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Cutting Edge: Optical
Microspectrophotometry Supports the
Existence of Gel Phase Lipid Rafts at the
Lamellipodium of Neutrophils: Apparent
Role in Calcium Signaling
Andrei L. Kindzelskii, Robert G. Sitrin and Howard R. Petty
J Immunol 2004; 172:4681-4685; ;
doi: 10.4049/jimmunol.172.8.4681
http://www.jimmunol.org/content/172/8/4681
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Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
OF
THE
JOURNAL IMMUNOLOGY
CUTTING EDGE
Cutting Edge: Optical Microspectrophotometry Supports
the Existence of Gel Phase Lipid Rafts at the
Lamellipodium of Neutrophils: Apparent Role in
Calcium Signaling1
Andrei L. Kindzelskii,* Robert G. Sitrin,‡ and Howard R. Petty2*†
ipid rafts are believed to be small (⬃50-nm) membrane
microdomains rich in cholesterol and sphingolipids.
Due to the high degree of saturated fatty acyl substitution in these sphingolipids, lipid rafts are thought to form a distinct gel-like ordered lipid (Lo)3 phase within cell membranes.
These domains have also been reported to be enriched in a variety of membrane proteins, especially GPI-linked proteins (1–
4). The cytoplasmic face of lipid rafts is thought to be enriched
in a variety of signaling molecules. These molecules, in turn,
may participate in certain signaling pathways of T cells, B cells,
and mast cells (1– 4). However, these conclusions are based
largely on fractionated cell components isolated by differential
detergent ultracentrifugation. To deflect concerns regarding
potential artifacts of cell fractionation, methyl-␤-cyclodextrin
L
Departments of *Ophthalmology and Visual Sciences, and †Microbiology and Immunology, and ‡Pulmonary and Critical Care Division, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48105
Received for publication January 2, 2004. Accepted for publication February 17, 2004.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Copyright © 2004 by The American Association of Immunologists, Inc.
(m␤CD) is frequently used, because it binds to cholesterol and
thereby disrupts lipid rafts in plasma membranes (5, 6). Because
m␤CD treatment provides no direct structural information regarding lipid rafts, it would be useful to have an independent
means of verifying and studying lipid rafts in cell membranes.
Recently, the fluorescent probe Laurdan has been used to study
lipid raft-like phase separations in artificial lipid bilayers (7),
although these are not necessarily representative of living cells.
Because lipid rafts in cell membranes are ⬃50 –100 nm in size,
which is much smaller than the wavelength of light, their analysis has been very limited. However, morphologically polarized
cells display large aggregates of lipid raft markers in the region of
the lamellipodium (8, 9). Thus, we have used polarized neutrophils to circumvent the size difficulties encountered in studying
lipid rafts in living cells. Using the lipid probe Laurdan in conjunction with an imaging monochrometer-based microscope
system, we have observed dramatic changes in the spectral properties of Laurdan at a cell’s lamellipodium, indicating a distinct
lipid-ordered structure at the leading edge of intact, living human neutrophils. This lipid raft-like structure disappears after
treatment with m␤CD. This is also the site of Ca2⫹ wave ignition in polarized neutrophils (10), and propagation of these
Ca2⫹ waves is abrogated by m␤CD. Thus, the existence of large
lipid raft-like domains participating in Ca2⫹ signaling can be
directly observed in living polarized neutrophils.
Materials and Methods
Materials
A polyclonal rabbit anti-transient receptor potential-like channel-1 (TRPC-1)
Ab was purchased from Chemicon International (Temecula, CA). Anti-GM3
was purchased from Seigagaku America (East Falmouth, MA). Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) was obtained from Molecular Probes
(Eugene, OR). m␤CD was obtained from Sigma-Aldrich (St. Louis, MO).
Cell preparation
Neutrophils were isolated from blood samples using Ficoll-Hypaque (SigmaAldrich) density gradient centrifugation (10). Neutrophil viability was ⬎95%
as assessed by trypan blue exclusion. Cells were suspended in HBSS (Life Technologies, Grand Island, NY). For Ca2⫹ studies, cells were prelabeled with indo1-AM at 5 ␮g/ml for 20 min at 37°C (10). The value of n given below is the
1
This research was supported by CA074120 (to H.R.P.) and HL58283 (to R.G.S.).
2
Address correspondence and reprint requests to Dr. Howard R. Petty, Department of
Ophthalmology and Visual Sciences, University of Michigan Medical School, 1000 Wall
Street, Ann Arbor, MI 48105. E-mail address: [email protected]
Abbreviations used in this paper: Lo, ordered lipid; m␤CD, methyl-␤-cyclodextrin;
SOC, store-operated Ca2⫹ channel; TRPC-1, transient receptor potential-like channel-1.
3
0022-1767/04/$02.00
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Although much progress has been made in elucidating the
biochemical properties of lipid rafts, there has been less
success in identifying these structures within living cell
membranes, which has led to some concern regarding their
existence. One difficulty in analyzing lipid rafts using optical microscopy is their small size. We now test the existence of lipid rafts in polarized neutrophils, which redistribute lipid raft markers into comparatively large
lamellipodia. Optical microspectrophotometry of Laurdan-labeled neutrophils revealed a large blue shift at lamellipodia relative to cell bodies. This blue shift disappeared after exposure to methyl-␤-cyclodextrin (m␤CD),
which disrupts lipid rafts. The Ca2ⴙ channel transient receptor potential-like channel-1, a lipid raft marker, traffics to lamellipodia, but redistributes uniformly about
cells after exposure to m␤CD. This is accompanied by disruption of Ca2ⴙ waves normally initiated at lamellipodia. Thus, m␤CD-sensitive lipid-ordered domains are
present at and participate in signaling from the lamellipodia of living neutrophils. The Journal of Immunology,
2004, 172: 4681– 4685.
4682
CUTTING EDGE: LIPID RAFTS IN NEUTROPHILS
number of days an experiment was repeated, with at least 50 cells evaluated for
each condition.
Lipid raft disruption
To disrupt lipid rafts, neutrophils were incubated with 5 mM m␤CD for 5 min
at 37°C as described (11).
Fluorescence microscopy
Cells were observed using an axiovert fluorescence microscope (Carl Zeiss, New
York, NY) (10). A narrow bandpass filter set (Omega Optical, Brattleboro, VT)
was used with excitation at 485/22 nm and emission at 530/30 nm for FITC,
and an excitation of 540/20 nm and emission at 590/30 nm for tetramethylrhodamine isothiocyanate. Long-pass dichroic mirrors of 510 and 560 nm were
used for FITC and tetramethylrhodamine isothiocyanate, respectively. The fluorescence images were collected with an intensified charge-coupled device camera (Princeton Instruments, Princeton, NJ).
Emission spectrophotometry
High-speed fluorescence microscopy
High-speed microscopy was performed as previously described (10). To detect
fluorescence changes in the short wavelength emission region of indo-1, a
355HT15 exciter, a 390LP dichroic reflector, and a 405DF43 emission filter
were used. A Gen-II intensifier tube was used to provide maximal detector efficiency at indo-1’s emission. For data management, a computer with dual
3.06-GHz xenon processors with 1-MB onboard cache each, 3.0-Gb RAM, and
3.2 Tb of hard drive space, was used. For data capture, a software-allocated
RAM disk was used.
Results
The neutrophil lamellipodium is enriched in ganglioside GM3 and
TRPC-1
Several previous studies have shown that the lamellipodia of
lymphocytes are enriched in lipid raft markers (8, 9). Therefore,
we first confirmed that lipid raft markers accumulate at the lamellipodium of neutrophils. When adherent to coverslips,
26 ⫾ 5% of the cells become polarized spontaneously. Neutrophils were labeled with anti-GM3 Ab. Representative micrographs are shown in Fig. 1. Although spherical cells did not display large clusters of GM3, 92 ⫾ 6% of the polarized cells
exhibited large aggregates of GM3 at lamellipodia (Fig. 1B).
Similarly, the Ca2⫹ channel TRPC-1, which also cofractionates
with lipid rafts (14), collects at lamellipodia of polarized cells
(94 ⫾ 5%) (Fig. 1F). Previous studies (15) have shown that
uPAR, a GPI-anchored protein, which associates with lipid
rafts, also collects at lamellipodia. Therefore, the neutrophil lamellipodium is highly enriched in lipid raft markers. Previous
studies have shown that lipid rafts can be disrupted by m␤CD,
which abstracts cholesterol from cell membranes (5, 6). Cells
were exposed to 5 mM m␤CD for 5 min at 37°C followed by
immunofluorescence microscopy. Although morphologically
polarized neutrophils were observed after m␤CD treatment
(Fig. 1, C and G), GM3 (D) and TRPC-1 (H) did not traffic to
the lamellipodium of treated cells; enriched labeling of the lamellipodium was noted in only 13 ⫾ 2 and 8 ⫾ 3% of the cells,
respectively. Thus, lipid raft markers collect at the lamellipodia
of polarized cells in an m␤CD-sensitive fashion.
Direct observation of Lo domains in morphologically polarized
neutrophils
Having demonstrated that lamellipodia possess lipid raft markers, we next sought to test the hypothesis that these domains
represent a distinct lipid environment. Although Lo phase lipids
have been observed in reconstituted lipid rafts using Laurdan
(7), they have not been demonstrated in living cells. One key
reason for this is that lipid rafts are often smaller than the wavelength of light, making identification of Lo phase rafts difficult.
As shown in Fig. 1, morphologically polarized neutrophils display ganglioside-enriched lipid raft-like regions at the lamellipodium. To test for differences in membrane fluidity in different regions of cells, neutrophils were labeled with Laurdan (16).
In fluid membranes, Laurdan exhibits a red shift in its steadystate emission spectrum compared with gel phase membranes.
Using high-sensitivity microspectrophotometry, we collected
emission spectra from the lamellipodia, uropods, and the lateral
cell body surfaces of morphologically polarized neutrophils. Although the emission spectra of spherical cells did not differ from
those of the lateral cell body surfaces (p ⬎ 0.3), the lamellipodia
were blue-shifted in comparison to spherical cells (Fig. 2), indicating that these regions are less fluid than other membrane
regions. The peak was shifted by ⬎12 nm (p ⬍ 0.001). Similar
observations were noted for the uropod (data not shown). The
blue shift of Laurdan in the lamellipodium is one of the most
pronounced spectral shifts (i.e., least fluid) reported for Laurdan or prodan in biological membranes. These studies provide
direct evidence in living cells for the presence of Lo phase domains, which spatially correspond to ganglioside-enriched domains and lipid rafts as defined by cell fractionation.
Having established the presence of Lo phase lipids in living
cell membranes, we next sought to test the ability of m␤CD to
influence these lipid domains. Although m␤CD affects the recovery of cold detergent-resistant membrane fractions, whether
or not artifacts of detergent solubilization contribute to these
observations cannot be resolved without confirmatory cell biology evidence. We found that morphologically polarized neutrophils remained polarized after treatment with 5 mM m␤CD.
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To study membrane phase properties, the fluorescent probe Laurdan, which
shows a significant shift to shorter emission wavelengths (blue shift) in gel phase
relative to liquid crystalline membranes (7), was used. However, if the surrounding lipid molecules move during its fluorescence lifetime, Laurdan’s excited state dipole will reorient neighboring molecules, and a red shift in its
steady-state emission spectrum will be observed. Laurdan emission spectra were
collected from specific regions of single cells using an imaging spectrophotomer
system. Cells were labeled with 2.5 ␮M Laurdan for 25 min at room temperature and then washed at least three times with buffer. To increase light collection efficiency, the microscope’s bottom port was fiber-optically coupled to the
input side of an Acton-150 (Acton Instruments, Acton, MA) imaging spectrophotometer. The exit side was connected to a liquid N2-cooled intensifier attached to a Peltier-cooled I-MAX-512 camera (approximately ⫺20°C) (Princeton Instruments) (12, 13). The camera was controlled by a high-speed
Princeton ST-133 interface and a Stanford Research Systems (Sunnyvale, CA)
DG-535 delay gate generator. Microspectrophotometry used a 355-nm bandpass discriminating filter for excitation and a 405-nm long-pass dichroic mirror.
Winspec software (Princeton Instruments) was used to analyze data.
FIGURE 1. Accumulation of lipid raft markers at neutrophil lamellipodia.
Cells were labeled with anti-GM3, a lipid raft ganglioside marker (A–D) and
TRPC-1, a Ca2⫹ channel associated with lipid rafts (E–H). Cells were examined without (A, B, E, and F) and with (C, D, G, and H) prior exposure to 5 mM
m␤CD for 5 min at 37°C. GM3 and TRPC-1 trafficked to the lamellipodium
of polarized neutrophils, but not on spherical cells (B and F). However, exposure to m␤CD blocked the ability of these molecules to accumulate at the lamellipodium (D and H). n ⫽ 4.
The Journal of Immunology
4683
FIGURE 3. Low-temporal resolution microfluorometry studies of neutrophil Ca2⫹ spikes. Photomultiplier tube recordings of Ca2⫹ signals were made in
untreated (A) and m␤CD-treated (B) polarized neutrophils. Note the dramatic decrease in the amplitude of the Ca2⫹ signal. Data were recorded for 6 min. n ⫽ 3.
We therefore examined m␤CD-treated cells using Laurdan.
We found no significant differences between the Laurdan emission spectra of spherical neutrophils and morphologically polarized neutrophils examined at the lamellipodium, lateral surfaces, and uropod (Fig. 2). Thus, the Lo phase lipid domains
disappear from the lamellipodium and uropod upon exposure
to m␤CD. These experiments provide novel insights into the
structural organization of lipid domains and further support the
identification of Lo domains as GM3-rich lipid rafts.
m␤CD-sensitive Lo lipid domains are required for Ca2⫹ wave
propagation, but not ignition
Early studies by Maxfield and coworker (17) showed that polarized neutrophils display intracellular Ca2⫹ spikes at regular
intervals, and we have replicated this finding in our laboratory
(e.g., Ref. 10). Neutrophils were labeled with the Ca2⫹-sensitive dye indo-1-AM, as described (10). When these cells were
observed using quantitative microfluorometry, a series of Ca2⫹
spikes were found (Fig. 3A). A recent study has suggested that
m␤CD affects Ca2⫹ signaling in neutrophils (5). When morphologically polarized m␤CD-treated neutrophils were examined, a much smaller Ca2⫹ bump was observed (Fig. 3B). The
time base of these experiments is very long (6 min), which gives
only a few pixels per spike. To obtain high temporal resolution
data, experiments were performed for ⬍1 s (Fig. 4). These experiments reveal the fine structure of these Ca2⫹ signals. The
Ca2⫹ bump appears to correspond to a shoulder at the leading
edge of the Ca2⫹ signal, which we interpret to be the ignition of
FIGURE 4. High temporal resolution microfluorometry studies of neutrophil Ca2⫹ spikes. Photomultiplier tube recordings of Ca2⫹ signals were made in
untreated (trace 1) and m␤CD-treated (trace 2) polarized neutrophils. The amplitude of the Ca2⫹ signal is dramatically decreased. The Ca2⫹ bump of
m␤CD-treated cells appears to correspond to an initial shoulder of the Ca2⫹
spike, which we interpret as the ignition of the Ca2⫹ signal. Data were recorded
for 0.8 s. n ⫽ 3.
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FIGURE 2. Demonstration of lipid domains in neutrophil membranes.
Cells were labeled with Laurdan, which undergoes a red shift in fluid membranes. Data from polarized cells were collected exclusively from the lamellipodium (lamel.), uropod, and the lateral aspects of the cell body. Data from spherical cells were collected from the entire cell. The emission spectrum of Laurdan
from the lamellipodium in controls (A) is dramatically shifted in comparison to
the cell body of controls (B) and spherical cells (B), and that of m␤CD-treated
lamellipodia (A). Thus, the lamellipodium is substantially more rigid than other
domains of the neutrophil plasma membrane. n ⫽ 4.
the Ca2⫹ signal. Thus, the Lo domain, which apparently contains important Ca2⫹ signaling machinery such as TRPC-1, appears to be necessary to generate Ca2⫹ signaling in morphologically polarized neutrophils.
We have recently developed high-speed optical microscopy,
which allows us to detect chemical signals within small cells
with unprecedented detail (10, 12, 13). A previous high-speed
microscopy study from this laboratory showed that each repetitive Ca2⫹ spike corresponded to a Ca2⫹ wave traveling around
the neutrophil’s periphery (10). We therefore sought to test the
concept that the lamellipodium plays a central role in Ca2⫹ signaling in an m␤CD-sensitive fashion. Fig. 5A shows an example of the traveling Ca2⫹ wave in an untreated cell. However,
after treatment with m␤CD, only a brief Ca2⫹ spark is observed
(Fig. 5B, arrow), which is the imaging equivalent of the bump
detected with microfluorometry. The advantage of this approach is that we can see that the signaling is disrupted at the
lamellipodium of polarized neutrophils. We speculate that
m␤CD treatment uncouples the intracellular Ca2⫹ signaling
apparatus (ignition) from the plasma membrane-bound signaling apparatus (propagation).
4684
CUTTING EDGE: LIPID RAFTS IN NEUTROPHILS
Discussion
The present study provides strong direct evidence for the existence of a lipid raft-like Lo domain at the lamellipodium of polarized neutrophils. The formation of lipid raft-like structures
has been previously noted for polarized lymphocytes (8, 9). The
neutrophil’s lamellipodium contains lipid raft markers such as
GPI-linked proteins (1– 6, 15), GM3 (Fig. 1B), and TRPC-1
(F). However, it is also possible that these molecules are delivered to the lamellipodium by a mechanism distinct from lipid
raft formation. Furthermore, although certain components
may be identified in lipid rafts on the basis of cell fractionation
and ultracentrifugation, it is always possible that an artifact
might be introduced during isolation procedures. Therefore,
we sought to confirm the presence of lipid rafts using an independent approach using intact cells. Although previous studies
have used the membrane probe Laurdan to examine the fluidity
of neutrophil membranes (16), these experiments did not provide spatially resolved spectral information at the single-cell
level and therefore do not address lateral variations in lipid
properties. Using imaging spectroscopy, we now show that
Laurdan undergoes a dramatic blue shift at the lamellipodium,
indicating that this region represents a highly ordered lipid domain. Because the lamellipodium is several micrometers in size,
we avoid the difficulties inherent in obtaining spatially resolved
information from lipid rafts. Our data clearly show that Laurdan samples a more ordered environment at the lamellipodium
than at other locations about the cell. While this manuscript
was in preparation, a study was reported using Laurdan in conjunction with other methods to examine spatially resolved domains on another cell type (18). These emerging studies of lipid
domains and their spatial identity with lipid raft markers in living cells gives renewed confidence in the existence of these
structures.
One crucial function of lipid rafts is believed to be their ability to serve as signaling platforms (1– 6). Cholesterol-rich lipid
rafts have recently been suggested to participate in neutrophil
Ca2⫹ signaling (5). Using previously established quantitative
microfluorometry and high-speed fluorescence microscopy
methods, we have studied the Ca2⫹ signaling events associated
with spontaneous neutrophil motility. Using an efficient optical system and photodetector capable of sensing single atoms,
we have been able to achieve electronic shutter speeds of ⬃0.1
␮s, which allows us to follow the intracellular trafficking of intracellular signals (10, 12, 13). Our recent study (10) and the
experiments described above indicate that the plasma membrane component of this signal originates at the lipid raft-like
lamellipodium. Thus, high-speed microscopy allows us to definitively establish the lipid raft as the source of the plasma
membrane signal. To further support this conclusion, cells were
studied after m␤CD treatment. Exposure to m␤CD dramatically reduced signal amplitude and its associated plasma membrane Ca2⫹ wave. Because lipid raft-associated TRPC-1 is a
type of store-operated Ca2⫹ channel (SOC) and SOCs have
been implicated in neutrophil raft signaling, the TRPC-1 clusters at the lamellipodium and their dissolution by m␤CD may
explain the changes in Ca2⫹ signaling that we have observed. In
a separate study, we have shown that SOC inhibitors block this
Ca2⫹ wave.4 Our data provide strong cell biological evidence
supporting the existence of lipid rafts in human neutrophils and
that these rafts are intimately associated with the Ca2⫹ signaling
apparatus. This signaling apparatus appears to be linked with
downstream processes, such as NADPH oxidase efficiency (19)
and cell motility (20).
4
A. Kindzelskii and H. Petty. Ion channel clustering enhances weak electric field
detection by neutrophils: apparent role of SKE96365-sensitive cation channels in
early events. Submitted for publication.
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FIGURE 5. m␤CD inhibits Ca2⫹ wave propagation in neutrophils. A, A Ca2⫹ wave traveling about the perimeter of untreated cells is shown. B, Shown is a brief
Ca2⫹ spark (arrow) in the same cell after 5-min exposure to m␤CD, without subsequent propagation of the Ca2⫹ wave. n ⫽ 3.
The Journal of Immunology
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