of ciliary neurons (Brusés et al.,
1995) and can be used as a marker of the overall distribution of
surface membrane.
On cells labeled with BTX and NCAM, BTX staining is distributed
over discrete areas of the cell surface (Fig.
2a, arrowheads). In
contrast, NCAM staining is found over both BTX-positive and -negative areas (Fig. 2b, arrowheads,
arrows, respectively). The difference in the distribution of
both markers is illustrated by the overlay of both images (Fig.
2c). To quantify the differential distribution of BTX and
NCAM over the cell surface, confocal midsection images of ciliary
neurons were analyzed using NIH Image software (Fig. 2j).
The average fluorescence intensity of a predetermined area was measured
within the BTX-positive region of the cell and divided by the
average fluorescence intensity of an area of the same size positioned
over an BTX-negative region of the cell, which corresponds to the
smooth surface of the membrane. The ratio for NCAM represents an even
distribution of the marker on the cell surface. The slight apparent
concentration of NCAM (~3) within the BTX-positive region is
because of a higher membrane density per surface area within the
aggregates of somatic spines. In contrast, BTX has a high ratio
(~25), indicating that the 7nAChR is highly concentrated in
discrete regions of the cell surface. A high ratio value for a
particular marker indicates an accumulation of the marker within
BTX-positive patches (see Materials and Methods)
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Figure 2.
Membrane lipid rafts are associated with
BTX-positive clusters and F-actin aggregates. Freshly dissociated St
40 ciliary neurons were double-labeled with RITC-BTX and anti-NCAM
antibodies followed by Cy2-conjugated secondary antibodies
(a-c), with RITC-BTX and FITC-CTX
(d-f), or with Alexa488-BTX and
RITC-phalloidin (g-i). Confocal midsections of
the cells were collected for analyses. a,
BTX-positive regions of the cell surface are indicated by
arrowheads, whereas arrows point to areas
negative for BTX. b, Same cell shown in
a is labeled with NCAM and indicates NCAM-positive
regions that are both NCAM positive (arrowheads)
and BTX negative (arrows). c, Overlay
of a and b is shown. d,
Arrowheads and arrows point to
BTX-positive and -negative regions, respectively. e,
The same cell areas are indicated on the CTX image, showing an uneven
distribution of the raft marker over the cell surface.
f, Overlay of d and f
indicates a high degree of colocalization of both markers.
g-i, A ciliary neuron double-labeled with BTX
(g) and phalloidin (h) is
shown; arrowheads in g-i point to the
same cell areas, which are positive for both markers
(i). j-l, Plots of the
fluorescence intensity ratio between BTX-positive and -negative
areas of the cell surface are shown. j, Comparison of
the fluorescence intensity ratio between BTX and NCAM
(n = 20 cells) is shown. k,
Comparison of the fluorescence intensity ratio between BTX and CTX
(n = 19 cells) is shown. l,
Comparison of BTX and phalloidin (n = 20 cells)
is shown. Error bars indicate SE. Scale bar, 5 µm.
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Similarly, CTX is localized to discrete areas of the cell surface that
correspond to the areas labeled with BTX (Fig. 2d,e, arrowheads). The high degree of colocalization for both
markers is shown in the overlay of both images (Fig.
2f). A comparison of the fluorescence intensity
between BTX-positive and -negative areas of the cell surface gave a
high ratio for both CTX and BTX (Fig. 2k). Because
7nAChRs are concentrated in the mats of somatic spines, these
results indicate that cholesterol-rich lipid microdomains are highly
concentrated on the surface of somatic spines of ciliary neurons
together with the 7nAChR.
To obtain ultrastructural evidence that somatic spines are enriched in
lipid rafts, dissociated ciliary neurons were incubated with
peroxidase-conjugated CTX followed by an anti-horseradish peroxidase
(anti-HRP) antibody coupled to 6 nm gold particles. Samples were
processed for transmission electron microscopy, and microphotographs
were taken from areas of the cell with somatic spines (Fig.
3a) and from smooth surface
membrane regions (Fig. 3b). To determine whether CTX is more
abundant on the surface of somatic spines than on the smooth surface of
the cell membrane, the number of gold particles over a distance of 20 µm of cell membrane was quantified for these two regions. The results
shown in Figure 3c indicate that the cell membrane on
somatic spines is enriched in gold particles as compared with the
smooth cell surface. Thus, both the confocal and EM analyses show that
somatic spines are enriched in membrane lipid rafts.
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Figure 3.
EM analysis of the distribution of lipid rafts on
the surface of ciliary neurons. Dissociated ciliary neurons were
incubated with HRP-conjugated CTX followed by anti-HRP antibodies
conjugated with 6 nm gold particles. a, b, Electron
micrographs of somatic spines (a) and smooth
membrane (b) show a higher density of gold
particles over somatic spines. The size of each gold particle has been
electronically enhanced to facilitate observation. c,
Plot of the number of gold particles per micrometer detected over 20 µm of smooth membrane and over somatic spines is shown
(*p < 0.0001, Student's t test).
Error bars indicate SE. Scale bar, 0.2 µm.
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Cholesterol-rich lipid microdomains are necessary to maintain
7nAChR clustering in somatic spines
Lipid rafts are enriched in cholesterol, and the removal of
cholesterol causes the dispersion of the components of lipid
microdomains into the phospholipid bilayer of the cell membrane
(Scheiffele et al., 1997; Harder et al., 1998; Keller and Simons,
1998). To determine whether rafts are required for the maintenance of
7nAChR macroclusters within somatic spine aggregates, cholesterol
was extracted from freshly dissociated St 40 ciliary neurons by
treating the cells with MBC, which selectively extracts 60-70% of the
cholesterol from the cell membrane (Keller and Simons, 1998). Cells
were then double-labeled with RITC-BTX and FITC-CTX and analyzed by
confocal microscopy.
As shown in Figure 4a-c,
lipid rafts and 7nAChR are colocalized on untreated ciliary neurons
(arrowheads). This colocalization is also illustrated in the
fluorescence intensity plot of the cell circumference (Fig.
4d), which shows three distinct peaks of fluorescence
corresponding to the areas of the cell indicated in the photograph
(Fig. 4c, arrowheads). After cholesterol is removed, CTX disperses over most of the cell membrane, indicating the
disruption of lipid microdomains (Fig. 4e). The surface
distribution of BTX is similarly affected by the removal of
cholesterol because the large clusters of 7nAChR were fragmented
into microclusters (Fig. 4f). Furthermore, whereas in
the cholesterol-depleted cells the aggregates of both CTX and BTX
become smaller and more diffusely distributed, they also become
spatially separated (Fig. 4g). This decrease in
colocalization suggests that the forces responsible for 7nAChR
association with the lipid raft component GM1 within somatic spines are
reduced as a consequence of the dispersion of the components of the
cholesterol-rich lipid microdomains. The effect of removing cholesterol
on the surface distribution of BTX and CTX is also illustrated in
the fluorescence intensity plot shown in Figure 4h, in which
several nonoverlapping peaks for each marker are detected
(arrows). To quantify the effect of MBC on receptor
clustering, two parameters were selected: the average number of BTX
clusters per cell section (Fig. 4i) and the average size of
each cluster (Fig. 4j). As a result of MBC treatment, the
average number of clusters per cell is significantly increased, whereas
the average size of each cluster is significantly reduced.
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Figure 4.
MBC treatment disrupts lipid rafts and causes the
dispersion of the 7nAChR. Freshly dissociated St 40 ciliary neurons
were left untreated (a-c) or were depleted of
cholesterol with MBC (e-g) and double-labeled with
RITC-BTX (b, f) and FITC-CTX (a,
e). a-c, Confocal sections of untreated cells
are shown. a, Arrowheads point to three
well defined clusters of lipid rafts as identified by CTX.
b, Arrowheads point to the same cell
region shown in a indicating that this area is also
BTX positive and contains 7nAChR. c, Overlay of
the images shown in a and b indicates in
yellow the colocalization of both markers.
e-g, Confocal sections of a cell after MBC treatment
and labeling with BTX (f) and CTX
(e) are shown. g, Overlay of the
images shown in e and f is given.
d, Plot of the fluorescence intensity on the
circumference of the cell shown in c is given.
Arrowheads point to the peaks of fluorescence
corresponding to the cell regions indicated by
arrowheads in c (solid
line, BTX; dotted line, CTX). Note the high
degree of overlap between both markers. h, Plot similar
to that shown in d from the MBC-treated cell is given.
The large numbers of peaks detected on the cell treated with MBC
correspond to the small clusters of BTX and CTX represented in
g. Arrows indicate nonoverlapping peaks
for each marker (solid line, BTX; dotted
line, CTX). i, Plot represents the average
number of BTX-positive clusters in control and MBC-treated cells
(n = 20 cells; *p < 0.003, Student's t test). A cluster was defined as an area of
20 or more (0.18 µm2) adjacent pixels positive for
the marker. j, Plot represents the average size of each
BTX-positive cluster in control and MBC-treated cells
(n = 20 cells; *p < 0.001, Student's t test). Scale bar, 5 µm.
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F-actin colocalizes with the 7nAChR, is depolymerized by
cholesterol depletion, and affects both receptor clusters and lipid
rafts
Although the above studies implicate rafts in receptor clustering,
they do not specify whether this relationship is direct or involves
effects transmitted via other components or mechanisms that influence
clustering. As indicated in the introductory remarks, the cytoskeleton
and its linkage to surface receptors represent relevant factors.
Somatic spines have a well organized cytoskeleton in the form of
polymerized F-actin (Shoop et al., 2000). To determine the spatial
relationship of F-actin to the 7nAChR, freshly dissociated ciliary
neurons were double-labeled with BTX and the F-actin marker
phalloidin. As shown in Figure 2g-i, phalloidin is highly colocalized with BTX clusters, and the ratio of the average
fluorescence intensity between BTX-positive and -negative areas is
very similar for these two markers (Fig. 2l). Because
of this distribution, it was important to determine the functional
relationship between F-actin and both receptor clustering and rafts.
Because the clustering of lipid rafts can induce F-actin polymerization
(Harder and Simons, 1999), we tested whether cholesterol removal
directly affects the F-actin cytoskeleton by treating cells with MBC
and staining with phalloidin. As shown in Figure 5a, control cells show a
discrete distribution of phalloidin in a few large patches on the cell
surface. In contrast, after MBC treatment, phalloidin staining is faint
and disperse, indicating the redistribution of the F-actin cytoskeleton
over the cell surface (Fig. 5b). Accordingly, the average
size of phalloidin-positive patches is substantially decreased in
MBC-treated cells (Fig. 5d). This effect is similar to that
found in cells treated with LAT-A, which selectively induces F-actin
depolymerization (Fig. 5c). Thus, cholesterol-rich lipid
microdomains appear to be required for the maintenance of both the
clusters of 7AChR and spine-associated F-actin filaments.
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Figure 5.
MBC or LAT-A treatment disrupts F-actin filaments.
Freshly dissociated St 40 ciliary neurons were left untreated
(a), treated with MBC (8 mM) for 1 hr
at 37°C (b), or treated with LAT-A (20 µg/ml)
for 2 hr at 37°C (c) and then labeled with
RITC-phalloidin. a, Arrowheads point to
well defined F-actin aggregates. b, MBC-treated cells
show disruption of the F-actin cytoskeleton as a result of cholesterol
removal. c, A similar effect is obtained with LAT-A
treatment. d, Plot indicates the average size of
phalloidin clusters found in control and treated cells. A cluster was
defined as an area of 20 or more (0.18 µm2)
adjacent pixels positive for the marker. Error bars indicate SE. Each
treatment was compared with control values independently
(*p < 0.001, Student's t test).
Scale bar, 5 µm.
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Because 7AChR clusters could be anchored to the cytoskeleton, these
experiments do not exclude the possibility that the dispersal of the
receptor clusters observed after cholesterol depletion is secondary to
the collapse of the spine produced by the depolymerization of the
F-actin filaments. Moreover, treatment with LAT-A converts well
organized 7nAChR macroclusters (Fig.
6a, arrowheads)
into small aggregates (Fig. 6d), so that the number of
clusters significantly increases and the size of each cluster is
significantly reduced (Fig. 6j,k, respectively). However, a
similar effect is simultaneously observed for CTX aggregates (Fig.
6e,j,k). Some colocalization of the receptor with GM1
persists after the actin depolymerization but appears less complete
than that in the control (Fig. 6, compare c, f).
Therefore, because F-actin depolymerization affects both the integrity
of lipid raft patches and 7nAChR clusters, this type of study does
not adequately determine whether there is a direct or indirect (via
F-actin) relationship between rafts and receptor clustering.
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Figure 6.
Patching of GM1 induces an F-actin-independent
clustering of the 7nAChR. a-c, Confocal images of an
untreated St 40 ciliary neuron double-labeled with RITC-BTX
(a) and FITC-CTX (b).
c, Overlay of images in a and
b. Arrowheads indicate marker
colocalization. d-f, St 40 ciliary neuron treated with
LAT-A and labeled with RITC-BTX (d) and
FITC-CTX (e). f, Overlay of images
in d and e illustrating the disappearance
of 7nAChR and CTX clusters after treatment with the toxin.
g-i, Confocal images of an St 40 ciliary neuron
incubated with FITC-CTX and an antibody against CTX before LAT-A
treatment and then labeled with RITC-BTX. g, BTX
staining identifying clusters of 7nAChR on the cell surface
(arrowheads) that are colocalized with CTX
(h). i, Overlay of the images
shown in g and h. j,
Number of BTX and CTX clusters per cell section on control cells
(black bars), LAT-A-treated cells (gray
bars), and cells incubated with anti-CTX antibodies and then
treated with LAT-A (white bars). k, Plot
of the size of BTX and CTX clusters under the same conditions shown
in j. Treated cells were compared with control cells
independently (*p < 0.001, Student's
t test). Note that the number and size of clusters in
cells incubated with anti-CTX antibodies and then treated with LAT-A
are not different from those of control cells. Scale bar, 5 µm.
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Antibody-stabilized lipid microdomains can maintain receptor
clusters independently of the F-actin cytoskeleton
To assess whether forces created by the interaction between lipid
rafts and 7nAChR can maintain receptor clustering independently of
F-actin filaments, dissociated ciliary neurons were incubated with
FITC-CTX followed by an antibody against CTX to help stabilize the
cholesterol-rich lipid microdomains. The addition of CTX and antibodies
against CTX has been shown to cross-link GM1 and thereby increase the
stability of lipid microdomains (Janes et al., 1999). Similarly,
patches of CTX and BTX on ciliary neurons were found to be more
resistant to MBC treatment in the presence of CTX and anti-CTX
antibodies, because some patches of smaller size remained after the
treatment (data not shown). CTX and anti-CTX antibodies by themselves
did not change the appearance of either 7nAChR, lipid rafts, or
F-actin filaments (Fig.
7a-d). When the cells were
subsequently treated with LAT-A to depolymerize F-actin, CTX patches
did in fact persist, although they tended to spread over a larger area
of the cell surface after the collapse of the actin-dependent spines
(Fig. 6h). The depolymerization of F-actin by LAT-A after
lipid rafts were patched with CTX and anti-CTX antibodies was
corroborated by staining with phalloidin (Fig. 7e,f).
Cells were then labeled with RITC-BTX and observed by confocal
microscopy to localize lipid rafts and the 7nAChR. As shown in
Figure 6h, the large patches of CTX on the surface of the
antibody/LAT-A-treated cells (arrowheads) are also positive for BTX (Fig. 6g,i, arrowheads). The GM1 and
7nAChR distribution in these stabilized patches was quantified by
determining the average number of BTX and CTX clusters per cell
section and the average size of each cluster (Fig. 6j,k,
respectively, white bars). The number and size of both CTX
and BTX clusters were undistinguishable from values obtained for the
untreated cells. Together, these findings indicate that the stabilized
lipid domains are able to retain their affinity for the 7nAChR in
the absence of F-actin, in accord with a direct interaction between the
domain and the receptor.
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Figure 7.
Anti-CTX antibodies do not affect the distribution
of 7nAChR, lipid rafts, or F-actin cytoskeleton. Freshly dissociated
ciliary neurons were incubated with RITC-BTX and FITC-CTX (a,
b) or with Alexa488-BTX and unconjugated CTX
(c-f) for 1 hr on ice followed by a 30 min
incubation with anti-CTX antibodies at 37°C. Thereafter cells were
either fixed (a, b) or treated with LAT-A for 2 hr at
37°C (e, f) or left untreated (c,
d) and stained with RITC-phalloidin after fixation
(c-f). a, b,
Arrowheads point to well defined patches of 7nAChR
and lipid rafts on the same cell, indicating that treatment with
antibodies against CTX does not change the distribution of either
marker. c, d, Arrowheads point to patches
of 7nAChR and F-actin, respectively, also indicating that the
antibody does not affect the organization of the cytoskeleton.
e, Arrowheads point to patches of
7nAChR, which remained clustered by the anti-CTX antibody after
treatment with LAT-A. f, Arrowheads point
to the same cell regions shown in e, indicating the
absence of F-actin cytoskeleton. Scale bar, 5 µm.
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The 7nAChR subunit is resistant to Triton X-100 solubilization
and partitions with the lipid raft fraction in density gradients
One of the properties of proteins associated with cholesterol-rich
membrane microdomains is their ability to remain insoluble in the
presence of Triton X-100 at 4°C. Because this material has a
high-lipid content, it floats during density gradient centrifugation (Brown and Rose, 1992). This method therefore can distinguish between
proteins that are detergent insoluble because of their association with
the cytoskeleton (they do not float) and those that are Triton X-100
resistant because of their association with lipid raft microdomains. To
determine whether 7nAChR is associated with membrane lipid rafts, St
40-42 CGs were homogenized, and the membrane fraction was isolated by
gradient centrifugation. Thereafter, the membrane-containing fractions
were extracted with Triton X-100 at 4°C, separated by density
gradient centrifugation, and analyzed by SDS-PAGE and Western blotting.
As shown in Figure 8, a sizable fraction
of the 7nAChR is found in the low-density (top) fractions of the
flotation gradient (fractions 1, 2), indicating the
association of the receptor subunit with detergent-resistant lipid
microdomains. Traces of the receptor could also be detected in the
higher density fractions (Fig. 8, fractions 3-6),
indicating that not all receptor subunits are or remain associated with
detergent-resistant membrane microdomains. The localization of the
majority of caveolin, a known lipid raft component, in the first two
fractions of the gradient confirms the position of the
detergent-resistant low-density fraction. Because caveolin has not been
reported to be expressed in substantial amounts in neurons, the
caveolin detected by Western blots in CG homogenates likely is derived
from Schwann and glial cells present in the CG and serves here only as
a convenient marker.
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Figure 8.
7nAChR is associated with detergent-resistant
membrane lipid microdomains. A membrane fraction from St 40-42 CGs was
prepared by gradient centrifugation, incubated with Triton X-100 on ice
for 30 min, and subjected to discontinuous gradient centrifugation
(35-30-0% Optiprep) at 285,000 × g for 4 hr at
4°C. Six fractions were collected from the top
(fraction 1) to the bottom
(fraction 6). 7nAChR was isolated from
each sample with BTX-conjugated Actigel, separated on 10% SDS-PAGE,
and analyzed by Western blotting with a monoclonal antibody against the
7nAChR. An aliquot of each fraction was also analyzed by Western
blotting with a polyclonal antibody against caveolin. The Western blots
indicate that the bulk of 7nAChR floats to the lower density
fractions (fraction 1, 2), indicating its
association with detergent-resistant membrane structures. The
accumulation of the integral lipid raft protein caveolin in the first
two fractions confirms the migration of the detergent-insoluble
components of the membrane to the low-density portion of the
gradient.
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DISCUSSION |
This study provides direct evidence that the 7nAChR is
associated with cholesterol-rich membrane lipid microdomains and that maintenance of these receptors within somatic spine-rich regions of
ciliary neurons is dependent on the presence of these microdomains within that structure. The results obtained also suggest that the
F-actin at somatic spines plays a less direct role in receptor redistribution. The following discussion relates these findings to
other work on lipid microdomains and then considers a series of issues
concerning the molecular nature and function of receptor-raft complexes in the somatic spine, as well as their relationship to the
actin-based cytoskeleton.
There is ample evidence in other systems that association of
transmembrane receptors and signaling molecules with cholesterol-rich microdomains provides a cellular mechanism for the concentration of
receptor proteins to particular functional domains of the cell surface
(Simons and Ikonen, 1997; Brown and London, 1998; Jacobson and
Dietrich, 1999). For example, ephrinB1 is localized in cholesterol-rich rafts in which it associates with various signaling molecules including
the glutamate receptor-interacting protein (GRIP). Stimulation of
ephrinB1 with EphB2 receptor causes the formation of large raft patches
that contain GRIP and induces serine/threonine kinase activity
(Bruckner et al., 1999). Similarly, the activation of CD28 leads to the
redistribution and clustering of kinase-rich lipid microdomains at the
site of T cell receptor (TCR) engagement (Montixi et al., 1998; Grakoui
et al., 1999; Harder and Simons, 1999). This redistribution of the TCR
is mediated by the reorganization of membrane lipid microdomains (Janes
et al., 1999), appears to involve the actin cytoskeleton (Viola et al.,
1999), and depends on myosin motor proteins (Wulfing and Davis,
1998).
In the present case, the fact that some of the 7nAChR did not float
with caveolin-rich fractions suggests either that not all receptor
molecules are incorporated into lipid rafts or that they partition
between raft-associated and -unassociated states. A number of proteins
are known to be functionally altered by a partial association with
lipid rafts. For example, GRIP variably associates with lipid rafts via
its interaction with ephrinB (Bruckner et al., 1999). A similar
phenomenon can occur via binding to a linker protein that is variably
associated with rafts. For example, the adhesion molecule CD44
associates with rafts via interaction with annexin II, which partitions
into the rafts in a calcium-dependent manner (Oliferenko et al.,
1999).
In addition to an association with lipids, some raft-associated
receptors also associate with the cytoskeleton via linker proteins. An
example is annexin II, which associates with actin filaments as well as
the cell membrane (Kube et al., 1992; Harder and Gerke, 1994). Thus, in
the ciliary neuron, which accumulates rafts and an actin-based
cytoskeleton in the same structure, an annexin II-like protein could
participate in both the aggregation of the 7nAChR-containing lipid
rafts and their coupling to the actin cytoskeleton.
A direct role for F-actin in receptor clustering, which would be
consistent with the observation that disruption of the actin filaments
with LAT-A causes dispersion of 7nAChR clusters, remains less clear.
The difficulty in demonstrating such a role stems from the fact that
the structure of the spine itself requires an intact actin cytoskeleton
(Shoop et al., 2000). Moreover, cell surface patching of the raft
component GM1 by CTX is known to cause accumulation of filamentous
actin (Harder and Simons, 1999), suggesting that the clustering of
lipid rafts leads to the recruitment of F-actin to help form the
somatic spines. Similarly, the integrity of actin cytoskeleton in
neurons has been shown to influence the association of both 7nACh
and NMDA receptors with spines (Allison et al., 1998; Shoop et al.,
2000); yet the localization of these receptors or associated molecules
to spines is independent of actin filaments and resistant to detergent
extraction (Allison et al., 2000; Shoop et al., 2000). These
observations would again suggest that the role of the cytoskeleton is
indirect and probably occurs via its ability to form or maintain the
spine. Nevertheless, the view that actin fiber recruitment lies
"downstream" of raft aggregation is probably overly simplistic,
because the cytoskeleton may well contribute to additional structuring
of this lipid domain and is known to be a factor in molding that cell
surface region into a mature somatic spine.
Finally, it is not yet clear what signals and processes underlie the
clustering of lipid rafts on ciliary neurons during synaptogenesis and
whether this process serves to induce as well as maintain the
distribution of the 7nAChR. In the case of the formation of the
neuromuscular junction, the clustering of the nAChR is induced by the
release of agrin from the presynaptic axonal terminal that binds to
MUSK, the muscle-specific tyrosine kinase, and activates a
tyrosine phosphorylation cascade that ends in the clustering of rapsyn
and the nAChR (Gautam et al., 1995, 1996; DeChiara et al., 1996; Glass
et al., 1996a,b; Apel et al., 1997; Sanes and Lichtman, 1999). Although
the molecular details of these events have not been completely
elucidated, it is clear from these studies that a nerve-derived signal
activates the machinery for receptor clustering. If we speculate that
rafts can be clustered on the surface of ciliary neurons via such an
extracellular signal, the progressive colocalization of lipid rafts and
7nAChR during synapse formation in the CG may not be merely
coincidental events. Instead the aggregation of lipid microdomains
would be a key early event both in the molecular organization of the
synapse as well as in the formation of somatic spines.
In summary, it is becoming evident that 7nAChR and other
neurotransmitter receptors are associated directly or via linker proteins with cholesterol-rich membrane microdomains. In addition, lipid rafts are required to maintain receptor clustering and may induce
the polymerization of F-actin bundles that constitute the spine
cytoskeleton. It will now be important to identify the nature of the
physical interaction between receptors and lipid rafts and to determine
whether linker molecules participate in this interaction.
| |
FOOTNOTES |
Received July 27, 2000; revised Oct. 25, 2000; accepted Oct. 30, 2000.
This study was supported by National Institutes of Health Grants
EY06107 and HD18369. N.C. was supported in part by the Institut National de la Santé et de la Recherche Médicale. We thank
Drs. William G. Conroy and Darwin K. Berg of the University of
California at San Diego for providing the monoclonal antibody 319 against the 7 subunit of the nicotinic acetylcholine receptor.
Correspondence should be addressed to Dr. Juan L. Brusés,
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 290, New
York, NY 10021. E-mail: j-bruses{at}ski.mskcc.org.
| |
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TOP
ABSTRACT
INTRODUCTION
