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. 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
Alon, R., and S. Feigelson. 2002. From rolling to arrest on blood vessels: leuko-
cyte tap dancing on endothelial integrin ligands and chemokines at sub-
second contacts. Semin. Immunol. 14:93–104.
Alon, R., P.D. Kassner, M.W. Carr, E.B. Finger, M.E. Hemler, and T.A.
Springer. 1995. The integrin VLA-4 supports tethering and rolling in
flow on VCAM-1. J. Cell Biol. 128:1243–1253.
Ambroise, Y., B. Yaspan, M.H. Ginsberg, and D.L. Boger. 2002. Inhibitors of cell
migration that inhibit intracellular paxillin/alpha4 binding: a well-docu-
mented use of positional scanning libraries. Chem. Biol. 9:1219–1226.
Benoit, M., D. Gabriel, G. Gerisch, and H.E. Gaub. 2000. Discrete interactions
in cell adhesion measured by single-molecule force spectroscopy. Nat.
Cell Biol. 2:313–317.
Berlin, C., E.L. Berg, M.J. Briskin, D.P. Andrew, P.J. Kilshaw, B. Holzmann,
I.L. Weissman, A. Hamann, and E.C. Butcher. 1993. ?4?7 integrin medi-
ates lymphocyte binding to the mucosal vascular addressin MAdCAM-1.
Berlin, C., R.F. Bargatze, J.J. Campbell, U.H. von Andrian, M.C. Szabo, S.R.
Hasslen, R.D. Nelson, E.L. Berg, S.L. Erlandsen, and E.C. Butcher.
1995. ?4 integrins mediate lymphocyte attachment and rolling under
physiologic flow. Cell. 80:413–422.
Brown, M.C., and C.E. Turner. 2004. Paxillin: adapting to change. Physiol. Rev.
Carman, C.V., and T.A. Springer. 2003. Integrin avidity regulation: are changes
in affinity and conformation underemphasized? Curr. Opin. Cell Biol.
Chen, C., J.L. Mobley, O. Dwir, F. Shimron, V. Grabovsky, R.L. Lobb, Y.
Shimizu, and R. Alon. 1999. High affinity VLA-4 subsets expressed on
T cells are mandatory for spontaneous adhesion strengthening but not for
rolling on VCAM-1 in shear flow. J. Immunol. 162:1084–1095.
Constantin, G., M. Majeed, C. Giagulli, L. Piccio, J.Y. Kim, E.C. Butcher, and
C. Laudanna. 2000. Chemokines trigger immediate ?2 integrin affinity
and mobility changes: differential regulation and roles in lymphocyte ar-
rest under flow. Immunity. 13:759–769.
Du, X.P., E.F. Plow, A.L. Frelinger 3rd, T.E. O’Toole, J.C. Loftus, and M.H.
Ginsberg. 1991. Ligands “activate” integrin alpha IIb beta 3 (platelet
GPIIb-IIIa). Cell. 65:409–416.
Dwir, O., G.S. Kansas, and R. Alon. 2000. An activated L-selectin mutant with
conserved equilibrium binding properties but enhanced ligand recogni-
tion under shear flow. J. Biol. Chem. 275:18682–18691.
Dwir, O., G.S. Kansas, and R. Alon. 2001. The cytoplasmic tail of L-selectin
regulates leukocyte capture and rolling by controlling the mechanical
stability of selectin:ligand tethers. J. Cell Biol. 155:145–156.
Evans, S.S., D.M. Schleider, L.A. Bowman, M.L. Francis, G.S. Kansas, and
J.D. Black. 1999. Dynamic association of L-selectin with the lympho-
cyte cytoskeletal matrix. J. Immunol. 162:3615–3624.
Feigelson, S.W., V. Grabovsky, E. Winter, L.L. Chen, R.B. Pepinsky, T. Yed-
nock, D. Yablonski, R. Lobb, and R. Alon. 2001. The src kinase p56Lck
upregulates VLA-4 integrin affinity: implications for rapid spontaneous
and chemokine-triggered T cell adhesion to VCAM-1 and fibronectin.
J. Biol. Chem. 276:13891–13901.
Feigelson, S.W., V. Grabovsky, R. Shamri, S. Levy, and R. Alon. 2003. The CD81
tetraspanin facilitates instantaneous leukocyte VLA-4 adhesion strengthen-
ing to VCAM-1 under shear flow. J. Biol. Chem. 278:51203–51212.
Firrell, J.C., and H.H. Lipowsky. 1989. Leukocyte margination and deformation
in mesenteric venules of rat. Am. J. Physiol. 256:H1667–H1674.
Geppert, T.D., and P.E. Lipsky. 1991. Association of various T cell-surface
molecules with the cytoskeleton. Effect of cross-linking and activation.
J. Immunol. 146:3298–3305.
Grabovsky, V., S. Feigelson, C. Chen, R. Bleijs, A. Peled, G. Cinamon, F.
Baleux, F. Arenzana-Seisdedos, T. Lapidot, Y. van Kooyk, et al. 2000.
Subsecond induction of ?4 integrin clustering by immobilized chemo-
kines enhances leukocyte capture and rolling under flow prior to firm ad-
hesion to endothelium. J. Exp. Med. 192:495–505.
Han, J., S. Liu, D.M. Rose, D.D. Schlaepfer, H. McDonald, and M.H. Ginsberg.
2001. Phosphorylation of the integrin alpha 4 cytoplasmic domain regu-
lates paxillin binding. J. Biol. Chem. 276:40903–40909.
Hynes, R.O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell.
Jiang, G., G. Giannone, D.R. Critchley, E. Fukumoto, and M.P. Sheetz. 2003.
Two-piconewton slip bond between fibronectin and the cytoskeleton de-
pends on talin. Nature. 424:334–337.
Kansas, G.S., K. Ley, J.M. Munro, and T.F. Tedder. 1993. Regulation of leuko-
cyte rolling and adhesion to high endothelial venules through the cyto-
plasmic domain of L-selectin. J. Exp. Med. 177:833–838.
Kassner, P.D., R. Alon, T.A. Springer, and M.E. Hemler. 1995. Specialized
functional properties of the integrin ?4 cytoplasmic domain. Mol. Biol.
Katagiri, K., N. Ohnishi, K. Kabashima, T. Iyoda, N. Takeda, Y. Shinkai, K. In-
aba, and T. Kinashi. 2004. Crucial functions of the Rap1 effector mole-
cule RAPL in lymphocyte and dendritic cell trafficking. Nat. Immunol.
Kim, M., C.V. Carman, W. Yang, A. Salas, and T.A. Springer. 2004. The primacy
of affinity over clustering in regulation of adhesiveness of the integrin
?L?2. J. Cell Biol. 167:1241–1253.
Kinashi, T. 2005. Intracellular signalling controlling integrin activation in
lymphocytes. Nat Rev Immunol. 5:546–559.
Kinashi, T., M. Aker, M. Sokolovsky-Eisenberg, V. Grabovsky, C. Tanaka, R.
Shamri, S. Feigelson, A. Etzioni, and R. Alon. 2004. LAD-III, a leukocyte
adhesion deficiency syndrome associated with defective Rap1 activation
and impaired stabilization of integrin bonds. Blood. 103:1033–1036.
Kucik, D.F., M.L. Dustin, J.M. Miller, and E.J. Brown. 1996. Adhesion-activat-
ing phorbol ester increases the mobility of leukocyte integrin LFA-1 in
cultured lymphocytes. J. Clin. Invest. 97:2139–2144.
Lin, K., H.S. Ateeq, S.H. Hsiung, L.T. Chong, C.N. Zimmerman, A. Castro,
W.C. Lee, C.E. Hammond, S. Kalkunte, L.L. Chen, et al. 1999. Selec-
tive, tight-binding inhibitors of integrin ?4?1 that inhibit allergic airway
responses. J. Med. Chem. 42:920–934.
Liu, S., and M.H. Ginsberg. 2000. Paxillin binding to a conserved sequence motif
in the ?4 integrin cytoplasmic domain. J. Biol. Chem. 275:22736–22742.
Liu, S., S.M. Thomas, D.G. Woodside, D.M. Rose, W.B. Kiosses, M. Pfaff, and
M.H. Ginsberg. 1999. Binding of paxillin to ?4 integrins modifies inte-
grin-dependent biological responses. Nature. 402:676–681.
Matthews, B.D., D.R. Overby, F.J. Alenghat, J. Karavitis, Y. Numaguchi, P.G.
Allen, and D.E. Ingber. 2004. Mechanical properties of individual focal
adhesions probed with a magnetic microneedle. Biochem. Biophys. Res.
Nishiya, N., W.B. Kiosses, J. Han, and M.H. Ginsberg. 2005. An ?4 integrin-
paxillin-Arf-GAP complex restricts Rac activation to the leading edge of
migrating cells. Nat. Cell Biol. 7:343–352.
Rose, D.M., P.M. Cardarelli, R.R. Cobb, and M.H. Ginsberg. 2000. Soluble
VCAM-1 binding to ?4 integrins is cell-type specific and activation de-
pendent and is disrupted during apoptosis in T cells. Blood. 95:602–609.
Rose, D.M., S. Liu, D.G. Woodside, J. Han, D.D. Schlaepfer, and M.H. Gins-
berg. 2003. Paxillin binding to the ?4 integrin subunit stimulates LFA-1
(integrin ?L?2)-dependent T cell migration by augmenting the activation
of focal adhesion kinase/proline-rich tyrosine kinase-2. J. Immunol.
Shamri, R., V. Grabovsky, J.M. Gauguet, S. Feigelson, E. Manevich, W. Kola-
nus, M.K. Robinson, D.E. Staunton, U.H. von Andrian, and R. Alon.
2005. Lymphocyte arrest requires instantaneous induction of an extended
LFA-1 conformation mediated by endothelium-bound chemokines. Nat.
Shimaoka, M., J. Takagi, and T.A. Springer. 2002. Conformational regulation
of integrin structure and function. Annu. Rev. Biophys. Biomol. Struct.
Sigal, A., D.A. Bleijs, V. Grabovsky, S.J. van Vliet, O. Dwir, C.G. Figdor, Y.
van Kooyk, and R. Alon. 2000. The LFA-1 integrin supports rolling ad-
hesions on ICAM-1 under physiological shear flow in a permissive cel-
lular environment. J. Immunol. 165:442–452.
Springer, T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte
emigration: the multistep paradigm. Cell. 76:301–314.
Tadokoro, S., S.J. Shattil, K. Eto, V. Tai, R.C. Liddington, J.M. de Pereda, M.H.
JCB • VOLUME 171 • NUMBER 6 • 20051084
Ginsberg, and D.A. Calderwood. 2003. Talin binding to integrin beta
tails: a final common step in integrin activation. Science. 302:103–106.
van Kooyk, Y., and C.G. Figdor. 2000. Avidity regulation of integrins: the driv-
ing force in leukocyte adhesion. Curr. Opin. Cell Biol. 12:542–547.
von Andrian, U.H., S.R. Hasslen, R.D. Nelson, S.L. Erlandsen, and E.C.
Butcher. 1995. A central role for microvillous receptor presentation in
leukocyte adhesion under flow. Cell. 82:989–999.
Yauch, R.L., D.P. Felsenfeld, S.K. Kraeft, L.B. Chen, M.P. Sheetz, and M.E.
Hemler. 1997. Mutational evidence for control of cell adhesion through
integrin recruitment, independent of ligand binding. J. Exp. Med. 186:
Yednock, T.A., C. Cannon, C. Vandevert, E.G. Goldbach, G. Shaw, D.K. Ellis,
C. Liaw, L.C. Fritz, and L.I. Tanner. 1995. ?4?1 integrin-dependent cell
adhesion is regulated by a low affinity receptor pool that is conforma-
tionally responsive to ligand. J. Biol. Chem. 270:28740–28750.
Zhang, X., S.E. Craig, H. Kirby, M.J. Humphries, and V.T. Moy. 2004. Molecular
basis for the dynamic strength of the integrin ?4?1/VCAM-1 interaction.
Biophys. J. 87:3470–3478.