T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 171, No. 6, December 19, 2005 1073–1084
The Rockefeller University Press$8.00
mechanical strain is regulated by paxillin
association with the
-dependent adhesion strengthening under
Donald E. Ingber,
Sara W. Feigelson,
and Mark H. Ginsberg
David M. Rose,
Benjamin D. Matthews,
Darryl R. Overby,
Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
Department of Medicine, University of California, San Diego, La Jolla, CA 9209
Center for Nano Science, Ludwigs-Maximilians-Universität, Munich D-80799, Germany
Department of Pathology and Department of Surgery, Vascular Biology Program, Children’s Hospital, and
Harvard Medical School, Boston, MA 02115
Department of Pediatrics, Massachusetts General Hospital,
he capacity of integrins to mediate adhesiveness is
modulated by their cytoplasmic associations. In this
study, we describe a novel mechanism by which
-integrin adhesiveness is regulated by the cytoskeletal
adaptor paxillin. A mutation of the
(Y991A), reduced talin association to
heterodimer, impaired integrin anchorage to the
cytoskeleton, and suppressed
and adhesion strengthening of Jurkat T cells to VCAM-1
tail that disrupts
under shear stress. The mutant retained intrinsic avidity to
soluble or bead-immobilized VCAM-1, supported nor-
mal cell spreading at short-lived contacts, had normal
-microvillar distribution, and responded to inside-out
signals. This is the first demonstration that cytoskeletal
anchorage of an integrin enhances the mechanical sta-
bility of its adhesive bonds under strain and, thereby,
promotes its ability to mediate leukocyte adhesion under
physiological shear stress conditions.
Circulating leukocytes rapidly develop firm adhesion to vessel
wall ligands through their various integrin receptors
son, 2002). Integrins bind their respective endothelial ligands
under shear flow at lower efficiency than selectins (Springer,
1994). Adhesive tethers form over a fraction of a second and
depend on the ability of the nascent adhesive bond to withstand
disruptive shear force. In contrast to selectins, all leukocyte in-
tegrins can undergo instantaneous up-regulation of their affinity
or avidity to endothelial ligands upon exposure to endothelial
chemokines (Kinashi, 2005). In addition, integrins can undergo
conformational changes upon ligand binding (Hynes, 2002).
Cytoskeletal constraints of integrins may also control integrin
adhesiveness (van Kooyk and Figdor, 2000). Previous studies
on leukocyte (L)-selectin function regulation have shown that
(Mac-1; Alon and Feigel-
preformed cytoskeletal associations of L-selectin with the actin
cytoskeleton control the ability of ligand-occupied selectin to
stabilize nascent tethers under shear flow and capture leuko-
cytes under physiological shear stresses (Kansas et al., 1993;
Dwir et al., 2001). This raised the possibility that specialized
subsets capable of interacting with their respective endothelial
ligands under physiological shear flow may also need to prop-
erly anchor to the cytoskeleton. Although selectins and inte-
grins are structurally distinct, we hypothesized that
bonds forming under disruptive shear stresses may share a
common regulatory mechanism with L-selectin bonds. How-
ever, as alterations in cytoskeletal constraints of integrins can
modify affinity, clustering, and ligand-induced conformational
rearrangements (Carman and Springer, 2003), the direct contri-
bution of integrin anchorage to adhesive outcome has been dif-
ficult to dissect.
In this study, we unraveled novel adhesive properties of
-tail mutant with disrupted association with the cytoskele-
tal adaptor paxillin (Liu et al., 1999). We found that blocking
–paxillin interaction markedly impaired the integrin’s
ability to anchor to the cytoskeleton in Jurkat T cells. Although
not essential for
affinity, ligand-induced conformational
Correspondence to Ronen Alon: firstname.lastname@example.org
D.R. Overby’s present address is Department of Biomedical Engineering, Tulane
University, New Orleans, LA 70118.
Abbreviations used in this paper: AFM, atomic force microscopy;
umbilical vein endothelial cell; siRNA, short inhibitory RNA; wt, wild type.
The online version of this article contains supplemental material.
JCB • VOLUME 171 • NUMBER 6 • 2005 1074
changes, surface clustering and topography, or redistribution at
short static contacts, paxillin association with
–VCAM-1 bonds to resist mechanical stress. These re-
sults suggest that subsecond stabilization of
on the ability of ligand-occupied
anchor to the cytoskeleton. This work also highlights the key
role of the
in postligand binding adhesion
strengthening of the integrin under mechanical strain.
integrins to properly
Paxillin association with the
-cytoplasmic domain is required for cell
resistance to detachment by shear stress
Paxillin binding to the
-cytoplasmic domain is important for
signaling but not for adhesion developed in shear-
free conditions (Rose et al., 2003). To examine the role of pax-
illin binding in
-mediated adhesion under shear stress, we
analyzed the resistance to shear-induced detachment from the
ligand VCAM-1 of
-deficient JB4 Jurkat T cells trans-
fected with either wild-type (wt)
(Y991A) mutant JB4-
al., 2003). JB4-
(Y991A) cells were less resistant to shear-
induced detachment than their JB4-wt counterparts (Fig. 1 A).
Notably, bivalent VCAM-1 (VCAM-1–Fc) was much more
potent than monovalent soluble VCAM-1 (sVCAM-1) in sup-
-specific adhesion (Fig. 1 A), but it still could not
rescue the adhesive defect of the
sults were confirmed with multiple clones expressing similar
subunits as well as the
15/7 (Fig. 1 B and not depicted). Nevertheless, resistance to de-
tachment from different densities of either ICAM-1–Fc or
(JB4-wt) or the paxillin
(Y991A) (Rose et
(Y991A) mutant. These re-
poor shear-resistant adhesion to VCAM-1. (A)
JB4 Jurkat cells expressing either wt ?4 (WT)
or the ?4(Y991A) mutant (Y991A) were settled
for 1 min on low density VCAM-1–Fc (80
CAM sites/?m2; left) or on sVCAM-1 coated
at medium (1,480 sites/?m2; middle) or high
density (3,700 sites/?m2; right), and their re-
sistance to detachment by incremented shear
stresses was analyzed. The fraction of cells
within initially settled populations remaining
bound at the end of each interval of shear in-
crease is shown for each cell population. (B)
FACS staining of ectopically expressed ?4, en-
dogenous ?1 and ?L subunits, as well as of the
?1 activation neoepitope 15/7 on wt- and
?4(Y991A)-expressing JB4 cells, depicted with
black and gray lines, respectively. (C) LFA-1–
dependent adhesion of both wt- and
?4(Y991A)-expressing JB4 cells to low (80
sites/?m2) or medium density ICAM-1–Fc
(160 sites/?m2) as well as to high density
ICAM-1 (7,600 sites/?m2), measured as in A.
In each panel, the mean ? range of two ex-
perimental fields is depicted. Results in A and
C are representative of six independent exper-
iments. (D) FACS staining of VCAM-1, ICAM-1,
and E-selectin on TNF?-stimulated HUVECs.
Dotted lines represent staining of isotype-
matched controls (left). VLA-4–dependent ad-
hesion of JB4 cells transfected with wt ?4 (WT)
or the ?4(Y991A) mutant to intact (left) or
E-selectin–blocked TNF?-stimulated HUVECs
(right). Resistance to the detachment of cells
settled for 1 min on the monolayer was as-
sessed as in A. Shown in parenthesis are the
fractions of adherent cells that maintained roll-
ing on the different HUVECs at 5 dyn/cm2.
LFA-1 blockage did not affect Jurkat resistance
to detachment, whereas pretreatment with the
?4?1-specific blocker Bio1211 (at 1 ?g/ml)
resulted in complete loss of shear resistance
(not depicted). Error bars represent SD.
The ?4(Y991A)?1 mutant mediates
INTEGRIN REGULATION UNDER SHEAR STRESS • ALON ET AL.1075
ICAM-1 was comparable between wt- and mutant ?4?1–
expressing cells (Fig. 1 C). In agreement with these results,
VLA-4–dependent adhesion to TNF?-stimulated human um-
bilical vein endothelial cells (HUVECs) was reduced in Jurkat
cells expressing the ?4(Y991A) mutant (Fig. 1 D), in particular
at shear stresses ?5 dyn/cm2, within the upper range of shear
stresses prevailing in postcapillary venules where the majority
of lymphocyte extravasation takes place (Firrell and Lipowsky,
1989). Whereas most cells expressing the wt ?4 firmly arrested
on the stimulated HUVEC via their VLA-4, a significant frac-
tion of ?4(Y991A) mutant–expressing cells failed to arrest and
established endothelial (E)-selectin–dependent rolling on the
HUVEC (Fig. 1 D). In the absence of functional E-selectin, the
shear resistance of cells expressing the ?4(Y991A) mutant was
much lower than the shear resistance of cells expressing wt ?4
(Fig. 1 D). Because the contribution of LFA-1 to Jurkat arrest
was minimal, these data suggest that the Y991A ?4 mutant is
deficient in establishing ?4?1-mediated shear resistance on en-
dothelial cells expressing VCAM-1 as well as on substrates
coated with isolated VCAM-1.
Notably, preformed clustering of wt and mutant ?4 sub-
units on JB4 cells was essentially identical (Fig. 2 A). Real
time imaging of JB4 cells that adhered on VCAM-1 also
showed identical cell spreading as well as the distribution of
both mutant and wt ?4 during 1-min cellular contacts before
shear application (WT: n ? 44, 16% round, 54% polarized
with uniform ?4, 30% polarized with patched ?4; Y911A: n ? 27,
18% round, 52% polarized with uniform ?4, 30% polarized
with patched ?4; Fig. 2 B). Notably, the strength of resistance
to detachment developed by wt ?4 did not correlate with the
degree of patching (Fig. 2 B) in contrast to reports on LFA-1–
dependent systems (Constantin et al., 2000; Kim et al., 2004).
Thus, a mutation of the ?4 tail defective in paxillin binding pre-
vents ?4?1-mediated resistance to shear-induced cell detachment
independent of cell spreading and ?4 patching on VCAM-1.
The ?4(Y991A) mutation blocks paxillin association with
the ?4 tail selectively (Liu et al., 1999). As an alternative test of
the role of the ?4–paxillin interaction, we exploited a recently
identified small molecule inhibitor of this interaction. The
compound, designated A7B7C7, blocks the ?4–paxillin inter-
action and interferes with ?4?1-dependent cell migration (Am-
broise et al., 2002). This inhibitor, but not a control compound
(A6B6C6), attenuated the shear resistance of wt ?4?1–mediated
Jurkat cell adhesion to VCAM-1 (Fig. 3 A, left) but had no
effect on the residual shear resistance developed by the JB4-
?4(Y991A) cells (Fig. 3 A, right). Adhesion mediated by the
?L?2–ICAM-1 interaction was also insensitive to the inhibitor
(not depicted). Knocking down paxillin expression by up to
75% using transient short inhibitory RNA (siRNA) silencing
(Fig. 3 B) resulted in reduced adhesiveness of wt ?4?1–medi-
ated Jurkat cell adhesion to VCAM-1 (Fig. 3 C), with no inhibi-
tion of adhesiveness mediated by the ?4(Y991A) mutant (Fig.
3 C). Notably, LFA-1–dependent adhesion to ICAM-1 was also
insensitive to identical paxillin silencing (not depicted). Thus,
both genetic and pharmacological approaches indicate that the
?4–paxillin interaction increases the resistance of ?4?1–VCAM-1
contacts to detachment by disruptive shear stresses.
Paxillin association with the ?4 subunit
promotes ?4?1 anchorage to the
Paxillin binds a number of actin-binding proteins such as
talin and vinculin (Brown and Turner, 2004) and does so at
sites distinct from the ?4-binding site (Liu and Ginsberg,
before and during early cell spreading on VCAM-1 in shear-
free conditions. (A) wt ?4 or ?4(Y991A) is evenly distributed
on the surface of JB4 cells. Confocal immunostaining of ?4 on
the surface of prefixed WT or Y991A cells using the non-
blocking B5G10 mAb. Three representative cells are shown
for each cell type. (B) Live imaging of wt ?4 or ?4(Y991A)
during short cellular contacts with VCAM-1. JB4 cells express-
ing wt or mutant ?4 were prelabeled with AlexaFluor488-con-
jugated B5G10 mAb and settled for 1 min on VCAM-1. wt
or mutant ?4 were each imaged on cells that had spread on
sVCAM-1 for 1 min (shear free) and were then subjected to
10 s of shear stress at 2 dyn/cm2. Cell morphology was mon-
itored in differential interface microscopy (DIC). The degree
of patching was calculated by Image J analysis and was de-
fined as having at least one region with a B5G10 staining
mean intensity threefold higher than another region on the
same cell. Note that shear stress on its own did not trigger wt
?4 redistribution. Shear direction is depicted by the arrow.
The ?4(Y991A)?1 mutant distributes normally
JCB • VOLUME 171 • NUMBER 6 • 20051076
2000). We next quantified the fraction of detergent-resistant
wt ?4 or ?4(Y991A) retained on NP-40–solubilized cells using
fluorescence-tagged integrin-bound ?4 mAb (Fig. 4 A). Re-
tention of intact wt and ?4(Y991A) was similar and low (20%
of the total surface ?4; Fig. 4 A). However, the addition of
anti–mouse Ig to cluster the mAb-bound wt ?4 markedly in-
creased the association of ?4?1 surface integrin with the deter-
gent-insoluble cytoskeleton (Fig. 4 B). In contrast, the same
treatment produced a negligible increase in the cytoskeletal
association of ?4(Y991A)?1 (Fig. 4 B). Thus, the ?4(Y991A)
mutant fails to anchor properly to the actin cytoskeleton in
Jurkat T cells.
The ?4(Y991A) mutant poorly associates
with talin and does not respond to talin
In light of this poor cytoskeletal anchorage of ?4(Y991A)?1,
we next compared the level of talin associated with the wt or
mutant ?4?1 complex in nonadherent Jurkat cells. Notably,
constitutive talin binding to the ?4(Y991A)?1 complex was sig-
nificantly reduced compared with the wt integrin, as was evi-
dent from coprecipitation analysis (Fig. 5 A). Knocking down
up to 65% of the total talin content in wt ?4?1–expressing Jurkat
cells (Fig. 5 B) retained integrin expression (not depicted) but
resulted in significant reduction in their ?4?1-mediated shear
resistance on both sVCAM-1 and VCAM-1–Fc (Fig. 5 C, left
and right insets, respectively). Notably, identical suppression
of talin expression in the ?4(Y991A)?1-expressing Jurkat cells
(Fig. 5 B) had no effect on their low shear resistant adhesion to
identical VCAM-1 substrates (Fig. 5 C, right). These results
collectively suggest that paxillin association with ?4?1 also re-
cruits talin to the ?4–paxillin complex and may enhance talin
association with the ?1 subunit tail. Thus, both paxillin and
talin associations promote ?4?1-dependent cell resistance to
detachment from VCAM-1 under shear stress.
?4-Paxillin association is not required for
?4?1 avidity for VCAM-1 but increases ?4
Although the affinity of integrin ?4(Y991A)?1 to soluble
VCAM-1–Fc is retained (Rose et al., 2003), we considered that
Jurkat cells expressing the ?4(Y991A)?1 mutant might fail
to develop shear resistant adhesion as a result of reduced
avidity for surface-bound VCAM-1. Comparing wt ?4?1 with
?4(Y991A)?1 adhesiveness to VCAM-1–coated beads in the
absence of applied shear stress, we found that JB4-wt and JB4-
resistance developed by wt ?4. (A) JB4 cells expressing either wt ?4 or
?4(Y991A) were pretreated for 15 min with A7B7C7, a cell-permeable in-
hibitor of paxillin binding to the ?4 tail, or with the control compound
A6B6C6, both present at 5 ?M. The shear resistance of carrier or com-
pound-treated cells developed after 1-min adhesion to sVCAM-1 (2,220
sites/?m2) was determined as in Fig. 1. Results are mean ? range of two
experimental fields. The experiments depicted are each representative of
four independent tests. *, P ? 0.001 (a two-tailed paired t test) for control
compared with A7B7C7-treated cells at 0.5 dyn/cm2. (B) JB4 cells ex-
pressing wt ?4 were transfected with either paxillin-specific or control lu-
ciferase siRNA. Total lysates of each group were immunoblotted with pax-
illin- or tubulin-specific mAbs. Densitometric analysis reveals a decrease of
70 and 75% in paxillin content in JB4 expressing either wt or ?4(Y991A),
respectively. (C) Paxillin silencing impairs resistance to detachment from
sVCAM-1 developed by wt ?4?1 but not ?4(Y991A). The shear resistance
of the indicated cells was determined as in A. Results are representative of
three independent experiments. Error bars represent SD.
Blockage of ?4?1 paxillin associations interferes with shear
cytoskeletal matrix. (A) Detergent removal of nonligated wt ?4 monitored
by FACS. Jurkat cells were reacted for 30 min at 4?C with FITC-conju-
gated anti-?4?1 mAb (HP1/2) or isotype-matched control (dotted line).
Cells were then incubated at RT in detergent-free buffer (black, ?NP-40)
or in buffer containing 0.05% NP-40 (gray, ?NP-40). The fraction of
?4-bound mAb remaining after detergent treatment assessed by flow cy-
tometry is shown relative to originally bound ?4 mAb. (B) The fraction of
mAb-bound wt or ?4(Y991A) resistant to detergent-induced removal was
compared before (white bars) and after ligation of the mAb by secondary
antibody (black bars). Results are a mean of three independent experi-
ments. Error bars represent SD.
Paxillin association with ?4 facilitates integrin anchorage to the
INTEGRIN REGULATION UNDER SHEAR STRESS • ALON ET AL. 1077
?4(Y991A) cells bound identically to magnetic beads coated
with increasing site densities of VCAM-1–Fc (Fig. 6 A), which
is supportive of the normal adhesion of ?4(Y991A)?1-express-
ing cells under static conditions. Nevertheless, when VCAM-1–
coated beads that bound to JB4-wt cells were exposed to
abrupt mechanical stress, these beads were displaced signifi-
cantly less than beads prebound to JB4-?4(Y991A) cells (Fig.
6 B and Fig. S1, available at http://www.jcb.org/cgi/content/
full/jcb.200503155/DC1). ?4 resistance to displacement re-
quired an intact actin cytoskeleton, as JB4-wt cells pretreated
with the F-actin–severing drug cytochalasin D exhibited even
greater displacement in response to abrupt magnetic stress (Fig.
6 C). These findings, together with the shear-based detachment
assays (Fig. 1), collectively suggest that paxillin association
with the ?4?1 heterodimer and an intact actin cytoskeleton are
both required for ligand-occupied ?4?1 to develop stress-resistant
Paxillin association with the ?4 tail
augments ?4-mediated T cell capture on
VCAM-1 and MadCAM-1 under
The ?4?1 and ?4?7 integrins mediate leukocyte capture under
physiological shear flow (Alon et al., 1995; Berlin et al., 1995).
Therefore, we compared the ability of mutant ?4(Y991A) versus
wt ?4 to support ?4?1-dependent T cell capture by monovalent
and bivalent VCAM-1 under continuous shear flow. Consistent
with its defective resistance to shear force, ?4(Y991A)?1 medi-
ated reduced cell capture and arrest on a large range of densi-
ties of either monovalent (sVCAM) or bivalent (VCAM-Fc)
VCAM-1 (Fig. 7, A and B). This differential behavior was also
manifested at different levels of shear stress that were tested
(Fig. 7 B, first two panels). Notably, whereas disruption of the
actin cytoskeleton by cytochalasin D resulted in marked inhibi-
tion of both cell capture and arrest mediated by wt ?4?1, cyto-
chalasin D had no effect on the residual adhesions mediated by
the ?4(Y991A)?1 mutant (Fig. 7 A). Interestingly, the duration
of individual ?4?1 tethers, which is a measure of integrin affin-
ity to the ligand (Feigelson et al., 2001), was not altered upon
the loss of paxillin binding (Fig. 7 A). Thus, for optimal cell
capture under shear flow, the ?4 tail of ?4?1 requires associa-
tions with the intact actin cytoskeleton.
Jurkat cells express low levels of the ?7 integrin subunit;
thus, ?95% of their ?4-integrin subunits are found in ?4?1 het-
erodimers. Nevertheless, JB4-wt cell capture on high density of
the bivalent ?4?7-integrin ligand MadCAM-1–Fc (Berlin et al.,
1993) was inhibited by the anti-?4?7 antibody Act-1 (unpub-
lished data). The JB4-?4(Y991A) cells formed fivefold fewer
tethers on MadCAM-1 than JB4-wt cells, with a diminished
fraction of tethers followed by immediate arrests (Fig. 7 B,
right). Thus, paxillin association with the ?4 subunit enhances
the ability of ?4 to promote adhesive tethers in the context of
both ?1 and ?7 integrins under continuously applied disruptive
Preferential localization of receptors to microvilli in-
creases their availability for interactions with counter ligands
under shear flow (von Andrian et al., 1995). Electron micro-
scopic analysis of wt ?4 and the ?4(Y991A)-tail mutant re-
vealed identical distribution of these variants to microvillar
compartments (82 ? 4% for wt ?4, n ? 222; 80 ? 6% for the
?4(Y991A) mutant, n ? 196; Fig. 5 C). Furthermore, a higher
ratio of the ?4(Y991A)-tail mutant localized on microvillar tips
than wt ?4 (a 4.5 tip/base ratio for the mutant vs. only 1.8 tip/
base ratio for wt ?4). The number and size of microvillar pro-
jections in JB4-wt and JB4-?4(Y991A) cells were also com-
parable (Fig. 7 C). Thus, the enhanced ability of wt ?4?1 to
associates with talin. (A) The ?4(Y991A)?1
complex does not properly recruit talin. Total ly-
sates (left) or talin coprecipitating with anti-?4,
anti-?1, or an irrelevant mouse IgG (right) from
lysates of either JB4 transfected with wt ?4 or
the ?4(Y991A) mutant (top). The blot was
stripped and reprobed for ?4 (bottom). (B)
Silencing of talin in JB4 cells expressing either
wt or ?4(Y991A). The indicated cells were
transfected with either talin1-specific or control
siRNA. Total lysates of each group were immu-
noblotted with talin or tubulin-specific mAbs.
Densitometric analysis reveals a decrease of
66 and 67% in talin content in JB4 expressing
either wt or ?4(Y991A), respectively. (C) Talin
suppression preferentially impairs wt ?4?1–
mediated resistance to detachment from
sVCAM-1 (2,220 sites/?m2). *, P ? 0.03 for
control compared with talin-silenced cells at
0.5 dyn/cm2. Where indicated, cells were pre-
treated with the ?4?1-specific blocker BIO1211.
(inset) Effect of talin suppression on resistance
to detachment from VCAM-1–Fc (30 CAM
sites/?m2) of JB4 cells expressing either wt or
?4(Y991A). In each panel, the mean ? range
of two experimental fields is depicted. Results
are representative of three independent experi-
ments. Error bars represent SD.
The ?4(Y991A)?1 mutant poorly
JCB • VOLUME 171 • NUMBER 6 • 20051078
promote adhesive tethers under shear flow was not the result of
its preferential distribution to cellular microvilli or to increased
localization on microvillar tips.
The ?4(Y991A)?1 mutant fails to
generate productive adhesive bonds with
VCAM-1 under disruptive forces
To examine the effects of disrupting the ?4–paxillin interac-
tion at a single molecule level and in the presence of an ex-
ternal force other than shear stress, we next measured the
force of unitary adhesive interactions between wt ?4?1 or the
?4(Y991A)?1 mutant and immobilized VCAM-1 by atomic
force microscopy (AFM). JB4-wt or JB4-?(Y991A) cells were
coupled to the end of an AFM cantilever (Fig. 8 A) and low-
ered onto a VCAM-1–Fc-coated surface. After a 0.5-s contact,
the frequency of productive adhesive events and their strength
were analyzed by the degree of deflection experienced by the
cantilever during its retraction from the adhesive substrate.
Both cell types detached from the VCAM-1 substrate through
single jumps, suggesting the breakage of individual bonds dur-
ing cantilever retraction (Fig. 8 B). The adhesion frequencies
of all experiments were maintained below 30%, a level as-
sumed to reflect single ?4?1–VCAM-1 interactions (Zhang et
al., 2004). The specificity of the adhesive events detected in
this system were confirmed by a similar 70% reduction in total
binding events by the blockade of wt ?4?1 with Bio1211 (Lin
et al., 1999) or by omission of VCAM-1 from the substrate
(Fig. 8 C and not depicted). As indicated by the force histo-
grams derived for JB4-wt or JB4-?4(Y991A) cells (Fig. 8 C),
the frequency of productive adhesive events developed by
?4(Y991A)?1 was up to 10-fold lower than those developed by
?4?1 after background subtraction. The distribution of unbind-
ing (rupture) forces measured for the two integrin variants was,
however, similar (Fig. 8 C). Thus, paxillin association with the
?4 subunit dramatically augments the ability of ?4?1 to form
adhesive tethers that resist disruptive forces irrespective to
whether these forces are applied during a vertical force loading
(AFM) or during cell rotation (shear stress).
Paxillin association with ?4 augments
shear resistance of integrin tethers
independent of ligand-induced
The aforementioned data suggest that paxillin binding to ?4 is re-
quired for mechanical stabilization of cell attachments rather than
for cytoplasmic induction of high affinity integrin conformations.
Ligand binding to integrins can induce conformational changes in
the integrin, resulting in high affinity conformations (Du et al.,
1991; Shimaoka et al., 2002). Therefore, we considered the possi-
bility that the reduced tether formation by the ?4(Y991A) mutant
could reflect defective, instantaneous ligand-induced conforma-
tional changes in the integrin under shear stress. We first verified
that the ?4?1 ligand Bio1211 provoked similar conformational
changes in ?4?1 and ?4(Y991A)?1 under shear-free conditions, as
indicated by the identical induction of the ?1 ligand–induced
binding site reporter 15/7 epitope by increasing doses of the
monovalent ?4?1-specific ligand Bio1211 (Fig. 9 A; Lin et al.,
1999). We next tested the intrinsic attachment efficacy of either
wt ?4 or the ?4Y991A mutant to surface-immobilized ?4 mAb in
the absence of ligand occupancy of the integrin. Notably, the
HP1/2 mAb binding to ?4 integrins is not sensitive to their affin-
free conditions but develops lower bond stiffness under applied force.
(A) Binding of either wt or Y991A ?4?1-expressing cells to M-280 protein
A Dynabeads coated with 2D VCAM-1–Fc. Relative bead binding was de-
termined by side scattering analysis. Bead binding in the presence of 1
?g/ml of the ?4?1-specific blocker Bio1211 is shown in gray squares.
Results are representative of three independent experiments. (B, top) Rep-
resentative bead displacement measured from an ?4(Y991A)-expressing
cell (open circles) and a wt ?4–expressing cell (closed circles) during a
500-ms force pulse of ?100 pN. (bottom) Electromagnetic current wave-
form corresponding to the displacement response. (C) VCAM-1–coated
magnetic bead displacement in response to magnetic force pulse. VCAM-1
beads bound on the surface of JB4 cells expressing wt or ?4(Y991A) as
well as on cytochalasin D–treated JB4 cells expressing wt ?4 were exposed
for 0.5 s to the force pulse as described in the supplemental Materials and
methods and Fig. S1 (available at http://www.jcb.org/cgi/content/full/
jcb.200503155/DC1). For each experimental group, 8–10 cells were
analyzed, and results are the mean ? SD (error bars) of all displacement
curves. All samples were confirmed by side scattering analysis to bind a
similar number of VCAM-1 beads. A two-tailed unpaired t test for mean
bead displacements on wt and ?4(Y991A)-expressing JB4 cells yielded
P ? 0.06. One representative experiment of three.
The ?4(Y991A)?1 mutant exhibits normal avidity under shear-
INTEGRIN REGULATION UNDER SHEAR STRESS • ALON ET AL. 1079
ity to or rearrangement by native ligands (Feigelson et al., 2001)
and, thus, should be insensitive to intrinsic or ligand-induced af-
finity changes under shear stress. Notably, in the presence of
shear flow, the ?4(Y991A) mutant formed adhesive tethers to im-
mobilized HP1/2 mAb much less efficiently than wt ?4 (Fig. 9 B),
as was observed for VCAM-1 (Fig. 1). In addition, adhesive con-
tacts generated by the ?4(Y991A) mutant after 1 min of static
contact also exhibited poor resistance to detachment by increas-
ing shear forces relative to wt ?4–mediated contacts (Fig. 9 C).
Thus, paxillin association with the ?4-integrin tail enhances the
ability of the integrin subunit to generate resistance to detachment
forces independently of ligand-induced conformational rearrange-
ments under shear stress conditions.
tethering mediated by ?4?1 and ?4?7 under shear flow without altering
?4 distribution on microvilli. (A) Tethering (transient or followed by imme-
diate arrest) of Jurkat cells expressing either wt ?4 (WT) or the
?4(Y991A) mutant (Y991A) to immobilized VCAM-1. The mean duration
of transient tethers is shown in parenthesis above bars. Where indicated,
cells were pretreated with 20 ?M cytochalasin D (cyto D) or carrier (carr).
Error bars represent SD. (B) Tethering under shear flow of Jurkat cells
mediated by either WT or Y991A to distinct ?4-integrin ligands. Tethers
(transient or arrest) were determined under the indicated shear stresses
on surfaces coated with either monomeric 7D VCAM-1 (sVCAM-1),
dimeric 7D VCAM-1 (VCAM-1–Fc), or high density MadCAM-Fc. In each
panel, the mean ? range of two experimental fields is depicted. All teth-
ers to VCAM-1 were blocked in the presence of the ?4-integrin mAb
HP1/2 (not depicted). All tethers to MadCAM-1 were blocked by the
anti-?4?7 antibody Act-I (not depicted). Results in A and B are representa-
tive of five and four independent experiments, respectively. (C) Surface
distribution of wt ?4 (WT) or the ?4(Y991A) mutant on JB4 Jurkat cells
monitored by immunoelectron microscopy. Insets show lower magnifica-
tion images. The boxed areas depict the cellular areas enlarged. Pre-
fixed cells were stained with the nonblocking ?4-specific mAb B5G10.
Paxillin association with the ?4-cytoplasmic tail facilitates
Washed cells were stained with rabbit anti–mouse Ig and 5 nm gold
particle–conjugated goat anti–rabbit as described in Materials and
methods. Gold particles are marked by arrowheads. Photomicrographs
are representative of 20–30 cells.
(A) Schematic representation of the experimental system. JB4 cells were
coupled to an AFM cantilever tip via an anti-CD43 mAb. VCAM-Fc was im-
mobilized onto the substrate as in previous figures. (B) Representative AFM
force–displacement curves acquired with wt ?4–expressing JB4 cells (top) or
?4(Y991A)-expressing cells (middle) approaching the VCAM-1–Fc-bearing
substrate. A force–displacement curve of wt ?4–expressing JB4 approaching
a control substrate devoid of VCAM-1 is indicated in the bottom curve. (C)
Force histograms of ?4?1–VCAM-1 unbinding forces measured under a
fixed loading rate of 0.33 nN/s. The number of productive adhesive inter-
actions and their unbinding force distribution are depicted. Background
binding is depicted by the dashed line. The mean unbinding force (UF)
values of 10 independent experiments are indicated near each histogram.
Pulling velocity was 3 ?m/s, and the cell–substrate contact time was 0.5 s.
A representative result of 10 independent experiments is depicted.
?4(Y991A)?1 fails to stabilize bonds ruptured by an AFM probe.
JCB • VOLUME 171 • NUMBER 6 • 20051080
The ?4(Y991A)?1 mutant responds to
inside-out stimulation but develops
weaker adhesions to VCAM-1 under
Cellular stimulation by various agonists increases integrin adhe-
siveness in various contexts (Hynes, 2002). We next examined
the effects of two prototypic agonists, PMA, a direct agonist of
diacyl glycerol–dependent PKCs, and the chemokine SDF-1
(CXCL12) on mutant and wt ?4. Exposure of JB4-?4(Y991A)
cells to soluble PMA or to immobilized SDF-1 resulted in en-
hanced resistance to detachment from VCAM-1–bearing sur-
faces (Fig. 10 A), although the overall adhesion strength
developed by the ?4(Y991A) mutant was lower than that
developed by the intact integrin. Further analysis of adhesive
tethers formed on VCAM-1 at subsecond contacts also indi-
cated that the ability of initial ?4(Y991A)?1-dependent Jurkat
tethers to convert to immediate firm arrests was enhanced by
PMA (Fig. 10 B). Likewise, JB4-?4(Y991A) cells efficiently
responded to in situ subsecond signals from SDF-1 with a
twofold elevated frequency of ?4?1-dependent tethers on
VCAM-1 (Fig. 10 B, right). However, overall SDF-1–stimu-
lated tethers mediated by ?4(Y991A) cells remained lower
than wt ?4–mediated tethers. Thus, although the mutant ?4
underwent robust activation in response to both chemokine
and PMA inside-out signals, its impaired cytoskeletal associ-
ations resulted in overall reduced adhesion to VCAM-1 under
This study shows that the disruption of paxillin binding to the
integrin ?4 tail abrogates its anchorage to the actin cytoskeleton
and impairs the ability of integrin ligand bonds to withstand
immediate rupture by shear stress, an AFM-pulling device,
or abruptly applied magnetic force. Despite normal distribu-
tion on the cell surface and retained avidity to immobilized
VCAM-1, in the presence of applied forces, this anchorage-
deficient mutant poorly mediates tether formation and rapid
adhesion strengthening on its ligand. Paxillin-dependent cy-
toskeletal anchoring of ligand-occupied ?4 integrins may thus
underlie their unique capacity to resist disruptive forces and
support leukocyte adhesion under shear flow. Thus, although
cytoskeletal constraints of integrins were predicted to restrict
mobility and clustering on the cell surface and reduce cell ad-
hesiveness (Kucik et al., 1996; Yauch et al., 1997; Kim et al.,
2004), we propose that ?4 integrins must retain correct cyto-
skeletal associations to resist immediate rupture by shear stresses
exerted at leukocyte contacts with target blood vessels. Our
findings indicate that ?4-integrin anchorage to the cell cyto-
skeleton is critical for nascent adhesive contacts to resist imme-
diate rupture by shear stress, but it is not required for integrin
binding to the ligand nor for ligand-induced conformational re-
arrangements in the absence of external force. The anchorage
deficiency of the ?4-tail mutant resulted in an inability to develop
to immobilized ?4-specific mAbs independent of ligand-induced rearrange-
ments. (A) Dose-dependent induction of the 15/7 epitope by the ?4?1-
specific ligand Bio1211 on wt ?4 or ?4(Y991A)–expressing Jurkat cells.
(B) Reduced tethering and firm adhesion of the ?4(Y991A) mutant to immo-
bilized ?4 mAb (HP1/2) under shear flow. Frequency of tethers and their
categories were determined as in Fig. 7. (C) Strength of adhesion devel-
oped by JB4 expressing either wt or ?4(Y991A) settled for 1 min on low or
high density mAb. Experiments in A and B are each representative of
three independent experiments. Error bars represent SD.
Paxillin association with ?4 integrins stabilizes adhesive tethers
inside-out signals but develops poor adhesiveness in stimulated T cells
under shear flow. Adhesion of JB4 cells expressing wt ?4 or ?4(Y991A)
mutant to sVCAM-1 (2,960 sites/?m2) left intact (?) or stimulated by 1
min PMA pretreatment or by cell encounter with SDF-1? coimmobilized at
2 ?g/ml. (A) Resistance to detachment after 1 min of static contact ana-
lyzed as in Fig. 1. Values are mean ? range of two experimental fields.
(B) Capture and arrest under continuous shear flow. Frequency of tethers
and their categories were determined as in Fig. 7. The experiments in A
and B are each representative of four independent tests.
The ?4(Y991A)?1 mutant responds to phorbol ester and SDF-1
INTEGRIN REGULATION UNDER SHEAR STRESS • ALON ET AL. 1081
adhesion to a high affinity mAb, which binds the integrin inde-
pendently of affinity to native ligands (Feigelson et al., 2001;
Kinashi et al., 2004). Altogether, these data suggest that paxil-
lin associations with the ?4 tail control (a postligand occupancy
anchorage step that is critical for tether stabilization under
stress), which is a mechanical property underlying the ability of
lymphocytes to capture and arrest on endothelial ?4-integrin
ligands under shear flow. Our experiments on cytokine-acti-
vated endothelial cells also predict an increased contribution of
this ?4–paxillin association to T cells interacting with endothe-
lial beds expressing ?4-integrin ligands in the absence of endo-
Integrin affinity is controlled by ?-subunit associations
with the talin head domain (Tadokoro et al., 2003) and by Rap1
(Kinashi, 2005) via effectors such as RAPL (regulator of adhe-
sion and cell polarization enriched in lymphoid tissues; Kata-
giri et al., 2004). The effect of RAPL requires ?-tail sequences
(Katagiri et al., 2004) that are distant from the paxillin-binding
site on ?4 (Liu and Ginsberg, 2000). The retained affinity of the
?4(Y991A) mutant and its capacity to mediate static adhesion
suggest that lack of paxillin association with the ?4-integrin tail
does not interfere with Rap1-dependent signals whether medi-
ated through RAPL or other Rap1 effectors. The reduction in
talin association with ?4(Y991A)?1 did not alter basal ?4?1 af-
finity for VCAM-1 (Rose et al., 2003), suggesting that the pax-
illin-mediated association of talin with ?4?1 does not contribute
to affinity modulation. On the other hand, the extent of these
cytoskeletal associations and an intact actin cytoskeleton criti-
cally determine the mechanical strength of ?4?1 VCAM-1
bonds (i.e., tether formation, adhesion strengthening, and resis-
tance to mechanical stress). Thus, we propose that the ability of
?4 integrins to translate ligand occupancy into immediate
mechanical stability of subsecond adhesive contacts requires
paxillin and talin-mediated linkages of ?4?1 to the actin cy-
toskeleton. The regulation of ?4?1 adhesiveness by talin has
never been addressed, especially not under shear stress condi-
tions. The finding that talin suppression impairs the strength-
ening of wt ?4?1 bonds under strain is reminiscent of results
reporting the involvement of talin1 in ligand-driven ?5?1-cyto-
skeletal bonds in fibroblasts (Jiang et al., 2003). Although dif-
ferent integrins may anchor differently to the cytoskeleton in
distinct cell types, this involvement of talin in both ?4- and ?5-
integrin associations with the actin cytoskeleton is consistent
with the notion that talin, apart from its role in integrin affinity
regulation (Tadokoro et al., 2003), is a key postligand occu-
pancy adaptor that promotes integrin bond stabilization in dis-
tinct mechanical contexts and cellular environments.
Our results highlight the role of the ?-integrin subunit
rather than the ? subunit in postligand binding adhesion strength-
ening of the ?4?1–VCAM-1 bond under mechanical strain. Pre-
vious findings suggested that a nearly complete truncation of
the ?4-cytoplasmic tail impairs ?4?1 adhesion strengthening
without altering initial cell capture to VCAM-1 under shear flow
(Alon et al., 1995; Kassner et al., 1995). This truncation of the
?4-integrin tail also reduced integrin mobility (Yauch et al., 1997)
and may have increased integrin cytoskeletal anchorage via the
intact ?1 subunit, although this was not experimentally demon-
strated. Therefore, in these earlier studies, it was impossible to
distinguish between the contributions of ?4 anchorage versus mo-
bility to rapid mechanical stabilization of ?4 integrin–mediated
tethers. Our present results provide the first direct evidence for
a positive role of ?4 anchorage for the earliest stabilization
events of ?4?1–VCAM-1 bonds subjected to mechanical strain.
In addition to the ?4 Y991 residue, the phosphorylation
level of the ?4 serine 988 has been shown to control the degree
of ?4 association with paxillin (Han et al., 2001). A dephosphor-
ylated serine variant mimicked by the phosphodeficient mutant
?4 S988A was reported to bind paxillin at enhanced levels
(Nishiya et al., 2005). Interestingly, this phosphodeficient mu-
tant did not properly anchor to the cytoskeleton and supported
reduced adhesiveness to VCAM-1 (unpublished data). These
findings, together with the paxillin-silencing data of this study,
suggest that paxillin binding to the ?4 subunit is required but is
insufficient to anchor ?4 to the cytoskeleton. Thus, ?4 phosphor-
ylation, which is postulated to attenuate paxillin binding to the
?4 subunit, is in fact required, at least at a basal level, for
proper cytoskeletal ?4 association and productive adhesiveness
under shear stress. Overphosphorylation of ?4, which reduces
paxillin association, may, on the other hand, attenuate both an-
chorage and adhesiveness. Studies are ongoing to address both
the positive and negative effects of serine phosphorylation on
?4 anchorage and function under strain.
?4 association with paxillin enhances the activation of the
focal adhesion kinases FAK and PYK-2 after ?4?1 ligation (Liu
et al., 1999; Rose et al., 2003). This association also restricts
Rac activation at the leading edge via recruitment of the Arf
GTPase-activating protein (Nishiya et al., 2005). Nevertheless,
at 1-min contacts with VCAM-1, cells expressing the ?4-tail
mutant spread normally on VCAM-1. Suppression of tyrosine
phosphorylation or inhibition of PYK-2 activity in Jurkat T cells
had no effect on ?4?1-mediated adhesion strengthening devel-
oped under shear stress (unpublished data). Thus, the ability of
paxillin association with ?4?1 to enhance integrin anchorage to
the cytoskeleton and promote mechanical stability of adhesive
tethers at short-lived contacts is distinct from its roles in focal
adhesion turnover and Rac deactivation during cell spreading on
VCAM-1–containing substrates (Nishiya et al., 2005). Alto-
gether, our findings suggest that modulating mechanical prop-
erties of ?4 integrins by the inhibition of specific associations
between ?4-cytoplasmic tails and the cytoskeleton may be a
selective strategy to fine tune integrin-mediated adhesion under
shear stress without altering integrin affinity.
Materials and methods
Reagents and antibodies
Recombinant seven-domain human VCAM-1 (sVCAM-1) was provided by
B. Pepinsky (Biogen, Cambridge, MA). VCAM-1–Fc fusion protein con-
taining seven-domain VCAM-1 fused to IgG was generated as described
previously (Rose et al., 2000). VCAM-1–Fc constructed from domains 1
and 2 of VCAM-1 fused to Fc, termed 2D VCAM-1–Fc, was provided by
B. Pepinsky. Affinity-purified human full-length spleen-derived ICAM-1 was
a gift from T. Springer (Harvard University, Boston, MA). ICAM-Fc and
SDF-1? were purchased from R&D Systems. BSA (fraction V), poly-L-lysine,
and Ca2?/Mg2?-free HBSS were obtained from Sigma-Aldrich. Human
serum albumin (fraction V) and PMA were purchased from Calbiochem.
JCB • VOLUME 171 • NUMBER 6 • 20051082
The ?4-integrin function–blocking HP1/2 mAb, the E-selectin–blocking
mAb BB11, the ?1-specific TS2/16 mAb, the ?4-specific nonblocking
5BG10 mAb (all provided by B. Pepinsky), the ?1-integrin subunit mAb
15/7 (provided by T. Yednock, Elan Pharmaceuticals, San Francisco, CA;
Yednock et al., 1995), and the anti-?4?7 Act-1 (a gift from M.J. Briskin,
Millennium Pharmaceuticals, Cambridge, MA) were all used as purified
Ig. Antitalin mAb (clone 8d4) was purchased from Sigma-Aldrich. Anti-
paxillin mAb (clone 349) was purchased from BD Transduction Laborato-
ries. Goat polyclonal anti-?4 Ab (clone C-20) was purchased from Santa
Cruz Biotechnology, Inc.
Cell culture and flow cytometry
Jurkat cells deficient in ?4 (JB4) were stably transfected with either wt ?4 or
?4(Y991A) cDNA as described previously (Liu et al., 1999). Cells were
subcloned, and multiple clones expressing identical levels of ?4 and ?1
subunits were taken for functional analysis. Clones were maintained in
RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 mM nonessential amino acids,
and antibiotics (Biological Industries). Primary HUVECs were established
as previously described (Shamri et al., 2005). HUVECs were left intact or
stimulated for 4 h with 0.1 ng/ml TNF-? (R&D Systems) before experi-
ments. Staining and FACS analysis were performed as previously described
(Feigelson et al., 2003).
Immunofluorescence staining and immunoelectron microscopy
For ?4-integrin immunostaining, Jurkat cells were washed in PBS and incu-
bated with 10 ?g/ml anti-?4 B5G10 mAb for 30 min at 4?C. Cells were
washed once with PBS ? 5 mM EDTA and twice with PBS/0.1% BSA,
and ?4 integrins were stained with AlexaFluor546-conjugated anti–mouse
Ab (Invitrogen) and fixed in 3% PFA in PBS (30 min at RT). Control cells
were fixed before mAb incubation steps. Cells were attached to poly-
L-lysine–coated glass slides, and coverslips were mounted with elvanol
overnight and analyzed with a confocal microscope (TE300; Nikon) and
a laser-scanning system (model 2000; Bio-Rad Laboratories).
?4 localization was assessed by immunoelectron microscopy as
previously described (Chen et al., 1999). In brief, cultured Jurkat cells
were washed and prefixed in 0.1 M phosphate buffer, pH 7.4, containing
2% PFA and 0.05% glutaraldehyde. Washed cells in H/H medium (HBSS
containing 2 mg/ml BSA and 10 mM Hepes, pH 7.4, supplemented with
1 mM CaCl2 and 1 mM MgCl2) were incubated with 10 ?g/ml anti-?4
B5G10 mAb for 40 min at 22?C. Washed cells were stained with 10 ?g/
ml rabbit anti–mouse Ig, washed, and incubated for 45 min with 5 nm
gold particle–conjugated goat anti–rabbit (Aurion). Ultrathin sections (70–
90 nm) of 40–60 cells for each experimental group were examined with
an electron microscope (Tecnai 12; FEI) under 120 kV, and images were
taken using a CCD camera (Megaview 3; Soft Imaging System). In each
experimental group analyzed, the number of ?4-specific gold particles on
microvillar projections was compared with that on adjacent cell body
compartments of identical dimensions.
siRNA-mediated silencing of paxillin and talin
Silencing of talin expression in Jurkat cells was achieved by a talin1-spe-
cific 21-nucleotide siRNA (Dharmacon) corresponding to positions 6,043–
6,063 relative to the talin1 mRNA start codon (Shamri et al., 2005).
Silencing of paxillin was conducted as described previously (Nishiya
et al., 2005). Control transfections were performed with a fluorescein-
labeled 21-nucleotide duplex directed to Luciferase GL2. Transfection of T
cells was performed by electroporation using the Nucleofection system
(Amaxa). Transfected cells were maintained in culture medium. Talin and
paxillin expression monitored by immunoblotting was maximally sup-
pressed 72 h posttransfection, and time points were chosen for subse-
quent functional assays. Immunoprecipitation of ?4 was performed as
previously described (Feigelson et al., 2003).
Quantification of integrin anchorage to the cytoskeleton
Cells were stained with 10 ?g/ml of the FITC-conjugated anti-?4 mAb
HP1/2 at 4?C for 30 min, washed twice with H/H medium supplemented
with 1 mM CaCl2 and 1 mM MgCl2, and left either untreated or cross-
linked with secondary antibodies at 4?C for 30 min followed by two
washes as described previously (Geppert and Lipsky, 1991; Evans et al.,
1999). All cells were then incubated at RT for 30 min with the cytoskeletal
stabilizing buffer (CSB; 50 mM NaCl, 2 mM MgCl2, 0.22 mM EGTA, 13
mM Tris, pH 8.0, 1 mM PMSF, 10 mM iodacetamide, and 2% FCS) alone
or supplemented with 0.1% NP-40. The intact cells or their recovered de-
tergent-insoluble cytoskeletal fractions were washed in detergent-free CSB,
fixed in 1% PFA/PBS, and analyzed by flow cytometry. Under these con-
ditions, the majority of detergent-treated cells retain their shape and size.
The ratio of mean fluorescence intensity recovered in detergent-treated
cells divided by that of cells not exposed to the detergent yields the frac-
tion of mAb-bound ?4 integrin that is resistant to detergent extraction; i.e.,
anchored to the (detergent resistant) cytoskeletal fraction of the cell.
VCAM-1 microbead–binding assays and integrin bond stiffness
Protein A–coated magnetic M-280 Dynabeads (Dynal) were coated at RT
with various concentrations (0.004–1 ?g/ml) of 2D VCAM-Fc in H/H
binding medium, washed according to the manufacturer’s instructions,
and stored on ice. Cells and VCAM-1–coated beads were mixed at RT for
1 min in binding medium at a concentration of 107 cells/ml at a cell/
bead ratio of 1:8 followed by a threefold dilution in binding medium. The
cellular side scatter, distinguishing between bead-bound and bead-free
cells, was analyzed immediately in a FACScan flow cytometer (Becton
Dickinson). Background binding determined with protein A–coated beads
was ?10% of the maximal binding observed at VCAM-1 saturation and
was subtracted from the total binding results.
The mechanical stiffness of ?4?1/VCAM-1 adhesions was mea-
sured by electromagnetic pulling cytometry using VCAM-1–coated
beads (Matthews et al., 2004). The detailed method is described in sup-
plemental material (available at http://www.jcb.org/cgi/content/full/
All force measurements were conducted at 35 ? 2?C using a previously
described AFM apparatus (Benoit et al., 2000). In brief, a microfabri-
cated Si3N4 cantilever tip (Park Scientific Instruments) was coated with 0.1
mg/ml of the anti-CD43 mAb (R&D Systems). The spring constants of the
cantilevers used were determined at ?4.7 ? 0.6 mN/m. A single cell
was immobilized on the cantilever tip shortly before experimentation. The
device was mounted with a piezo-actuator (Piezosystem Jena) on an in-
verted optical microscope (Carl Zeiss MicroImaging, Inc.) containing a
heating stage. A diode laser beam focused on the sensor was used to
measure the displacement of the cantilever by the laser beam deflection
on a two-segment photodetector. The cell adhering to the cantilever was
positioned above an adhesive substrate coated with 2D VCAM-1–Fc cap-
tured via human IgG Fc mAb (Jackson ImmunoResearch Laboratories). The
cantilever was lowered until the sensor detected a contact force equal to a
preselected value (typically 50 pN). After the contact was established for
a dwelling time of 500 ms, the cell-bearing cantilever was lifted up by the
piezo-actuator, and the de-adhesion force was monitored by a force–dis-
tance plot (Fig. 5 B). From this plot, the last detectable de-adhesion force
was calculated. For each cell, ?50–200 force–distance plots were col-
lected within ?30 min. All de-adhesion events collected in at least 10
independent experiments were presented in histograms (Fig. 5 C).
Laminar flow adhesion assays
Purified ligands or mAbs were coated on polystyrene plates as previously
described (Grabovsky et al., 2000). Site densities of coated sVCAM-1
and VCAM-1–Fc were determined as previously described (Grabovsky et
al., 2000; Sigal et al., 2000). The polystyrene plates were each assem-
bled on the lower wall of the flow chamber (260-um gap) as previously
described (Dwir et al., 2000; Feigelson et al., 2001). Cells were washed
with cation-free H/H medium, resuspended in binding medium (H/H me-
dium supplemented with 1 mM CaCl2 and 1 mM MgCl2), and perfused
through the flow chamber at the desired shear stress. To disrupt actin cy-
toskeleton, cells were pretreated for 15 min with 20 ?M cytochalasin D
(Calbiochem) or carrier solution (0.1% DMSO). All flow experiments were
conducted at 37?C. Tethers were defined as transient if cells attached
briefly (?2 s) to the substrate and as arrests if they immediately arrested
and remained stationary for at least 5 s of continuous flow. Frequencies of
adhesive categories within differently pretreated cells or rates of cell ac-
cumulation on adhesive substrates were determined as a percentage of
cells flowing immediately over the substrates, as previously described
(Grabovsky et al., 2000). To assess rapid development of integrin avidity
to the ligand at 1-min stationary contacts, cells were allowed to settle onto
the substrate for 1 min at stasis. Flow was then initiated and increased
step-wise every 5 s by a programmed set of rates. At the indicated shear
stresses, the number of cells that remained bound was expressed relative
to the number of cells originally settled on the substrate. Over 95% of teth-
ers to VCAM-1 were blocked by pretreating cells with 10 ?g/ml of the ?4-
blocking mAb HP1/2. Live imaging of ?4 on Jurkat cells prelabeled with
AlexaFluor488-conjugated B5G10 mAb that settled on VCAM-1 was con-
ducted with Delta Vision Spectris RT (Applied Precision). ?4 patching was
quantified using Image J software (National Institutes of Health).
INTEGRIN REGULATION UNDER SHEAR STRESS • ALON ET AL. 1083
Online supplemental material
Fig. S1 shows the analysis of VCAM-1–coated bead displacement during
a magnetic force pulse applied on wt Jurkat cells. The supplemental Mate-
rials and methods section describes the experimental setup.
We thank S. Schwarzbaum for editorial assistance.
R. Alon is the Incumbent of The Tauro Career Development Chair in
Biomedical Research. This research was supported by the Fogarty Interna-
tional Research Collaboration Awards and was partially supported by the Is-
rael Science Foundation and MAIN, the EU6 Program for Migration and In-
flammation. This work was also supported by National Institutes of Health
(NIH) grants AR27214 and HL57009 to M.H. Ginsberg, NIH grant
CA45548 to D.E. Ingber, and NIH grant P30AR47360 to D.M. Rose. The
work of D.M. Rose was additionally supported by the Department of Veterans
Affairs Merit Review Entry Program Award and by the Arthritis Foundation.
Submitted: 28 March 2005
Accepted: 8 November 2005
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