From the George W. Woodruff School of Mechanical
Engineering and Department of Biomedical Engineering, Georgia Institute
of Technology, Atlanta, Georgia 30332-0363 and the ¶ Department of
Pathology and Laboratory Medicine, Emory University School of Medicine,
Atlanta, Georgia 30322
Received for publication, November 17, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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Just as interactions of soluble proteins are
affected by the solvent, membrane protein binding is influenced by the
surface environment. This is particularly true for adhesion receptors because their function requires tightly apposed membranes. We sought to
demonstrate, and further, to quantify the possible scale of this
phenomenon by comparing the effective affinity and kinetic rates of an
adhesion receptor (CD16b) placed in three distinct environments: red
blood cells (RBCs), detached Chinese hamster ovary (CHO) cells, and
K562 cells. Effective affinity reflects both the intrinsic
receptor-ligand kinetics and the effectiveness of their presentation by
the host membranes. Expression of CD16b, a low affinity Fc Cell-cell bond formation must be preceded by the creation of a
contact zone, a region in which surface-bound receptors and ligands are
able to bridge narrow gaps, properly aligning by lateral and rotational
diffusion (1). Rough cells initially may form isolated point contacts
that over time might be broadened and connected through active membrane
processes (2). Alternatively, the cells may simply possess inherently
smooth surfaces capable of broad initial contacts. A smooth membrane
will not enhance binding to a soluble ligand because the soluble
protein can diffuse freely into the membrane folds of rough cells.
However, in adhering to immobilized ligand, the ability to form an
expansive tight contact area is a distinct advantage. Therefore,
cell-cell adhesion depends not only on the intrinsic kinetic rates of
the receptor-ligand interaction but also on how effectively the host
membranes present these molecules (3).
In this study we quantified the effects of various microtopological
presentations on cell adhesion by comparing the effective affinity of
the same receptor (CD16b) in three distinct host environments: erythrocytes (RBCs),1 Chinese
hamster ovary (CHO) cells, and erythroleukemic K562 cells. Although all
other Fc receptors have intracellular domains, CD16b (Fc The adhesive character of CD16b in each environment was quantified by
using a recently developed micropipette method (4). This assay is
unique in its yield of estimates for the effective kinetic rate
constants for binding of membrane-bound ligand, so-called two-dimensional parameters. Earlier results from the application of
this assay to the binding of CD16b-transfected CHO and K562 cells to
IgG-coated RBCs produced similar two-dimensional binding parameters for
both transfectants (5, 6). The objective of the present study was to
examine an additional environment, the RBC membrane, that has a
markedly smoother morphology and to compare these results with those
from the earlier CHO and K562 studies. We also visually compared the
structure of the different cell to cell contacts with scanning electron
microscopy (SEM). In this manner we were able to demonstrate a major
role for membrane microtopology in cell adhesion and suggest an
expanded role for the cytoskeleton in the regulation of adhesion.
Cells and Proteins--
Erythrocytes were isolated from whole
blood and stored in EAS45 RBC additive solution (5). Transfected
cell lines and murine monoclonal antibodies were produced in house
except for UPC-10 (IgG2a, Sigma). CD16b (NA2 allotype) was
detergent-extracted from transfected K562 cells and purified by
affinity chromatography (7) with the addition of 1,10-phenanthroline (5 mM) to the lysis buffer. Purity was confirmed by
SDS-polyacrylamide gel electrophoresis.
Reconstitution and Chromium-Chloride
Coupling--
Reconstitution, or transfer of purified CD16b to target
membranes, was accomplished by coincubation at 37 °C according to Nagarajan et al. (7). Reconstituted RBCs were kept in
ice-cold EAS45 for up to 2 weeks (5). Covalent coupling of human
IgG to the surfaces of RBCs employed a modified chromium-chloride method (5). Site densities of protein on RBCs
(CrCl3-coupled IgG or reconstituted CD16b) were determined
by quantitative indirect fluorescent immunoassay using LFA-3 expression
as in Ref. 5. Site densities of CD16b on K562 and CHO cells were
quantified by radioimmunoassay using 125I-CLBFcgran1 Fab fragments.
The Micropipette Adhesion Frequency Assay--
Details of the
micropipette system and the adhesion frequency assay have been
described (4). Briefly, the assay involves using a computer-controlled
piezoelectric actuator to move a pair of cells (one receptor-bearing
and one ligand-bearing) into contact, hold them for a prescribed
duration, and then quickly retract one. Direct observation of any
deflection in the flexible RBC membrane indicates the presence of at
least one bond at the moment of retraction (Fig.
1). A video demonstration of the
micropipette assay is available online (Video 1). For each cell line
40-80 cell pairs were examined over a range of contact durations and receptor-ligand expression levels, with at least 100 consecutive adhesion cycles performed for each cell pair. Adhesion frequencies were
either directly compared or used to generate characteristic binding
parameters. In the latter cases, estimates for the effective affinity
and kinetics were determined (4, 8) by fitting the adhesion frequency
data to a probabilistic model using a nonlinear regression method based
on the Levenberg-Marquardt algorithm (5).
Scanning Electron Microscopy--
Samples intended for CD16b
localization studies were labeled with CLBFcgran1 followed by goat
anti-mouse IgG polyclonal antibody conjugated to 12-nm gold particles
(Jackson Immunologicals; Ref. 3). Conjugated samples were prepared by
mixing equal amounts of receptor and ligand-bearing cells, centrifuging
(5 min, 200 × g), incubating on ice for 30 min, and
gently resuspending with a wide mouth pipette. Normal RBCs replaced the
reconstituted RBCs in negative controls. Samples were examined for
conjugate formation under light microscopy prior to fixation.
Subsequent sample processing and imaging were performed by R. Apkarian
(Emory University Integrated SEM Facility). Specimens were staged in
the lens of an ISI DS-130 SEM operated at 5 kV. Localization images
were obtained below the lens using a 4-mm working distance.
CD16b and IgG Expression Levels on the Host Cell
Surfaces--
CD16b-expressing K562 and CHO cells, transfected from
the same vectors, were used as independent cellular hosts for the
adhesion receptor. Because CD16b utilizes a hydrophobic GPI anchor, it can be reinserted, or reconstituted, into the lipid bilayer of any
available membrane (7). Accordingly, CD16b was purified from the K562
transfectant membranes and reconstituted into RBCs and wild-type CHO
cells. The ligand, human IgG, was covalently coupled to RBCs by a
chromium-chloride method. Expression levels for all cells are shown in
Fig. 2. Flow cytometry before and after each experiment series confirmed expression stability.
RBC-reconstituted CD16b Binds Specifically to RBC-bound
IgG--
Specificity was established with the micropipette assay (Fig.
3a). RBCs that underwent the
sham reconstitution (i.e. without CD16b present) showed
minimal binding to IgG+ RBCs. Preincubation of CD16b+ RBCs with the
function-blocking monoclonal antibody CLBFcgran1 (10 µg/ml, 30 min,
4 °C) suppressed the binding seen when preincubating these cells
with an irrelevant mIgG2a. Similarly, preincubation of IgG+ RBCs with
soluble dimeric CD16a, a moderate affinity Fc The Time-dependent Adhesion Frequency Provides
Estimates for the Two-dimensional Kinetic Rates Ackf
and kr and the Effective Affinity
AcKa--
The micropipette assay was used to generate
time-dependent adhesion frequency curves from three sets of
RBC pairs expressing various levels of CD16b and IgG. An additional
series of experiments was performed with cells coated with ovalbumin
rather than IgG. Results are shown in Fig. 3b. All data were
fit simultaneously to a previously described probabilistic adhesion
model (4) to determine the effective affinity and kinetic rates for the RBC-RBC system.
The adhesion model (and its generalized extension (8)) is premised on
the idea that, once membranes become close enough for receptors and
ligands to bridge the gap, the actual event of forming or breaking each
incremental bond is probabilistic. The kinetic rates influence this
probability in a predictable way. The initial slope of the adhesion
frequency curve is proportional to the forward kinetic rate
kf, whereas the equilibrium frequency reflects the
affinity Ka. The equilibrium adhesion frequency and
initial slope also depend on the product of the receptor and ligand
population densities (as is evident in Fig. 3b) and the
contact area Ac. Because acceptable contact gaps
must be on the order of tens of nanometers, the true contact area is not determinable in the micropipette assay (although a constant
apparent contact area, as seen under light microscopy, is
maintained by the piezo throughout all experiments). The contact area
therefore is embedded with Ka to form what we have termed the effective affinity
AcKa. Because it includes
information regarding both the intrinsic reaction kinetics of the
molecules and the effectiveness of their presentation, effective
affinity will determine the membrane-specific level of bond formation
for any given level of receptor-ligand expression. Note that these are
two-dimensional binding parameters describing the association of
membrane-bound receptors and ligands. The commonly used
three-dimensional parameters, governing the binding of soluble ligand to membrane-bound receptors, do not necessarily predict the
two-dimensional behavior (5).
Best fits of the data to the model produced estimates for the effective
affinity as well as the reverse kinetic rate constant for IgG+ RBC to
CD16b+ RBC adhesion. (The effective forward rate constant is derivable
from Ackf = AcKa × kr.)
Using this single pair of parameters, all three curves shown in Fig.
3b were generated, illustrating the high quality of the estimates.
RBC Effective Affinity Is 50-fold Higher than That of CHO or K562
Cells, but There Are No Differences in Reverse Kinetic Rates--
The
effective affinity and reverse rate for CD16b+ RBC are compared in Fig.
4a with those for CHO cells
(5) and K562 cells (6). When hosted by RBCs, the receptor displayed a
tremendous 50-fold increase in effective affinity over the CHO and K562
cell hosts. However, no significant differences in reverse kinetic rates were found, indicating that the affinity enhancement was due
solely to a much increased effective forward rate on the RBC host.
Increased Binding Efficiency Is Not Due to
Reconstitution--
Because the receptor was expressed on RBCs via
reconstitution (protein transfer) and by transfection (gene transfer)
in the other hosts, we investigated whether reconstitution itself could account for the enhanced binding. Using the same aliquot of purified protein, CD16b was reconstituted onto both RBCs and wild-type CHO
cells. With a fixed contact time of 5 s, cells were assayed for
binding frequency against IgG-coated RBCs. Sham-reconstituted cells
were negative controls. Results are shown in Fig. 4b. Just as with the transfected CHO cells, the reconstituted CHO cells exhibited a low level of binding relative to the RBCs. In fact, using
the parameters obtained with the transfected CHO cells (Fig. 4a), a predicted adhesion frequency was calculated and found
to be quite similar to the adhesion frequency actually observed here using reconstituted CHOs, thus establishing the equivalency
of the reconstitution and transfection modes of expression. It should be noted also that the excellent correspondence between the predictions based on earlier experiments and results from new experiments with a
different CD16b preparation suggests a high degree of reproducibility for all reagents and protocols employed.
Enhanced Binding to RBCs Is Not Evident in the Three-dimensional
Association of Soluble Antibody to the F/G Loop of Membrane-bound
CD16b--
Unlike two-dimensional interactions, microtopological
differences should not impact three-dimensional binding because a
soluble ligand can easily access both peaks and valleys on the cell
surface. To test this in our system we used Scatchard analysis to
measure the three-dimensional affinity of the monoclonal antibody
CLBFcgran1 for RBC-reconstituted CD16b. CLBFcgran1 binds CD16b in the
area of the F/G loop on the membrane-proximal globular domain of the receptor (9). The F/G loop also hosts critical epitopes for IgG binding
(10). The CD16b-CLBFcgran1 dissociation constant, Kd
(= 1/Ka), was found to be 8 ± 2 nM
with reconstituted RBCs and 12 ± 5 nM with
transfected CHO cells (11), indicating the lack of any significant
binding enhancement for RBCs with an antibody in solution. It is also
further evidence of the equivalency of the two expression modes.
RBC Doublets Form Broad Tight Contacts, Whereas CHO-RBC Conjugates
Do Not--
The surface microtopology of RBCs, CHO cells, and K562
cells was examined by scanning electron microscopy (Figs. 2 and
5). Although the RBC surfaces were quite
smooth, CHO cells (detached with EDTA) were densely covered with long
microvilli, and K562 cells displayed bulbous extensions averaging
200-400 nm in height. Still, even on the red cell intermembrane gaps
greater than about 50 nm are in effect infinite, precluding any
possibility for receptor-ligand interaction. Quantifying true contact
area formation by direct means has proven challenging (12), although
Dustin et al. (13) has demonstrated a promising approach. In
this paper we have presented effective affinity as an indirect measure
when intrinsic affinity can be controlled. For a more direct
examination, CD16b-IgG-mediated RBC-RBC and CHO-RBC conjugates were
prepared and viewed under SEM (Fig. 5, a and b).
High magnification imaging of the region of contact between RBC-RBC
conjugates revealed broad areas in which the membrane gap would likely
accommodate receptor-ligand interactions. In contrast, the CHO-RBC
conjugates were characterized by widely distributed point contacts
between CHO microvilli and the RBC membrane. This was consistent with
Mege et al. (12), who presented transmission electron
microscopy images of RBCs showing sparse small contacts with
rough-surfaced macrophages.
The effective forward kinetic rate,
Ackf, was found to be about
50-fold higher when the receptor CD16b was hosted by RBCs rather than
CHO or K562 cell membranes. Purification and reconstitution of the
protein did not create any alterations leading to increased binding.
The evidence points toward the host cell environment as the source of
this effect rather than any variation in the protein itself.
These results suggest the presence of a 50-fold greater true contact
area in RBC-RBC pairings over those in CHO-RBC or K562-RBC pairings,
all with similar apparent contact areas. A larger true contact area
will increase the rate at which bonds form but will have no influence
on their reverse rates because the latter governs the dissociation of
preexisting bonds. This pattern is evident in the data presented here.
The enhanced RBC binding was isolated to the forward kinetic rate and
two-dimensional assays only, consistent with our hypothesis that
microtopology was the underlying mechanism for the enhancement.
Cell-dependent Differences in Lateral Diffusion Should
Not Affect the Outcomes of Short Duration Low Affinity
Contacts--
There has been considerable recent discussion regarding
the effects of lateral diffusion on receptor-ligand interactions. Although the lateral diffusion of lipid-anchored proteins on RBCs may
differ from that on K562 or CHO cells, such differences are unlikely to
play a significant part in the micropipette assay employed here. There
are two elements of higher lateral diffusion that may affect adhesion:
(i) faster depletion-driven diffusion of free molecules into
the contact area as bonds form, and (ii) more frequent free
receptor-ligand interactions within the contact. In the
first case, bond formation lowers the concentration of free molecules
in the contact area relative to the noncontact surfaces, therefore
driving the flux of additional free molecules into the contact area
(13). In high affinity cases this initial flow might be rapid. However,
in very low affinity (and very brief) interactions such as those
described here, only a few bonds are expected to form despite the
presence of hundreds to thousands of free receptors and ligands in the
contact area. (This can be seen in Fig. 3b, in which the
frequency of having no adhesion at all is considerable.) Depletion
therefore is so limited that even if bound molecules were immediately
replaced the increase in population density (and hence, adhesion
frequency) would be negligibly small. This is in contrast with long
duration, actively spreading contacts, where lateral diffusion has been
demonstrated to have a role in the rate of adhesion strengthening
(14).
Note that the mechanism described above can potentially affect bond
formation rate but not the ultimate equilibrium adhesion level. This is
also true for the second rapid diffusion feature, increased molecular
interaction frequency. In a fixed amount of time, a highly mobile
receptor may encounter a larger number of free ligands than a less
mobile molecule. In a high affinity system this may result in a
noticeable acceleration of bond formation and, hence, an earlier
equilibrium, but it will also cause an increase in the dissociation
rate. Diffusion is fundamentally a process that can alter the rate at
which a system reaches equilibrium but cannot alter the final
equilibrium state. Therefore the 50-fold equilibrium affinity
difference described in this paper cannot be explained by dissimilar diffusivities.
It is interesting to note that even the kinetic rates will be
independent of diffusivity if the adhesion is reaction-limited rather
than diffusion-limited. This, in fact, is the case here, again because
of very low affinity. With kr = 0.5/s and kf = 3 × 10 Cell-specific Localization or Clustering Patterns Could
Contribute--
Although the simplest explanation for the 50-fold
difference in the CD16b effective affinity appears to be the distinct
microtopology of the RBCs as compared with CHO and K562 cells, there
may be other cell-specific factors playing a contributory role that
will require additional studies to clarify. For example, localization of molecules into regions of low or high adhesive potential has been
observed in several other cases. On neutrophils, L-selectin is
localized preferentially on microvilli tips, whereas integrin
Clustering (distinct from localization) of receptors could increase
their effective affinity by lowering the entropic barrier to ligand
binding. GPI-rich lipid microdomains, termed rafts, have been proposed,
although they remain controversial (16, 17). At present their cellular
distribution patterns and the proportion of GPI molecules likely
associated with them is unclear. Our preliminary SEM studies have shown
a seemingly random distribution of CD16b on RBC membranes (Fig.
5c). To be consistent with the binding data presented in
this paper, receptor clustering would have to be extensive on RBCs
while being nearly absent on both K562 and CHO cell membranes. This
state would have to prevail despite very low CD16b densities on the
RBCs and quite high CD16b densities on the transfected K562 and CHO
cells. Although such a state cannot be excluded, the evidence suggests
it is unlikely to be a factor in the present studies.
An Expanded Role for the Cytoskeleton in Adhesion
Regulation--
Modifications to protein expression, conformation, and
localization are well recognized mechanisms of adhesion regulation. This study suggests that modulation of surface smoothness also may be
an important regulatory mechanism, implying an expanded role for the
cytoskeleton in cell adhesion. There is a growing body of evidence
supporting this hypothesis. Treatment with cytochalasin increases the
contact area in lectin-mediated adhesion of rat hepatocytes (18) and
adherent erythroleukemia cells (19). Although similar treatment
disrupts integrin-mediated focal contacts, it appears to enhance weaker
nonfocal adhesions. Cytochalasin treatment leads to the arrest of
neutrophils undergoing P-selectin-mediated rolling (20) and increases
the strength of rolling adhesion on both E- and P-selectin (21). In the
latter study, hypo-osmotic swelling of neutrophils produced similar
results on P-selectin surfaces, and electron microscopy showed that
microvilli had been nearly abolished, strongly implicating surface
smoothing in the promotion of adhesion.
Localized close contact formation is beneficial in several important
cases. Reflection interference contrast microscopy reveals close
contacts on the upstream side of endothelial cells exposed to flow
(22), thereby maximizing their stability. Total internal reflection and
reflection interference contrast microscopy of rapidly moving
keratocytes displayed a rim of tight contact at the leading
edge, with speed closely correlated with uniformity of this contact
region (23). The common model for locomotion involves the transport of
adhesion molecules from posterior to anterior in superposition with
cycles of leading edge extension/whole-cell retraction. The performance
of the transport process is expected to be reflected in the cell
crawling speed. Through microtopological manipulation alone, the
cytoskeleton may be able to shift the effective affinity of the local
receptor population (even those with no cytoskeletal associations) by
as much as 50-fold. This would lower the transport threshold required
for locomotion substantially or might even replace the transport
process altogether. This would explain intriguing cases of normal
motility despite suppression of integrin transport (24).
receptor,
was established by either transfection or spontaneous insertion via its
glycosylphosphatidylinositol anchor. Binding to IgG-coated RBCs,
measured using a micropipette method, indicated a 50-fold increase in
effective affinity for receptors on RBCs over CHO and K562 cells,
whereas the off rates were similar for all three. Electron microscopy
confirmed that specific tight contacts were broad in RBC-RBC conjugates
but sparse in CHO-RBC conjugates. We suggest that through modulation of
surface roughness the cytoskeleton can greatly impact the effectiveness of adhesion molecules, even those with no cytoplasmic structures. Implications for locomotion and static adhesion are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RIIIb)
terminates at the lipid bilayer with a glycosylphosphatidylinositol (GPI) moiety and so has no direct cytoskeletal interface. Currently there are no data to suggest the existence of multiple activation states in CD16b. These simplifications permit a more focused
investigation into the role of the membrane environment in the
functionality of this receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (91K):
[in a new window]
Fig. 1.
a, RBCs bearing CD16b (left
cell) and IgG (right cell) are repeatedly brought into
contact (b) for 1-16 s using a computer-driven
piezoelectric actuator. A transient deflection of the weakly aspirated
RBC indicates the presence of an adhesion at the moment of retraction.
c, no adhesion; d, adhesion. A supplemental video
is available online.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (40K):
[in a new window]
Fig. 2.
The receptor CD16b was presented on
three cell types (a) using two different mechanisms
(b), transfection (CHO, K562) and
reconstitution (RBC, CHO). The ligand IgG
was coated on RBC membranes via chromium-chloride chemistry. Mean site
densities were determined by radioimmunoassay or quantitative indirect
fluorescent immunoassay. Several RBC site densities were examined
within the ranges shown.
receptor, partially
blocked binding, whereas soluble dimeric B7, a non-IgG-associating
protein, had no effect.
View larger version (22K):
[in a new window]
Fig. 3.
a, adhesion frequencies during
controlled contacts between RBC pairs presenting CD16b and IgG. Either
the receptor- or ligand-presenting RBC was preincubated with an
adhesion-blocking or structurally similar irrelevant protein.
b, results from micropipette adhesion frequency assays show
how adhesion approaches equilibrium as the contact times increase. A
single fit (curves) to the model of all the data shown
generated an estimate for AcKa
and kr. Total n = 40 pairs, 100 contacts each. Individual points are means ± S.E. Site densities
are in molecules/µm2.
View larger version (21K):
[in a new window]
Fig. 4.
a, comparison of
AcKa and kr
(means ± S.E.) of RBC-reconstituted CD16b with CHO- and
K562-transfected CD16b illustrates the 50-fold higher effective
affinity of RBCs despite similar reverse rates. b, using
reconstitution only, K562-purified CD16b was transferred to the
surfaces of RBCs and wild-type CHO cells from the same aliquot. Testing
for adhesion frequency during 5-s contacts confirmed that the
reconstituted CHO cells performed as poorly as the transfected CHOs,
relative to the RBCs. For multialiquot comparison, the frequencies
predicted by the binding parameters in a under these
conditions (CD16b/µm2 = 115 for RBC, 80 for CHO) are
shown in italics.
View larger version (90K):
[in a new window]
Fig. 5.
Scanning electron microscopy of
receptor-ligand-mediated RBC-RBC (a) and CHO-RBC
(b) conjugates illustrates the effects of microtopology on
initial contact area formation. c, RBCs labeled with 12 nm
anti-CD16 immunogold prior to SEM prep suggest a random distribution of
this GPI-anchored receptor when reconstituted into RBC membranes.
CD16b/µm2 = 21 from quantitative indirect fluorescent
immunoassay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 µm2/s
(assuming at least half of the apparent contact area is true contact in
RBC-RBC adhesions) and a very slow diffusion coefficient of 0.01 µm2/s, the procedure of Lauffenburger and Linderman (15)
decomposes kf into a diffusion-driven
k+ of 2 × 10
2
µm2/s and a reaction-driven kon of
3 × 10
6 µm2/s. So the diffusion step
is conservatively four orders faster than the reaction step, strongly
suggesting reaction-limited diffusion-independent kinetic rate
constants and accounting for the nearly identical kr
values in Fig. 4a. We have used fluorescence recovery after
photobleaching to estimate the actual diffusion coefficient of CD16b on
CHO cells to be 0.16 µm2/s, an even more
reaction-limiting value than above.2
M
2 is predominantly on the cell body (3).
In this study, a surface distribution that localized the CD16b near the
cell body and away from promontories on both CHO and K562 cells would contribute to the diminished binding seen relative to RBCs. Because GPI
proteins lack a cytoplasmic domain, nonrandom distributions would rely
on either an association with a transmembrane protein linked to the
cytoskeleton or lipid interactions.
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ACKNOWLEDGEMENTS |
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We are grateful to Mike Dustin and Rodger McEver for helpful discussions. We also thank Ping Li and Scott Chesla for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Science Foundation Grants BCS 9210648 (to C. Z.) and BCS 9350370 (to C. Z.) and National Institutes of Health Grants AI38282 (to C. Z.), AI30631 (to P. S.) and GM08433 (to T. E. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a video.
To whom correspondence should be addressed. Tel.:
404-894-3269; Fax: 404-385-1397; E-mail:
cheng.zhu@me.gatech.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010427200
2 S. Chesla and C. Zhu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: RBC, red blood cell; CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; SEM, scanning electron microscopy.
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REFERENCES |
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