By
From the * Division of Tumor Virology, and the Division of Molecular and Cellular Biology,
Dana-Farber Cancer Institute, Boston, Massachusetts 02115; and the § Department of Cell Biology,
Duke University Medical Center, Durham, North Carolina 27710
Previous studies have shown that integrin chain tails make strong positive contributions to
integrin-mediated cell adhesion. We now show here that integrin
4 tail deletion markedly impairs static cell adhesion by a mechanism that does not involve altered binding of soluble vascular cell adhesion molecule 1 ligand. Instead, truncation of the
4 cytoplasmic domain caused a
severe deficiency in integrin accumulation into cell surface clusters, as induced by ligand and/
or antibodies. Furthermore,
4 tail deletion also significantly decreased the membrane diffusivity of
4
1, as determined by a single particle tracking technique. Notably, low doses of cytochalasin D partially restored the deficiency in cell adhesion seen upon
4 tail deletion. Together, these results suggest that
4 tail deletion exposes the
1 cytoplasmic domain, leading to
cytoskeletal associations that apparently restrict integrin lateral diffusion and accumulation into
clusters, thus causing reduced static cell adhesion. Our demonstration of integrin adhesive activity regulated through receptor diffusion/clustering (rather than through altered ligand binding affinity) may be highly relevant towards the understanding of inside-out signaling mechanisms for
1 integrins.
Cell adhesion is a critical event in the initiation and
maintenance of a wide array of physiological processes, including embryogenesis, hematopoiesis, tumor cell
metastasis, and the immune response. The integrin protein
family, which consists of 22 distinct Here, we have used an Cells.
K562 erythroleukemia cells and Chinese hamster ovary
(CHO) cells transfected with cDNAs representing the wild-type
human Reagents and Antibodies.
The antibodies used in this study include anti- Flow Cytometry.
Flow cytometric assays were performed as
described (21). For determination of 15/7 epitope expression,
K562 cells were preincubated (10 min) with 2 mM EDTA (in
PBS), washed and suspended in assay buffer (24 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 7.4 [Tris-buffered saline; TBS], 5%
BSA, 0.02% NaN3) with or without MnCl2 and/or CS-1 peptide. Then, mAb 15/7 or negative control mAb J-2A2 was added
and mean fluorescence intensities were determined. Results for
15/7 expression are given as a percent of VCAM-Ig-AP Direct Ligand Binding Assay.
A detailed description of a high sensitivity, direct ligand binding assay has been described elsewhere (15). In brief, cells in 96-well porous plates
were incubated with a VCAM-Ig fusion protein conjugated with
AP (VCAM-Ig-AP), and then washed using a Millipore Multiscreen filtration manifold. Bound VCAM-Ig-AP was then detected by colorimetric assay using p-nitrophenyl phosphate.
VCAM- Cell Adhesion.
The effects of cytochalasin D on cell adhesion
were performed as previously described (7), with minor modifications. In brief, BCECF-AM (Molecular Probes, Eugene, OR)
-labeled cells were pretreated with various doses of cytochalasin
D (Sigma Chemical Co., St. Louis, MO) for 15 min at 37°C.
Cells were added to 96-well plates previously coated overnight
with Confocal Microscopy.
K562 cells were incubated on ice for 10 min in PBS containing 2 mM EDTA, washed, and resuspended
in assay buffer (TBS, 5% BSA, 0.02% NaN3). For examination of
VCAM-induced clustering of Analysis of and
heterodimers,
mediates cell adhesion to extracellular matrix proteins, serum proteins, and counterreceptors on other cells (1).
Through inside-out signaling, integrin adhesive activity can
be triggered by multiple agonists, and integrins display multiple activation states within different cell types, independent
of changes in integrin expression levels (2). Many studies of
integrin regulation have focused on conformational changes,
altered ligand binding affinity, and/or modulation of postligand binding events (e.g., cell spreading) (3). However, a novel mechanism was recently put forth, suggesting that
activation of adhesion may involve release of cytoskeletal
constraints, leading to increased integrin lateral mobility (7,
8). Implicit is the assumption that increased mobility is
proadhesive because it leads to increased integrin accumulation at an adhesive site, and thus greater adhesion strengthening.
4 integrin cytoplasmic domain
mutant to provide strong evidence for this hypothesis.
Upon truncation of the
4 cytoplasmic domain, the
4
1
integrin shows severe impairments in both constitutive and phorbol ester-induced static cell adhesion (9, 10), and also shows deficient adhesion strengthening under shear (11,
12). However, the reason for these defects was not previously understood. Because other integrin cytoplasmic domain mutations cause altered ligand binding (3, 13, 14), we
closely examined binding of soluble vascular cell adhesion
molecule (VCAM)-11 (15) to mutant and wild-type
4
1
integrin. Not finding any alterations in ligand binding, we then examined receptor accumulation into cell surface
clusters, and integrin lateral mobility. The results strongly
support the hypothesis that integrin diffusion/clustering,
independent of alterations in ligand binding, can play a major role in regulating integrin adhesive functions.
4 integrin (
4wt), chimeric
4 containing the extracellular and transmembrane domains of
4 with the cytoplasmic domain of
2 (-X4C2), and a truncated
4 integrin lacking a cytoplasmic domain (-X4C0), have been described elsewhere (9).
Untransfected or mock-transfected K562 and/or CHO cells were
used as negative controls. K562 transfectants were maintained in
RPMI-1640 containing 10% fetal bovine serum (FBS), 1 mg/ml
G418 sulfate (GIBCO BRL, Gaithersburg, MD), and antibiotics, whereas CHO transfectants were maintained in MEM
media
containing 10% dialyzed FBS, 0.5 mg/ml G418 sulfate, and antibiotics.
4, B5G10 (16), and A4-PUJ1 (17); anti-CD32 (anti-Fc
RII), 4.6.19 (18); fluorescein-conjugated goat anti-mouse
IgG (Cappel, Westchester, PA); fluorescein-conjugated goat anti-
mouse
(Caltag, San Francisco, CA); negative control mAb J-2A2
(19); and mAb 15/7, recognizing a
1 epitope induced by manganese or ligand (20). Fluoresceinated B5G10 was produced using
N-hydroxy succinimide (NHS)-fluorescein (Pierce, Rockford, IL),
as described by the manufacturer. Recombinant soluble VCAM
(rsVCAM) and alkaline phosphatase (AP)-conjugated VCAM-Ig
(VCAM-Ig-AP) were a gift from Dr. Roy Lobb (Biogen, Inc., Cambridge, MA) and prepared as described elsewhere (15). The VCAM-Ig-AP contains the two NH2-terminal domains of human VCAM fused to the hinge, CH2, and CH3 domains of human IgG1. A purified VCAM-mouse C
chain fusion protein
(VCAM-
) was a gift from Dr. Philip Lake (Sandoz Co., East
Hanover, NJ). VCAM-
was produced as a soluble protein from
sf 9 cells and contains all seven human VCAM domains, except
the transmembrane and cytoplasmic domains, which have been
replaced by a 100-amino acid mouse C
segment. The CS-1
peptide (GPEILDVPST) derived from fibronectin was synthesized at the Dana-Farber Molecular Biology Core facility (Boston, MA).
4
1 levels (% 15/7 = [15/7
J2A2]/[A4-PUJ1
J2A2] × 100). Untransfected K562
cells (expressing the
5
1 integrin) showed no constitutive or divalent cation-induced 15/7 expression.
Indirect Ligand Binding Assay.
Transfected K562 cells
were incubated for 10 min on ice with TBS containing 2 mM
EDTA, washed three times with assay buffer (TBS, 2% BSA), and
resuspended in assay buffer containing the desired concentrations
of VCAM-
and either MnCl2 or 5 mM EDTA. Cells were incubated at 4°C for 30 min, washed two times in assay buffer containing 2 mM MnCl2, and subsequently incubated for 30 min at
4°C with assay buffer containing fluorescein-conjugated goat
anti-mouse
antibodies. Cells were washed two times and fixed
with 3% paraformaldehyde. VCAM-
binding on K562 cells was analyzed using a FACScan® flow cytometer to give mean fluorescence intensity units. Background binding of VCAM-
(i.e.,
VCAM-
binding in the presence of 5 mM EDTA) was subtracted and data were also corrected for
4 surface expression, if
applicable.
4 ligands and blocked with 0.1% heat-denatured BSA for
45 min at 37°C. Plates were centrifuged at 500 rpm for 2 min and
analyzed in a Cytofluor 2300 measurement system (Millipore
Corp., Bedford, MA). Plates were incubated for an additional 15 min at 37°C, washed 3-4 times with adhesion media, and fluorescence was reanalyzed. Background binding to heat-denatured
BSA alone was typically <5% and was subtracted from experimental values. Data is expressed as fold induction in cell adhesion,
and calculated (adhesion in the presence of cytochalasin D/adhesion in the absence of cytochalasin D) from triplicate cultures.
4, cells were incubated with 5 µg/ml of mAb 4.6.19 to block Fc
RII sites, and then with 500 nM rsVCAM and 2 mM MnCl2 in assay buffer for 45 min. Cells
were washed two times in assay buffer containing 2 mM MnCl2,
incubated an additional 30 min in assay buffer containing fluoresceinated B5G10 mAb, washed, and fixed with 4% paraformaldehyde in PBS. For detection of
4 clustering induced by secondary
antibodies, K562 cells were incubated for 30 min in assay buffer
(PBS substituted for TBS) containing purified B5G10, washed,
incubated an additional 30 min with fluorescein-conjugated goat
anti-mouse IgG, washed, and fixed as above. All procedures were
done at 4°C in the presence of 0.02% NaN3 to prevent internalization. Fixed cells were resuspended in Fluorosave reagent (Calbiochem Novabiochem, La Jolla, CA), mounted onto slides, and
fluorescence was analyzed using a Zeiss model LSM4 confocal laser scanning microscope equipped with an external argon-krypton laser (488 nm). To evaluate cell surface fluorescence, optical
sections of 0.5-µm thickness were taken at the center and at the
cell membrane of representative cells. Images of 512 × 512 pixels
were digitally recorded within 4 s and printed with a Kodak 8650 PS color printer, using Adobe Photoshop software (Adobe Systems, Mountain View, CA).
4
1 Diffusion.
40-nm colloidal gold particles (EY
Laboratories, San Mateo, CA) were coated with antibody using a
biotin-avidin linkage as described (22). In brief, gold particles
were coated with ovalbumin (20 µg/ml gold suspension) at pH
4.7, followed by blocking with 0.05% PEG 20K. After washing
(three times with 0.05% PEG 20K/PBS; 16.5K g for 10 min),
particles were reacted with NHS-LC-biotin (20 µg/ml gold;
Pierce) overnight on ice. Particles were subsequently washed
three times (0.05% PEG 20K in PBS) and incubated with avidin neutralite (Molecular Probes; 1 mg/ml gold, starting volume) for 3 h on ice. The gold solution was then washed three times as described above and incubated for 3 h with biotin-B5G10 (60 µg/
ml gold, starting volume) and blocked with 1 mg BSA biotinamido caproyl (Sigma) overnight on ice.
supplemented with 2 mM
L-glutamine, 10% FCS, and 20 mM Hepes. Gold particles were
added to cell-conditioned culture medium and culture dishes were
sealed before mounting on a Zeiss Axiovert 100 TV inverted microscope equipped with Nomarski optics and a NA 1.3 100×
plan neofluar objective. Serial, recorded video frames were digitized and analyzed for particle centroid position using previously
published nanometer-resolution techniques (24).
Previously, it was shown that deletion of the 4 cytoplasmic domain markedly decreased
4
1-dependent adhesion
of several cell types to multiple ligands (9). Here, we
sought to determine whether this mutation also altered the
ability of
4
1 to bind soluble ligand. Wild-type
4 (-
4
wt), truncated
4 (-X4C0), and a chimeric
4 containing
the cytoplasmic domain of
2 (-X4C2) were stably expressed at comparable levels on the surface of both K562
erythroleukemia and CHO cells (Fig. 1). In a direct ligand
binding assay (Fig. 2 A), comparable binding of an AP-conjugated VCAM-Ig fusion protein was seen for cells expressing wild-type
4, truncated
4, or chimeric
4. The
concentration of VCAM-Ig-AP yielding half-maximal direct ligand binding activity (ED50) was 1-1.5 nM for all
three K562 transfectants, consistent with previously published results showing ED50 values of ~1 nM (15). Again,
no essential difference between wild-type and mutant
4
was obtained in an indirect binding assay, using fluorescein-conjugated goat anti-mouse
antibodies to detect bound
VCAM-
(Fig. 2 B). Minimal nonspecific binding of either
VCAM-Ig-AP or VCAM-
was detected on mock-transfected K562 cells, confirming that binding is
4 integrin-dependent (Fig. 2, A and B).
Ligand binding was carried out in 2 mM manganese, because calcium and magnesium (either alone, or together, at ~ 1-2 mM) fail to support binding of soluble VCAM (15,
26). To alleviate concern that manganese might mask differences in VCAM binding by inducing high affinity 4
1
(15, 26, 27), manganese was titrated over a range of concentrations, whereas VCAM was held constant at 4 nM
VCAM-Ig-AP (Fig. 2 C), or 500 nM VCAM-
(Fig. 2 D).
Manganese stimulated VCAM binding that was dose-dependent and
4-specific, but again no differences were
apparent between K562-
4wt, K562-X4C2 and, K562-
X4C0 cells (Fig. 2, C and D).
In CHO cells, compared with K562 cells, 4
1 is constitutively more active with respect to mediating cell adhesion
(9, 10). Nonetheless, in the CHO cellular environment,
there were again no differences in direct VCAM binding to
4wt and X4C0 integrins at either optimal (Fig. 3 A; 4 nM
VCAM-Ig-AP) or suboptimal (Fig. 3 B; 1 nM VCAM-
Ig-AP) doses of ligand. Half-maximal direct VCAM-Ig-
AP binding occurred at ~100 µM manganese for all transfectants examined, consistent with previously published
manganese ED50 values for VCAM-
4
1 binding (15). In
contrast with ligand binding, cell adhesion to immobilized
VCAM was markedly diminished for CHO-X4C0 cells, compared with
4wt cells (Fig. 3 C). For example, adhesion at 0.1 and 1 µM Mn2+ was reduced by 88 and 69%,
respectively.
To examine 4 tail deletion effects on very late antigen 4 conformation, we used the mAb 15/7, which recognizes a
1 integrin conformation induced by ligand occupancy or
manganese. When 15/7 epitope is induced by manganese,
it correlates with increased ligand binding affinity (20).
However, the 15/7 epitope also appears when the
1 cytoplasmic domain is deleted, and ligand binding is diminished
(28). Notably, 15/7 epitope expression is most readily induced on
4
1, as compared with other
1 integrins (Bazzoni, G., L. Ma, M.L. Blue, and M.E. Hemler, manuscript
submitted for publication), and thus is an especially useful
tool for evaluating altered
4
1 conformations.
Negligible 15/7 epitope expression was seen for 4wt
and mutant
4 integrins in K562 cells in the absence of
stimulation (Table 1). However, the percentage of
4
1
molecules expressing the 15/7 epitope increased dramatically upon addition of CS-1 peptide or manganese or both
together to the K562-
4wt and K562-X4C2 cells. Importantly, stimulation with manganese and/or CS-1 peptide
also resulted in comparably increased 15/7 epitope on
K562-X4C0 cells (Table 1). No 15/7 epitope was detected in mock-transfected K562 cells (stimulated or unstimulated), demonstrating that 15/7 was specifically reporting
4
1 conformational changes.
|
Having found that 4 tail deletion does not alter ligand
binding or integrin conformation, we then sought alternative explanations for why tail deletion impairs cell adhesion. To this end, confocal laser microscopy was used to
examine
4 tail deletion effects on accumulation of
4
1 in
clusters. As illustrated, wild-type
4 (Fig. 4 d) and X4C2
(Fig. 4 c) were detected in clusters on the surface of K562
cells after addition of recombinant soluble VCAM in the
presence of manganese. In sharp contrast, X4C0 showed
hardly any VCAM-induced accumulation in clusters (Fig. 4
b). The X4C0 subunit was present on the cell surface at
levels comparable to
4wt and X4C2 (see Fig. 1), suggesting that differences in signal strength reflect aggregated receptor and not differences in total receptor number. Cell
surface staining was specific for
4, as shown by the lack of
staining on mock-transfected K562 cells (Fig. 4 a). The distribution of
4 into clusters was dependent upon the addition of VCAM, because manganese alone (at 2 mM) did
not induce clustering of
4 (data not shown).
Experiments were carried out at 4°C in the presence of
sodium azide to prevent receptor internalization. Transverse
sections of K562-4wt cells (Fig. 4 e) showed peripheral,
but not intracellular staining, consistent with cell surface
clustering without receptor internalization. Also, transverse
sections of K562-X4C0 cells showed no evidence for intracellular staining (data not shown). Furthermore, levels of
cell surface
4 (X4C0) were unaltered after incubation with
VCAM, as determined by flow cytometry (data not shown).
To extend our findings, we also examined 4 clustering
induced by anti-
4 mAb, followed by polyclonal secondary
antibody (Fig. 4, f-j). As indicated, clustering was again
pronounced on K562-
4wt (Fig. 4, i and j) and K562-
X4C2 (Fig. 4 h) cells, whereas minimal clustering was observed when the
4 tail was deleted (K562-X4C0 cells;
Fig. 4 g) or when no
4 was present (Fig. 4 f ). Results in
Fig. 5, showing 9-15 cells/panel, confirm the single cell results shown in Fig. 4. As indicated, nearly all of the K562-
4wt cells exhibit pronounced clustering, induced either by
VCAM (Fig. 5 a) or by antibody (Fig. 5 c). In contrast, the
X4C0 mutant was much less clustered (Fig. 5, b and d ), despite being expressed on the cell surface at levels nearly
equivalent to
4wt (see Fig. 1).
The failure of truncated 4 to form cell surface clusters
raises the possibility that increased or altered associations
with the underlying cytoskeleton may impair the lateral
mobility of truncated
4, restricting its redistribution into a
cluster. Because restricted lateral movement of integrin receptors will likely be reflected by a lower integrin diffusion
rate (22, 23), next we directly measured the diffusion coefficients of wild-type and truncated
4 in CHO transfectants
at 37°C. The two-dimensional diffusivity of 40-nm gold
particles, coated with anti-
4 mAb, was measured on the
lamellipodia of CHO-
4wt and CHO-X4C0 cells spread
on an
4-independent substrate, vitronectin. Movement of
gold particles was viewed by high magnification, video-enhanced differential interference contrast microscopy and
particles were tracked by computer with nanometer-level
accuracy (23). A nonperturbing anti-
4 mAb, B5G10, was
used because this mAb neither blocks nor stimulates
4
1-mediated functions (29). It was shown elsewhere that nonperturbing antibodies coupled to 40-nm gold can report
the random diffusion of integrins without stimulating the
cross-linking and directed movement of these receptors (22).
As illustrated in Fig. 6, A and C, gold particles bound to
the lamella of CHO-4wt cells diffused freely with a mean
diffusion coefficient of 0.03 µm2/s (Fig. 6 E), consistent
with the diffusion rate observed for other
1 integrins (22),
as well as other cell surface glycoproteins (30). However,
truncation of the
4 cytoplasmic domain resulted in a significant decrease in the
4
1 diffusion rate (P <0.01). Particles bound to CHO-X4C0 cells exhibited reduced lateral
mobility (Fig. 6, B and D), with a diffusion coefficient that
was sixfold lower (0.005 µm2/s) than wild-type
4
1. No
binding of gold particles was detected on mock-transfected CHO cells, demonstrating that the binding is
4
1 specific
(data not shown).
The association of integrins with cytoskeletal elements
can restrain the random diffusivity of integrins and thus
contribute to a diminished adhesive state (7). To examine
whether the actin cytoskeleton may contribute to the deficiency in adhesion mediated by truncated 4, we disrupted
actin filament organization with cytochalasin D and measured its effect on
4
1-mediated adhesion. At high doses
(>10 µg/ml) of cytochalasin D, adhesion of both CHO-
4wt and CHO-X4C0 to
4 ligands was dramatically reduced (data not shown), as seen many times previously.
However, at low doses, cytochalasin D stimulated markedly the adhesion of CHO-X4C0 cells to two different
4
ligands, FN40 (Fig. 7 A) and VCAM (Fig. 7 B), without
much increasing the adhesion of wild-type
4 transfectants.
Adhesion was
4 specific, as mock-transfected CHO cells
did not adhere under these conditions (data not shown).
Although 4 tail deletion has a profound negative effect
on cell adhesion (9, 10; Fig. 3 C ), and on adhesion
strengthening under shear conditions (11, 12), we show
here that it does not alter ligand binding. Ligand binding
was unaltered by
4 tail deletion (a) as measured either directly or indirectly, (b) as measured on either K562 cells or
CHO cells, and (c) as shown either by manganese titration
(at constant ligand) or by ligand titration (at constant manganese). Previous results also suggested that
4 tail deletion
did not alter ligand binding, but that study was done only
indirectly, and under single cation conditions, on a single
cell line (18). In addition,
4 tail deletion was shown previously not to alter cell tethering in hydrodynamic flow (11,
12), a function that is likely dependent on univalent integrin-ligand bond formation. Thus, our results argue strongly
against affinity modulation as a mechanism for
4 tail regulation of cellular adhesion. Consistent with these findings,
4 tail deletion also did not decrease the ability of divalent
cations or ligand to induce
4
1 conformations detected by
mAb 15/7. Similarly,
4 tail deletion was shown previously
to have no effect on induction of an epitope defined by
mAb 9EG7 (10), that maps to a
1 site distinct from the
15/7 site (28, 31).
It is, perhaps, not surprising that replacement of the 4
tail with the
2 tail had no effect on ligand binding or integrin conformation, because previously that mutation had
no effect on cell adhesion, or tethering under flow (9, 12).
Notably, replacement of the
IIb cytoplasmic domain with
that of
2 did cause an increase in
IIb
3 integrin ligand
binding (14), suggesting that different rules may apply to
regulation of the
IIb
3 integrin.
The defect in cell adhesion seen for the X4C0 mutant is not due to altered ligand binding, but rather appears to arise from a reduced diffusion rate. Presumably, a lower rate of diffusion prevents the passive accumulation of integrin receptors into clusters. After initial cell contact with immobilized ligand, a dynamic, diffusion-dependent accumulation of clustered integrins may be needed to augment the overall cellular avidity for the ligand-coated surface. Notably, clustering deficiencies for the X4C0 integrin, directly measured here at 4°C, are consistent with an indirectly measured deficiency in X4C0 clustering seen previously at 37°C (18). In that case, X4C0 was defective in mediating antibody-redirected cell adhesion, a process dependent on mAb bridging between Fc receptors and clustered integrins (18).
How might tail deletion cause decreased diffusivity
leading to reduced clustering? We propose that the
chain
cytoplasmic domain covers a negative site in the integrin
chain tail. Consistent with this model, it was previously
shown that various integrin
chain tails can shield
chain
tails from critical interactions with cytoskeletal proteins
(32), whereas at the same time,
chains tails often
make positive contributions to cell adhesion (9, 10, 35).
Most likely, the unshielded and unregulated interactions of
tails with cytoskeletal proteins may lead to increased constitutive cytoskeletal anchoring, and thus diminished diffusion and clustering at adhesive sites. Supporting this notion, low doses of cytochalasin D markedly increased adhesion of
truncated
4
1, but not wild-type
4
1. The range of cytochalasin D concentrations that promoted X4C0 adhesion
(0.01-1 µg/ml) is consistent with previously published cytochalasin D concentrations that stimulated
L
2-mediated
adhesion (7).
The overall importance of both diffusion and clustering
to cell adhesion has been noted previously. For example,
increases in the diffusion and lateral mobility of L
2 (7)
and LFA-3 (38) correlate with increases in cell adhesion and adhesion strengthening, respectively. Furthermore, integrin clustering is necessary for full integrin signaling (39),
and clustering of
M
2 and
L
2 integrins promoted by
phorbol ester or calcium also correlates with increased integrin-mediated adhesion (40, 41).
Regulation of integrin diffusion/clustering may be highly
relevant towards the understanding of inside-out signaling
mechanisms for 1 and
2 integrins, especially when affinity modulation is not involved. For example, stimulation of
4
1-mediated adhesion with macrophage inflammatory
protein-1
or with anti-CD3 or anti-CD31 antibodies did
not detectably induce binding of soluble VCAM (26), and
phorbol esters stimulated adhesion mediated by
5
1,
M
2, and
L
2 without affecting soluble ligand binding
(42). Notably, the effects of phorbol ester stimulation
and integrin
4 tail deletion show a striking parallel. Like
4 tail deletion, phorbol esters also (a) fail to alter integrin affinity for ligand, (b) fail to alter
4
1-dependent tethering
under shear (11, 12), but (c) markedly regulate static cell adhesion and adhesion strengthening under hydrodynamic
flow (11), and (d) regulate integrin diffusion rates (7).
However, whereas
4 tail deletion leads to increased cytoskeletal restraints and diminished lateral diffusion, phorbol
ester appears to release active cytoskeletal restraints, thereby
increasing lateral diffusion of the
L
2 integrin (7). Together, these results emphasize that a diffusion/clustering mechanism may be of general importance for regulating
adhesion, especially in the absence of changes in ligand
binding (26). Also, impaired integrin diffusion/clustering
may at least partly explain loss of cell adhesion observed
upon the deletion of other integrin
chain cytoplasmic
domains (35).
In conclusion, this report demonstrates that an integrin mutation can alter cell adhesion by a selective effect on receptor diffusion and clustering. In addition, the results strongly suggest that integrin cytoplasmic domains are critical for control of integrin diffusivity and clustering. We propose that control of cell adhesion at the level of integrin clustering is likely to be an important component of inside-out signaling, especially in cases when ligand binding is not altered.
Address correspondence to Martin E. Hemler, Rm. M-613, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115.
Received for publication 26 November 1996 and in revised form 11 July 1997.
1 Abbreviations used in this paper: AP, alkaline phosphatase; CHO, Chinese hamster ovary; FBS, fetal bovine serum; MSD, mean square displacement; rsVCAM, recombinant soluble vascular cell adhesion molecule; TBS, Tris-buffered saline; VCAM, vascular cell adhesion molecule.We thank Dr. Roy Lobb (Biogen, Inc., Cambridge, MA) for providing recombinant soluble VCAM and
VCAM-Ig-AP, Dr. Philip Lake (Sandoz Co., East Hanover, NJ) for providing VCAM-, and Dr. Ted Yednock (Athena Neurosciences, San Francisco, CA) for providing mAb 15/7.
This work was supported by a research grant (GM46526 to M.E. Hemler) and a postdoctoral fellowship (AI09490 to R.L. Yauch) from the National Institutes of Health. D.P. Felsenfeld was supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship.
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