Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts

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Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts Ronen Alon and Sara W Feigelson The arrest of rolling leukocytes on target vascular beds is mediated by specialized leukocyte integrins and their endothelial ligands. In the circulation, these integrins are generally maintained as inactive ‘clasped’ heterodimers. Encounter by leukocytes of specialized endothelialpresented chemoattractants termed arrest chemokines drive these integrins to undergo force-regulated biochemical conformational changes in response to signals from chemokine-stimulated Gi-protein coupled receptors (GPCRs) and actin remodeling Rho GTPases. To arrest rolling leukocytes, integrin:ligand bonds must undergo stabilization by several orders of magnitude within quantal submicron contacts that consist of discrete integrin:ligand bonds. We present a unifying three step model for rapid integrin activation by chemokines in the quantal arrest unit, the smallest firm adhesive contact formed by a rolling or a captured leukocyte: integrin extension triggered by talin, integrin headpiece opening driven by surface-immobilized ligand and stabilized by low force, and full heterodimer unclasping requiring integrin tail associations with actinconnected talin and Kindlin-3. Specialized GPCRs and their Gi-protein signaling assemblies drive these and other adaptors to specifically bind integrin cytoplasmic tails possibly in conjunction with de novo actin remodeling, thereby optimizing bi-directional activation of ligandoccupied integrins. Address Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding author: Alon, Ronen ([email protected])

Current Opinion in Cell Biology 2012, 24:670–676 This review comes from a themed issue on Cell-to-cell contact and extracellular matrix Edited by Carl-Phillip Heisenberg and Reinhard Fa¨ssler For a complete overview see the Issue and the Editorial Available online 5th July 2012 0955-0674/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2012.06.001

Introduction Leukocyte arrest on specific target vascular cells involves adhesive cascades mediated by sequential but overlapping steps, initiated by reversible selectin-mediated adhesions, followed by firm integrin-mediated arrest on Current Opinion in Cell Biology 2012, 24:670–676

Ig superfamily integrin receptors such as ICAM-1, VCAM-1 and MadCAM-1 [1]. These arrests involve an abrupt activation of leukocyte integrins by specialized chemokines displayed on the endothelial surface [2–4]. Chemokines like CXCL12, CCL21, CXCL1, CCL2 and CCL25 are the most potent physiological inducers of integrin-dependent leukocyte arrest, and subsequent leukocyte crawling over and diapedesis through vascular barriers. Upon binding their cognate GPCRs, these chemokines, collectively termed arrest chemokines, activate integrin adhesiveness within subsecond time frames by triggering heterotrimeric G-proteins, primarily of the Gi/ o subtype (for earlier reviews, please refer to [4,5]). Some lymphocyte integrins can also support weak rolling adhesions in the absence of chemokine or selectin signals [6], but require chemokines to promote firm adhesions. Thus, with the exception of effector lymphocytes that express constitutively active integrins [7], leukocyte arrest on all known endothelial integrin ligands involves a critical series of chemokine-dependent activation events.

Initial selectin-mediated leukocyte rolling on blood vessels – a means of increasing encounter with endothelial chemokines and integrin ligands Selectins are the main receptors that mediate the initial capture of circulating leukocytes to vascular endothelial surfaces and their subsequent rolling adhesions [8], critical steps in the ability of leukocytes to encounter integrin-activating chemokines displayed on the endothelium. L-selectin, expressed on most circulating leukocytes, is critical mainly for lymphocyte capture and rolling on high endothelial venules (HEVs) of lymph nodes, whereas P-selectins and E-selectins function in almost all acutely and chronically inflamed endothelial beds. Selectin bonds are exceptionally shear resistant and display catch bond properties since their lifetimes can be prolonged by low force application [9]. Selectins and their ligands are enriched on microvillus-like projections, preferred sites for leukocyte–endothelial collisions [10]. Selectin-mediated rolling leads to microvillus flattening that may enhance both chemokine and integrin encounters with ligand [11]. In rolling myeloid leukocytes, but not in memory or naı¨ve T cells [2,3], engagement of L-selectin [12], as well as of selectin ligands like PSGL-1, can stimulate weak LFA-1:ligand interactions that can retard rolling, but cannot substitute for chemokine dependent arrest signals [13,14,15]. www.sciencedirect.com

Chemokine signals for integrin-dependent leukocyte arrest Alon and Feigelson 671

Firm leukocyte adhesions require integrin activation via coordinated occupancy by both external and cytoplasmic ligands: the quantal arrest unit Chemokine-triggered leukocyte arrest is mediated by high affinity integrin bonds often stabilized by oligomerization in submicron focal dots [16]. Both in vitro and in vivo studies suggest that prior encounter with chemokines during the rolling phase is not necessary for this rapid integrin activation [2,3,17]. As opposed to the slow assembly of integrin macroclusters [18], large focal zones of motile leukocytes [19], and other supramolecular assemblies of leukocytes adhered to low density integrin ligands, the focal integrin dots that rapidly arrest rolling leukocytes rearrange within less than 0.5 s [3,20] and form independently of each other. It is also believed that each integrin molecule within a microcluster undergoes full activation independently of its neighboring integrins [21,22]. Based on accumulating evidence in the literature, we propose a universal model for full activation of leukocyte integrins in this arrest unit that involves three major overlapping steps. Full integrin activation by chemokine arrest signals appears to involve simultaneous bi-directional activation of integrin heterodimers through conformational changes induced from the cytoplasmic side, coupled with ligand-reinforced (outside-in) rearrangements of the integrin headpiece [15]. Leukocyte integrins exist in an inactive clasped conformation, stabilized by inter-subunit associations between the transmembrane and cytoplasmic integrin domains. The integrin clasps must be disrupted by the binding of the key integrin activating adaptor, talin, to the integrin b subunit tail [23–25]. The exclusive talin isoform expressed in leukocytes, talin-1, is the main cytoskeletal integrin activating adaptor in all hematopoietic cells [26]. Indeed, knock down of talin-1 results in almost total loss of adhesiveness in multiple leukocytes integrin [3,15,27–29,30,31]. Talin-1 binding to the b subunit tail requires local elevation of PI(4,5)P2 (PIP2) [30,32] near the talin head, which drives the binding of its F3 domain to a membrane proximal NPXY motif on the b integrin tail. Talin associations with other negatively charged phospholipids may also facilitate this integrin tail binding [33]. Talin can then anchor ligand-bound integrins to the cortical actin cytoskeleton via its numerous actin binding sites and its association with a second adaptor, vinculin [25]. Based on a reconstituted in vitro system, in which talin head domain binding induces aIIbb3 integrin extension [34], it appears that this initial talin–integrin association can drive only partial unclasping of the integrin heterodimer resulting in integrin extension. In the case of LFA-1 and Mac-1, the ectodomains can extend up to 10–25 nm above the cell surface [35], whereas in the case of VLA-4, much more modest unbending occurs [36]. These conformational switches www.sciencedirect.com

variably increase the association rate of the integrin headpieces with immobilized ligands [37], especially if the integrins are pre-anchored to the leukocyte cytoskeleton [38]. Yet, if these integrin headpieces remain in a closed conformation with low affinity to ligand, only very shortlived integrin–ligand bonds can form [22,39]. A critical bond duration therefore appears necessary to allow ligand-induced opening of the integrin headpiece to enable the b subunit hybrid domain to swing out, resulting in separation of the b and a subunits [37]. Subsequent allosteric rearrangements pull apart the transmembranal and cytoplasmic domains of the a and b subunit, and stabilize the unclasped integrin heterodimer [40]. Mere associations of talin with the b subunit tail, the plasma membrane and the actin cytoskeleton (Figure 1) are probably insufficient to accommodate all of these conformational transitions. Thus, to maximally disrupt the integrin clasp, both talin and a second b subunit interacting adaptor, Kindlin, need to co-associate with the same integrin b tail [26,41]. Kindlin-3 is the exclusive Kindlin family member expressed in hematopoietic cells with critical function in nearly all platelet and leukocyte integrin adhesions studied to date [42–44]. Notably, Kindlin-3 does not directly bind actin or talin, and may need to associate with one or more of its partners (e.g. migfilin or ILK) to co-activate a target integrin [45,46]. Recent data in neutrophils support this notion by demonstrating that talin requires Kindlin-3 to drive full LFA-1 activation by chemokine signals [15]. Mutations introducing a stop codon in Kindlin-3 underlie a rare leukocyte and platelet integrin dependent adhesion deficiency syndrome, called LAD-III [44,46,47], which is associated with severe bleeding defects and impaired integrindependent adhesion of leukocytes to inflamed endothelia, though normal integrin-independent motility [48]. Consistent with their role as mechanical connectors, conversion of transient integrin–ligand bonds into stable, high affinity bonds requires the extracellular integrin ligands to be immobilized [22,49], and the integrin cytoplasmic tails to be properly anchored to an intact cortical cytoskeleton either before or shortly after chemokine stimulation [3,20,50]. Immobilization of both the integrin and its extracellular ligand seems necessary to allow low external forces to pull on the integrin–ligand interface at the leukocyte–endothelial contact and thereby drive rapid ligand-induced integrin headpiece activation through the opening of the b I-domains [51,52], and in the case of the b2 integrins, through the a I domain, as well [53]. These force-facilitated headpiece rearrangements induced by extracellular integrin ligands might be able to sufficiently prolong integrin:ligand bond lifetimes, characteristic of ‘catch’ bonds [54,55] to allow sufficient duration of the nascent bond that is necessary for the final integrin unclasping events critical for leukocyte arrest. Surface-immobilized integrin ligands might also rebind integrins after bond dissociation more frequently than Current Opinion in Cell Biology 2012, 24:670–676

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Figure 1

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Rap-1

2

3

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Force

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actin fiber talin dimer RhoA

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Gi PIP5Kγ Leukocyte membrane

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immobilized CAM

chemokine

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high affinity integrin bond soluble CAM

Current Opinion in Cell Biology

Stimulatory chemokines signal to integrins at the quantal arrest focal unit. A proposed mechanism for bi-directional conformational integrin activation in a single microvillus necessary for leukocyte arrest on vascular endothelium. Arrest may involve several simultaneously activated focal units, but it is assumed that the chemokine signal and the target integrin reside in the same quantal submicron unit. Integrin mediated arrest depends on simultaneous co-occupancy of each integrin heterodimer by multiple cytoplasmic and extracellular ligands and optimal allosteric force-regulated changes in the integrin headpiece. These changes are facilitated by force transmission to the cortical actin cytoskeleton, underlying both the leukocyte and endothelial plasma membranes. In the example shown here, the Giprotein signals are co-transmitted to RhoA and Rap-1 GTPases. These upstream regulators may share the ability to recruit two integrin co-activators, talin-1 and Kindlin-3, and/or drive their associations with the cytoplasmic tail of the b integrin subunit of the target integrin by as yet unknown mechanisms. High affinity bonds require the full unclasping of the integrin heterodimer, which appears to depend on stable binding of activated talin and Kindlin-3 to adjacent NPXY motifs on the b subunit tail. Talin-1 recruitment can be facilitated by Rap-1 or by Gi-protein dependent phosphorylation of ZAP-70 or other PTKs (not shown). Talin-1 activity depends on PIP2 (purple balls) generated by submembranal PIP5Kg (purple rectangle), possibly stimulated by chemokine-stimulated RhoA and Rac1 (not shown) GTPases or by other Gi-protein stimulated effectors. 1: Inactive integrin. 2: Chemokine triggered talin-1 binds the integrin b tail and drives its unbending or extension. In the absence of a proper chemokine signal, Kindlin-3 binding to the same b integrin tail does not take place. The semiactivated talin-bound integrin binds its extracellular ligand without undergoing a full bidirectional activation. Such partial activation can take place also when global Rap-1 activation and talin-1 recruitment occurs through chemokineindependent signals (e.g. by PSGL-1 ligation in rolling neutrophils or by other PLC dependent signaling events). 3: Strong chemokine signals recruit and reinforce the stable binding of the two cytoplasmic ligands, talin-1 and Kindin-3 to their b subunit tail motifs. The extracellular ligand and the cytoplasmic tail ligands form an integrin–cytoskeletal complex that can properly deliver force-facilitated allosteric changes to the integrin headpiece and the two integrin subunits, and stabilize the unclasped integrin with maximal separation of the a and b subunits from each other. Additional cytoplasmic partners of Kindlin-3 may modulate the stability of this canonical talin-1-Kindlin-3 integrin complex. The potential role of force generated by de novo elongated actin filaments that pull the talin-1-occupied and Kindlin-3-occupied b subunit, relative to the fixed actin-linked a subunit, was suggested but its relevance to the formation and stability of the quantal arrest unit is still largely unclear. Rapid integrin dimerization can take place to further stabilize the quantal focal adhesion unit (not shown). 4: Integrin activated by a strong chemokine signal. GTPases and their talin-1 and Kindlin-3 targets are properly activated, but a soluble ligand does not exert sufficient force and a stable unclasped integrin heterodimer does not form. Soluble multivalent ligands that bind such inside-out activated integrin molecules may be able to induce only partial outside-in headpiece rearrangements owing to the absence of force application on these ligand-occupied integrins.

their soluble counterparts, since reactants in viscous media are more likely to recombine than to diffuse apart. Notably, in the presence of soluble rather than immobilized integrin ligands, even when both talin and Kindlin-3 Current Opinion in Cell Biology 2012, 24:670–676

and their partners are stimulated by strong chemokine signals, unclasped integrin heterodimers are short-lived (Figure 1) and are very difficult to detect by saturating levels of mAbs specific for integrin epitopes associated www.sciencedirect.com

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with integrin extension and headpiece activation [27]. Thus, integrin activating arrest chemokines must correctly position both talin and Kindlin-3 together with one or more of their cytoskeletal partners (e.g. vinculin, a-actinin, paxillin, filamin, migfilin or ILK [56,57]) near the integrin cytoplasmic tails with which they associate and at the same time allow integrin binding with its immobile extracellular ligand to further anchor the unclasped integrin to the cortical cytoskeleton [38]. In light of these multiple requirements of integrin activation by surface bound ligands, it is not surprising that only surface immobilized chemokines, in close proximity to the integrin ligands can efficiently and accurately drive integrin co-occupancy by both extracellular and cytoplasmic partners within 0.5 s time frames, the minimal duration for the formation of a quantal arrest unit [3,20].

How do particular chemokine-stimulated GTPases contribute to rapid activation of distinct integrins? Different Rho and Rap GTPases have been implicated in various chemokine triggered integrin activation processes. In vitro and in vivo dissection of chemokinetriggered integrin activation in different types of leukocytes strongly support the existence of multi-molecular GTPase complexes, signalosomes, which are either preassembled near the plasma membrane or become rapidly assembled by the chemokine-stimulated GPCRs. The notion that major leukocyte integrins like VLA-4 and a4b7 are topographically segregated from LFA-1 [58,59] has raised the possibility that chemokine occupied GPCRs, their Gi-proteins and GTPase-containing signalosomes may operate differently within distinct cell surface compartments. In support of this possibility, a recent study on a monocyte cell line indicated that both LFA-1 and talin-1 are largely excluded from microvilli, whereas Kindlin-3 is present [59]. Thus, distinct GPCR and GTPase assemblies at different locations on the leukocyte surface are likely to also vary in their Guanine Exchange Factors (GEFs), talin and Kindlin-3 content, as well as the enzymatic machineries critical for their membrane localization [60] and phosphorylation [25]. These signalosomes probably also vary between different immune cell types. Indeed, a particular signalosome essential for chemokine triggered activation of a particular integrin in monocytes (e.g. chemokine triggered Rap-1 and VLA-4 [61]) is not utilized by the same integrin and activating chemokine in T cells [62]. We will now provide brief examples of how distinct GTPases or PTKs may translate signals from chemokine stimulated GPCRs into either LFA-1 or VLA-4 arrest units in these leukocytes. LFA-1 activation by chemokine-triggered GTPases

In T cells, GPCR-stimulated RhoA, Rac1 and Rap-1 appear to additively trigger high LFA-1 adhesiveness at quantal arrest units by directly recruiting and activating talin-1 [30,63,64] (Figure 1). Both RhoA and Rac1 can www.sciencedirect.com

locally raise plasma membrane levels of the talin activating phosphatidyl inositol, PtdIns(4,5)P2 (PIP2), possibly by the co-stimulation of phospholipase D1 (PLD1) and the PIP2 generating talin-1 associated kinase, PIP5Kg (PIPK1g90) [30,60] (Figure 1). Chemokine activated Rho and its downstream effector, mDia1, may also generate new actin bundles that help anchor the integrin–talin– Kindlin-3 complex to the cortical cytoskeleton. RhoA dependent elongation of actin filaments might also generate lateral forces to drive the talin-occupied and Kindlin-occupied integrin b subunit apart from a preanchored a subunit (Figure 1). Theoretical models of b3 integrin activation predict that ligand-occupied integrins at the leading edge of motile cells can be unclasped by such internally applied actin driven forces [65]. This raises an attractive explanation for the multi-functional involvement of chemokine stimulated RhoA and Rac1 in stabilization of high affinity LFA-1 binding to ICAM-1 (Figure 1), although these GTPases do not participate in all leukocyte integrin activation events (see below). Notably, PIP2 is also a substrate for PLC, and pharmacological blockage of total PLC activity impairs integrin activation by multiple chemokines [61], which activate Rap-1 via the PLC dependent GEF, CalDAG-GEFI [66]. Chemokine-activated Rap-1 can recruit talin to the plasma membrane via its interacting adaptor, RIAM [67], and reinforce the binding of RAPL to the cytoplasmic interface of the LFA-1 a chain, contributing to LFA-1 clustering and adhesion strengthening following arrest [64].

VLA-4 activation by chemokine-stimulated effectors

Interestingly, neither RhoA nor its PLD effector are involved in chemokine triggered VLA-4 activation in T cells [31], in contrast to PIP5Kg [31], reflecting the universal role of its product, PIP2 in integrin activation and function at focal arrest units. Instead of using the RhoA-PLD axis, chemokines activate the VLA-4 dependent arrest units by activating Zap-70. This protein tyrosine kinase (PTK) phosphorylates the Rho GTPase GEF, VAV1 [31], but rather than activating its target Rho GTPases, the phosphorylated VAV1 drives talin association with VLA-4 [31]. This mechanism may replace talin recruitment by chemokine-activated Rap1, since in the same T cells, VLA-4 activation by arrest chemokines does not involve Rap-1 [62]. Rather, VLA-4 activation by chemokines is driven by PLC and DAGdependent PKC activities [62], possibly in parallel to the Zap-70–talin-1 axis. In contrast to T cells, VLA-4 activation by arrest chemokines in monocytes requires Rap-1 activation, and in these leukocytes, chemokine triggering of VLA-4 depends on a PLC-triggered CalDAG-GEF-Imediated signaling [61], most probably to talin-1. In line with the large diversity of GPCR-integrin signalosomes operating in different cellular backgrounds, chemokine stimulated PI3K signaling may also trigger VLA-4 activation in some leukocytes (e.g. monocytes, but not T cells) Current Opinion in Cell Biology 2012, 24:670–676

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[20,68], possibly via PIP3 dependent VAV-1 recruitment to the plasma membrane.

Conclusions and perspectives In this review, we have tried to present a unifying multistep model for rapid integrin activation by chemokines in quantal arrest units and postulated how talin and Kindlin3 may coordinate chemokine-stimulated integrin activation events within quantal arrest units mediated by distinct integrins, Our model integrates the functions of the cytoplasmic integrin ligands talin and Kindlin with those of the external (endothelial) ligands and highlights the potential roles of shear forces and actin remodeling in this potentially universal mechanism used by endothelial chemokines to induce abrupt integrin-mediated arrest of rolling leukocytes. In spite of considerable progress on the critical roles played by these two integrin coactivators in leukocyte arrest, and of their upstream chemokine-stimulated Rho and Rap-1 GTPases, it is still unclear how, when, and where these GTPases and their downstream effectors recruit and activate talin and Kindlin-3 to conformationally trigger bi-directional integrin activation in focal arrest units. In fact, it is still not known why arrest chemokines exclusively use Gi/o proteins and not other G-proteins (like Gq and G12/13) to activate leukocyte integrins. Another intriguing question is whether distinct integrin molecules co-expressed on the same leukocyte compete for Kindlin-3 and talin-1 during initial responses to endothelial chemokine signals. Such competition may explain why certain chemokine signals fail to optimally drive integrin unclasping, and why a4 integrins are less sensitive to conformational activation than b2 integrins [27]. Another pressing issue is how GPCRs specialized in integrin activation are regulated by other GPCRs occupied by blood borne chemoattractants before leukocyte encounter of endothelial chemokines [69]. A related issue is why global chemokine-independent rolling interactions can be integrated by only certain types of leukocytes, and what advantage these alternative systems provide for optimizing integrin responsiveness to subsequent chemokine signals [13]. A further open question is whether and how de novo actin polymerization triggered by chemokine activated Rho GTPases contributes to full integrin unclasping in the quantal arrest unit or in a later stage of an adhesion strengthening of this unit. Since chemokine stimulated Rho GTPases involved in integrin affinity triggering also stimulate de novo actin polymerization, it is possible that the unclasping of the first integrin molecule that generates the nascent arrest unit, or its neighboring integrin heterodimer involves chemokinetriggered RhoA dependent elongation of actin filaments near ligand-occupied integrins (Figure 1). Addressing these questions is likely to provide us with many missing links accounting for the large variations in integrin activation modalities used by different types of leukocytes, integrins and chemokine receptors [4]. This information Current Opinion in Cell Biology 2012, 24:670–676

will be critical for the future design of multifunctional GPCR and integrin inhibitors with stronger yet more selective capacity to interfere with specific leukocyte– endothelial associations underlying distinct inflammatory pathologies.

Acknowledgements R. Alon holds The Linda Jacobs Chair in Immune and Stem Cell Research and his research is supported by the Israel Science Foundation, the German Israeli Foundation, the Flight Attendant Medical Research Institute (FAMRI), and the Minerva Foundation.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest

1.

Springer TA: Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994, 76:301-314.

2.

Campbell JJ, Hedrick J, Zlotnik A, Siani MA, Thompson DA, Butcher EC: Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 1998, 279:381-384.

3.

Shamri R, Grabovsky V, Gauguet JM, Feigelson S, Manevich E, Kolanus W, Robinson MK, Staunton DE, von Andrian UH, Alon R: Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat Immunol 2005, 6:497-506.

4.

Laudanna C, Alon R: Right on the spot. Chemokine triggering of integrin-mediated arrest of rolling leukocytes. Thromb Haemost 2006, 95:5-11.

5.

Ley K: Arrest chemokines. Microcirculation 2003, 10:289-295.

6.

Berlin C, Berg EL, Briskin MJ, Andrew DP, Kilshaw PJ, Holzmann B, Weissman IL, Hamann A, Butcher EC: Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 1993, 74:185-195.

7.

Shulman Z, Cohen SJ, Roediger B, Kalchenko V, Jain R, Grabovsky V, Klein E, Shinder V, Stoler-Barak L, Feigelson SW et al.: Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots. Nat Immunol 2012, 13:67-76.

8.

Zarbock A, Ley K, McEver RP, Hidalgo A: Leukocyte ligands for endothelial selectins: specialized glycoconjugates that mediate rolling and signaling under flow. Blood 2011, 118:6743-6751.

9.

Marshall BT, Long M, Piper JW, Yago T, McEver RP, Zhu C: Direct observation of catch bonds involving cell-adhesion molecules. Nature 2003, 423:190-193.

10. von Andrian UH, Hasslen SR, Nelson RD, Erlandsen SL, Butcher EC: A central role for microvillous receptor presentation in leukocyte adhesion under flow. Cell 1995, 82:989-999. 11. Chen S, Springer TA: An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear. J Cell Biol 1999, 144:185-200. 12. Green CE, Pearson DN, Camphausen RT, Staunton DE, Simon SI: Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of highavidity beta2-integrin on neutrophils. J Immunol 2004, 172:7780-7790. 13. Zarbock A, Lowell CA, Ley K: Spleen tyrosine kinase Syk is necessary for E-selectin-induced alpha(L)beta(2) integrinmediated rolling on intercellular adhesion molecule-1. Immunity 2007, 26:773-783. www.sciencedirect.com

Chemokine signals for integrin-dependent leukocyte arrest Alon and Feigelson 675

14. Stadtmann A, Brinkhaus L, Mueller H, Rossaint J, BolominiVittori M, Bergmeier W, Van Aken H, Wagner DD, Laudanna C, Ley K et al.: Rap1a activation by CalDAG-GEFI and p38 MAPK is involved in E-selectin-dependent slow leukocyte rolling. Eur J Immunol 2011, 41:2074-2085. 15. Lefort CT, Rossaint J, Moser M, Petrich BG, Zarbock A,  Monkley SJ, Critchley DR, Ginsberg MH, Fassler R, Ley K: Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation. Blood 2012, 119:4275-4282. The first evidence for differential roles of talin-1 and Kindlin-3 in integrin inside-out activation in rolling and arrested neutrophils. In the absence of talin-1 neutrophil LFA-1 does not undergo extension, fails to arrest rolling neutrophils in response to arrest chemokine signals, and cannot slow down selectin-mediated rolling adhesions. In the absence of Kindlin-3, LFA-1 undergoes extension and can slow neutrophil rolling on E-selectin and ICAM-1 but fails to arrest these neutrophils in response to chemokine signals. 16. Shulman Z, Shinder V, Klein E, Grabovsky V, Yeger O, Geron E, Montresor A, Bolomini-Vittori M, Feigelson SW, Kirchhausen T et al.: Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin. Immunity 2009, 30:384-396. 17. Huo Y, Schober A, Forlow SB, Smith DF, Hyman MC, Jung S, Littman DR, Weber C, Ley K: Circulating activated platelets exacerbate atherosclerosis in mice deficient in apolipoprotein E. Nat Med 2003, 9:61-67. 18. Dustin ML: The cellular context of T cell signaling. Immunity 2009, 30:482-492. 19. Smith A, Carrasco YR, Stanley P, Kieffer N, Batista FD, Hogg N: A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes. J Cell Biol 2005, 170:141-151. 20. Grabovsky V, Feigelson S, Chen C, Bleijs DA, Peled A, Cinamon G, Baleux F, Arenzana-Seisdedos F, Lapidot T, van Kooyk Y et al.: Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J Exp Med 2000, 192:495-506. 21. Luo BH, Carman CV, Takagi J, Springer TA: Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering. Proc Natl Acad Sci USA 2005, 102:3679-3684. 22. Schurpf T, Springer TA: Regulation of integrin affinity on cell  surfaces. EMBO J 2011, 30:4712-4727. A comprehensive measurements of LFA-1 affinity to monovalent ICAM-1 that reveals only minor conformational switches of this main leukocyte integrin in the absence of immobilized ligands even in the presence of strong chemokine and other inside-out activation signals. Interaction of primed LFA-1 with immobilized but not soluble ICAM-1 triggers affinity maturation of LFA-1 to an adhesive, high affinity state. This work, together with refs [37] and [48] collectively suggest that the ability of immobilized ligand to stabilize high affinity binding to activated LFA-1 is a force dependent process that requires both LFA-1 and ICAM-1 anchorage on interacting counter-surfaces. 23. Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, de Pereda JM, Ginsberg MH, Calderwood DA: Talin binding to integrin beta tails: a final common step in integrin activation. Science 2003, 302:103-106. 24. Wegener KL, Basran J, Bagshaw CR, Campbell ID, Roberts GC, Critchley DR, Barsukov IL: Structural basis for the interaction between the cytoplasmic domain of the hyaluronate receptor layilin and the talin F3 subdomain. J Mol Biol 2008, 382:112-126. 25. Critchley DR: Biochemical and structural properties of the integrin-associated cytoskeletal protein talin. Annu Rev Biophys 2009, 38:235-254. 26. Moser M, Legate KR, Zent R, Fassler R: The tail of integrins, talin, and kindlins. Science 2009, 324:895-899. 27. Manevich E, Grabovsky V, Feigelson SW, Alon R: Talin 1 and paxillin facilitate distinct steps in rapid VLA-4-mediated adhesion strengthening to vascular cell adhesion molecule 1. J Biol Chem 2007, 282:25338-25348. 28. Simonson WT, Franco SJ, Huttenlocher A: Talin1 regulates TCRmediated LFA-1 function. J Immunol 2006, 177:7707-7714. www.sciencedirect.com

29. Lammermann T, Bader BL, Monkley SJ, Worbs T, WedlichSoldner R, Hirsch K, Keller M, Forster R, Critchley DR, Fassler R et al.: Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 2008, 453:51-55. 30. Bolomini-Vittori M, Montresor A, Giagulli C, Staunton D, Rossi B,  Martinello M, Constantin G, Laudanna C: Regulation of conformer-specific activation of the integrin LFA-1 by a chemokine-triggered Rho signaling module. Nat Immunol 2009, 10:185-194. The most comprehensive dissection of the individual GTPases critical for LFA-1 conformational activation by chemokine signals in T cells. The paper assigns new opposite functions for two Rho family members, Rac1 and Cdc42 in the regulation of high LFA-1 affinity to ICAM-1. PLD and PIP5Kg are identified as major downstream effectors of RhoA and Rac1 critical for high affinity LFA-1 conformational switches in response to chemokine signals. 31. Garcia-Bernal D, Parmo-Cabanas M, Dios-Esponera A,  Samaniego R, Hernan-P de la Ossa D, Teixido J: Chemokineinduced Zap70 kinase-mediated dissociation of the Vav1-talin complex activates alpha4beta1 integrin for T cell adhesion. Immunity 2009, 31:953-964. The first comprehensive description of VLA-4 activation by chemokine signals in T cells. A first indication that this VLA-4 activation, although depending on both PIP5Kg and talin activities uses entirely different regulatory elements than LFA-1 to recruit and activate talin association with VLA-4 in response to chemokine signals. 32. Martel V, Racaud-Sultan C, Dupe S, Marie C, Paulhe F, Galmiche A, Block MR, Albiges-Rizo C: Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides. J Biol Chem 2001, 276:21217-21227. 33. Anthis NJ, Wegener KL, Ye F, Kim C, Goult BT, Lowe ED,  Vakonakis I, Bate N, Critchley DR, Ginsberg MH et al.: The structure of an integrin/talin complex reveals the basis of inside-out signal transduction. EMBO J 2009, 28:3623-3632. The first high resolution structure of talin in complex with a b integrin tail indicating talin1 formation of a salt bridge with an Asp residue in the b tail and may therefore directly disrupt the salt bridge that stabilizes the cytoplasmic integrin clasp. 34. Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA,  Sligar SG, Taylor KA, Ginsberg MH: Recreation of the terminal events in physiological integrin activation. J Cell Biol 2010, 188:157-173. The paper investigated the terminal events in platelet integrin activation and presents conclusive evidence that talin-1 on its own can drive aIIb3 integrin activation in the absence of Kindlin–integrin binding. 35. Phan UT, Waldron TT, Springer TA: Remodeling of the lectinEGF-like domain interface in P- and L-selectin increases adhesiveness and shear resistance under hydrodynamic force. Nat Immunol 2006, 7:883-889. 36. Chigaev A, Waller A, Zwartz GJ, Buranda T, Sklar LA: Regulation of cell adhesion by affinity and conformational unbending of alpha4beta1 integrin. J Immunol 2007, 178:6828-6839. 37. Luo BH, Carman CV, Springer TA: Structural basis of integrin regulation and signaling. Annu Rev Immunol 2007, 25:619-647. 38. Cairo CW, Mirchev R, Golan DE: Cytoskeletal regulation couples LFA-1 conformational changes to receptor lateral mobility and clustering. Immunity 2006, 25:297-308. 39. Salas A, Shimaoka M, Kogan AN, Harwood C, von Andrian UH, Springer TA: Rolling adhesion through an extended conformation of integrin alphaLbeta2 and relation to alpha I and beta I-like domain interaction. Immunity 2004, 20:393-406. 40. Kim M, Carman CV, Springer TA: Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 2003, 301:1720-1725. 41. Ma YQ, Qin J, Wu C, Plow EF: Kindlin-2 (Mig-2): a co-activator of beta3 integrins. J Cell Biol 2008, 181:439-446. 42. Moser M, Nieswandt B, Ussar S, Pozgajova M, Fassler R: Kindlin3 is essential for integrin activation and platelet aggregation. Nat Med 2008, 14:325-330. 43. Manevich-Mendelson E, Feigelson SW, Pasvolsky R, Aker M, Grabovsky V, Shulman Z, Kilic SS, Rosenthal-Allieri MA, Ben-Dor S, Current Opinion in Cell Biology 2012, 24:670–676

676 Cell-to-cell contact and extracellular matrix

Mory A et al.: Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions. Blood 2009, 114:2344-2353.

extracellular domain in determining membrane localization. J Cell Biol 1997, 139:563-571.

46. Malinin NL, Zhang L, Choi J, Ciocea A, Razorenova O, Ma YQ, Podrez EA, Tosi M, Lennon DP, Caplan AI et al.: A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans. Nat Med 2009, 15:313-318.

59. Hyduk SJ, Rullo J, Cano AP, Xiao H, Chen M, Moser M, Cybulsky MI: Talin-1 and kindlin-3 regulate alpha4beta1  integrin-mediated adhesion stabilization, but not G proteincoupled receptor-induced affinity upregulation. J Immunol 2011, 187:4360-4368. The first evidence for exclusion of talin 1 and LFA-1 from cell surface microvilli/microridges and for uniform distribution of Kindlin-3 in a leukocyte cell line. Both talin1 and Kindlin-3 are necessary for optimal LFA-1 activation but each one alone is sufficient for VLA-4 activation. Although focused on monocytes, this novel proteomic analysis of surface molecules and cortical cytoskeleteal elements in microvilli and cell body compartments opens up new opportunities to identification of specific GPCR adaptors, GTPases and GEFs in distinct cellular compartments.

47. Kuijpers TW, van de Vijver E, Weterman MA, de Boer M, Tool AT, van den Berg TK, Moser M, Jakobs ME, Seeger K, Sanal O et al.: LAD-1/variant syndrome is caused by mutations in FERMT3. Blood 2009, 113:4740-4746.

60. Legate KR, Takahashi S, Bonakdar N, Fabry B, Boettiger D, Zent R, Fassler R: Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis. EMBO J 2011, 30:4539-4553.

48. Feigelson SW, Grabovsky V, Manevich-Mendelson E, Pasvolsky R, Shulman Z, Shinder V, Klein E, Etzioni A, Aker M, Alon R: Kindlin-3 is required for the stabilization of TCRstimulated LFA-1:ICAM-1 bonds critical for lymphocyte arrest and spreading on dendritic cells. Blood 2011, 117:7042-7052.

61. Hyduk SJ, Chan JR, Duffy ST, Chen M, Peterson MD, Waddell TK, Digby GC, Szaszi K, Kapus A, Cybulsky MI: Phospholipase C, calcium, and calmodulin are critical for alpha4beta1 integrin affinity up-regulation and monocyte arrest triggered by chemoattractants. Blood 2007, 109:176-184.

49. Feigelson SW, Pasvolsky R, Cemerski S, Shulman Z, Grabovsky V, Ilani T, Sagiv A, Lemaitre F, Laudanna C, Shaw AS et al.: Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state. J Immunol 2010, 185:7394-7404.

62. Ghandour H, Cullere X, Alvarez A, Luscinskas FW, Mayadas TN: Essential role for Rap1 GTPase and its guanine exchange factor CalDAG-GEFI in LFA-1 but not VLA-4 integrin mediated human T-cell adhesion. Blood 2007, 110:3682-3690.

44. Svensson L, Howarth K, McDowall A, Patzak I, Evans R, Ussar S, Moser M, Metin A, Fried M, Tomlinson I et al.: Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation. Nat Med 2009, 15:306-312. 45. Meves A, Stremmel C, Gottschalk K, Fassler R: The Kindlin protein family: new members to the club of focal adhesion proteins. Trends Cell Biol 2009, 19:504-513.

50. Alon R, Feigelson SW, Manevich E, Rose DM, Schmitz J, Overby DR, Winter E, Grabovsky V, Shinder V, Matthews BD et al.: Alpha4beta1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain. J Cell Biol 2005, 171:1073-1084. 51. Puklin-Faucher E, Gao M, Schulten K, Vogel V: How the headpiece hinge angle is opened: new insights into the dynamics of integrin activation. J Cell Biol 2006, 175:349-360. 52. Alon R, Dustin ML: Force as a facilitator of integrin conformational changes during leukocyte arrest on blood vessels and antigen-presenting cells. Immunity 2007, 26:17-27. 53. Astrof NS, Salas A, Shimaoka M, Chen J, Springer TA: Importance of force linkage in mechanochemistry of adhesion receptors. Biochemistry 2006, 45:15020-15028. 54. Kong F, Garcia AJ, Mould AP, Humphries MJ, Zhu C: Demonstration of catch bonds between an integrin and its ligand. J Cell Biol 2009, 185:1275-1284. 55. Chen W, Lou J, Zhu C: Forcing switch from short- to  intermediate- and long-lived states of the {alpha}A domain generates LFA-1/ICAM-1 catch bonds. J Biol Chem 2010, 285:35967-35978. An elegant demonstration of LFA-1 catch bonds in vitro. Using force probes, this work nicely demonstrates how low force application on the LFA-1-ICAM-1 interface prolongs bond lifetime. Since the integrin legs are fixed, this study suggests that mechanochemical allosteric communications between ICAM-1-occupied a I-domain and the b I-domain, on their own, stabilize the LFA-1 headpiece association with ICAM-1, independently of full subunit separation and unclasping of the LFA-1 heterodimer. 56. Stanley P, Smith A, McDowall A, Nicol A, Zicha D, Hogg N: Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell. EMBO J 2008, 27:62-75. 57. Legate KR, Fassler R: Mechanisms that regulate adaptor binding to beta-integrin cytoplasmic tails. J Cell Sci 2009, 122:187-198. 58. Abitorabi MA, Pachynski RK, Ferrando RE, Tidswell M, Erle DJ: Presentation of integrins on leukocyte microvilli: a role for the

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63. Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O, Kinashi T: Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow. J Cell Biol 2003, 161:417-427. 64. Ebisuno Y, Katagiri K, Katakai T, Ueda Y, Nemoto T, Inada H, Nabekura J, Okada T, Kannagi R, Tanaka T et al.: Rap1 controls  lymphocyte adhesion cascade and interstitial migration within lymph nodes in RAPL-dependent and -independent manners. Blood 2010, 115:804-814. The first comprehensive analysis of the role of Rap-1 and RAPL in different steps of lymphocyte arrest on and migration through lymph node HEVs and the extravascular lymph node tissue. Using lentiviral shRNA knockdown of Rap1, RapL, and talin in a B cell line, they demonstrated that initial arrest events require Rap-1 but not RapL or talin, wheras the latter two are required for post arrest adhesion strengthening. Consistent with this, they showed that primary RapL / murine lymphocytes fail to form stable attachments to ICAM-1 expressing cells in vitro. In vivo RapL / murine lymphocytes arrested normally on HEVs but readily detatched consistent with a role of RapL in post arrest integrin mediated adhesion strengthening. 65. Zhu J, Luo BH, Xiao T, Zhang C, Nishida N, Springer TA: Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces. Mol Cell 2008, 32:849-861. 66. Bergmeier W, Goerge T, Wang HW, Crittenden JR, Baldwin AC, Cifuni SM, Housman DE, Graybiel AM, Wagner DD: Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. J Clin Invest 2007, 117:1699-1707. 67. Lee HS, Lim CJ, Puzon-McLaughlin W, Shattil SJ, Ginsberg MH: RIAM activates integrins by linking talin to ras GTPase membrane-targeting sequences. J Biol Chem 2009, 284:5119-5127. 68. Gerszten RE, Friedrich EB, Matsui T, Hung RR, Li L, Force T, Rosenzweig A: Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions. J Biol Chem 2001, 276:26846-26851. 69. Arnon TI, Xu Y, Lo C, Pham T, An J, Coughlin S, Dorn GW, Cyster JG: GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood. Science 2011, 333:1898-1903.

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