Chemokine triggered integrin activation and actin remodeling events guiding lymphocyte migration across vascular barriers

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Review

Chemokine triggered integrin activation and actin remodeling events guiding lymphocyte migration across vascular barriers Ronen Alon⁎, Ziv Shulman Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100 Israel

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

Chemokine signals activate leukocyte integrins and actin remodeling machineries critical for

Received 18 August 2010

leukocyte adhesion and motility across vascular barriers. The arrest of leukocytes at target blood

Revised version received

vessel sites depends on rapid conformational activation of their α4 and β2 integrins by the

7 December 2010

binding of endothelial-displayed chemokines to leukocyte Gi-protein coupled receptors (GPCRs).

Accepted 7 December 2010

A universal regulator of this event is the integrin-actin adaptor, talin1. Chemokine-stimulated GPCRs can transmit within fractions of seconds signals via multiple Rho GTPases, which locally

Keywords:

raise plasma membrane levels of the talin activating phosphatidyl inositol, PtdIns(4,5)P2 (PIP2).

Adhesion

Additional pools of GPCR stimulated Rac-1 and Rap-1 GTPases together with GPCR stimulated PLC

Transendothelial migration

and PI3K family members regulate the turnover of focal contacts of leukocyte integrins, induce the

Crawling

collapse of leukocyte microvilli, and promote polarized leukocyte crawling in search of exit cues.

Endothelium

Concomitantly, other leukocyte GTPases trigger invasive protrusions into and between endothelial

Cytoskeleton

cells in search of basolateral chemokine exit cues. We will review here major findings and open

Shear stress

questions related to these sequential guiding activities of endothelial presented chemokines, focusing mainly on lymphocyte-endothelial interactions as a paradigm for other leukocytes. © 2010 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integrin affinity regulation by talin underlying chemokine triggered leukocyte arrest . . . Roles of chemokine-stimulated Rho GTPases during the earliest integrin activation events Chemokine triggered integrin microclustering and macroclustering . . . . . . . . . . . . The PLC-Rap-1 axis in chemokine-dependent control of integrin adhesiveness . . . . . . The rapid turnover of focal adhesive contacts in crawling lymphocytes . . . . . . . . . . Chemokine triggered integrin macroclustering at the leading edge . . . . . . . . . . . .

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⁎ Corresponding author. Fax: +972 8 934 2724. E-mail address: [email protected] (R. Alon). Abbreviations: Cdc42, cell division cycle 42; GEF, Guanine nucleotide exchange factor; GPCR, G-protein coupled receptor; LFA-1, Leukocyte function-associated antigen-1; LTB4, leukotriene B4; PAF, platelet activating factor; PI3K, Phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol 4,5 bisphosphate; PIP5K, Phosphatidylinositol-4-phosphate 5-kinase; PKC, Protein kinase C; PLC, phopholipase C; RhoA, Ras homology gene family member A; Rac, Ras-related C3 botulinum toxin substrate; Rap-1, Ras-proximate-1; RAPL, regulator of adhesion and cell polarization enriched in lymphoid tissues; RIAM, RAP-1-GTP-interacting adaptor molecule; TEM, transendothelial migration; VLA-4, Very late activation antigen-4 0014-4827/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2010.12.007

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Chemokine triggered collapse of leukocyte microvilli: a checkpoint of lateral motility . . . . . Triggering of ventral invasive filopodia by chemokine and integrin cues during lymphocyte TEM . Importance of chemokine presentation for leukocyte activation at distinct vascular synapses . . Conclusions and open questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction

Integrin affinity regulation by talin underlying chemokine triggered leukocyte arrest

Firm adhesion of leukocytes to blood vessels is tightly regulated by integrins and their cognate endothelial ligands [1,2]. These include the α4 integrins, VLA-4 (α4β1) and α4β7, as well as the β2 integrins, LFA-1 (αLβ2) and Mac-1 (αMβ2). Integrins comprise the major adhesion receptor family for both extracellular matrix proteins as well as cell surface ligands. These heterodimers are composed of large extracellular domains short TM domain and usually short cytoplasmic tails. As many other integrin heterodimers, the leukocyte integrins are maintained in inactive states and can undergo rapid and extensive conformational changes over a time period of fractions of seconds [3–5]. Accumulated data suggest that these counter-receptors are optimally activated by blood-derived shear forces or by tensile forces, driven by appropriate cytoskeletal attachments of integrins [6–8]. The most robust signals for leukocyte integrins are transduced by chemokines displayed by glycosamine glycan (GAG) constituents on blood vessel endothelial cells [9,10]. A growing body of evidence suggests that specific leukocyte Gi-protein coupled receptors for these endothelial chemokines, mainly of the Gi2 subtype [11], elicit diverse signaling pathways which coregulate both integrin-dependent leukocyte adhesiveness and actomyosin dependent motility over and across specific endothelial barriers [2]. Accumulating data highlight the key roles of two cytoskeletal adaptors crucial for the co-activation of all major leukocyte integrins: talin1 and Kindlin-3[12]. These two coactivators can be regarded as ligands for the integrin cytoplasmic domains, since upon appropriate transmission of an inside out signal, they bind, respectively, to membrane proximal and distal NPXY motifs on the integrin beta subunit tails. This co-occupancy is believed to be necessary for driving maximal stabilization of the integrin heterodimer as it is being occupied by its extracellular ligand. The chemokine signals that regulate the function of these key adaptors are highly diversified, and vary considerably between cell types and types of integrins. A subset of these signals must also modulate the actin cytoskeleton in the vicinity of functional integrins to allow the initially arrested leukocyte to spread and crawl in search for transendothelial crossing (diapedesis) cues. This review will focus on the functions and mode of activities of key chemokine regulated effectors in integrin-mediated adhesions of T lymphocytes as a paradigm for other leukocytes. We will not cover chemokine functions in conferring leukocyte polarity and motility underlying interstitial leukocyte motility, processes recently shown to involve various integrin-independent Gi-protein-dependent actomyosin mediated mechanisms. These mechanisms are discussed by excellent reviews by Friedl [13], Lammermann [8], and Thelen [14].

Accumulating structural and biophysical studies (reviewed by Luo et al. [5]) suggest that leukocyte integrins can alternate between inactive bent conformers, which are clasped heterodimers, and unclasped heterodimers with extended ectodomains exhibiting variable intermediate or high affinity to extracellular ligands. Most leukocyte integrins are maintained in an inactive resting state, whereas in situ chemokine-stimulated integrins unfold and can extend up to 10–25 nm above the cell surface [3,4]. In order to arrest rolling leukocytes on blood vessel walls, these extended integrins must undergo additional allosteric rearrangements of their headpiece domains following the occupancy of these domains by endothelial-displayed ligands [5,15]. Although these canonical switches are very short-lived, the simultaneous bi-directional occupancy of the integrin by both its extracellular ligand and by one or more cytoplasmic partners results in high affinity integrin binding to its ligand [16,17]. Full integrin activation is thought to occur when ligand-driven rearrangements of the integrin headpiece are coupled to force transduction via the integrin heterodimer [6,18,19] and its individual cytoplasmic tails [17,20]. Ligand occupancy indeed anchors integrins to the cortical cytoskeleton [21], and so proper integrin anchorage to the actin cytoskeleon [22] may be critical for optimal force transduction and full stabilization of integrin-ligand bonds. How chemokine signals recruit and temporally activate specific integrin-actin adaptors to the vicinity of their integrin targets and thereby trigger integrin conformational activation and force transduction are therefore major open questions in the field of chemokine regulated leukocyte adhesion. A key integrin adaptor essential for chemokine regulated integrin activation is talin. Talin directly induces integrin extension and headpiece activation in many cell types, including leukocytes [23]. Structural studies show that the talin head FERM domain (F for band 4.1 protein, ezrin, radixin, and moesin adaptors [24]) binds to a membrane proximal helix and to an NPxY motif shared by all major β integrin subunit cytoplasmic domains. This interaction unclasps the integrin heterodimer and allosterically increases the affinity of the integrin extracellular domain for ligand [25,26]. Talin may also anchor ligand-occupied integrins to the cortical actin via its numerous actin binding sites, and thereby facilitate force transduction and full stabilization of integrin-ligand bonds [6,17]. Knockdown of talin1, the predominant talin member in leukocytes and platelets, results in nearly total loss of integrin activation by all major physiological integrin agonists [27,28]. Furthermore, partial talin1 suppression is sufficient to abrogate the earliest chemokine triggered LFA-1 and VLA-4 mediated lymphocyte arrest events on endothelial ligands [3,29,30]. To activate its target integrins, talin must bind the integrin β subunit tail, and this association can be locally increased by the plasma membrane signaling lipid, PI(4,5)P2 (PIP2) [31]. This

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phosphoinositide presumably binds the talin head, enhances its binding to the β integrin tail, and drives an unclasping of the integrin heterodimer [32,33]. Mere talin association may be insufficient to unclasp and anchor the integrin heterodimer to the actin cytoskeleton, and therefore the β subunit tail may need to become co-occupied by a second co-activator of the Kindlin family [34,35]. In murine leukocytes, chemokine triggered adhesiveness of both β1 and β2 integrins have been shown to require both talin1 and the hematopoietic specific Kindlin, Kindlin-3 [35]. Mutations introducing a stop codon in Kindlin-3 underlie a rare human leukocyte and platelet integrin-dependent adhesion deficiency syndrome, called LAD-III [36,37], which is associated with severe bleeding defects and impaired integrin-dependent adhesion of leukocytes to inflamed endothelia [38].

Roles of chemokine-stimulated Rho GTPases during the earliest integrin activation events The mechanisms by which talin and Kindlin-3 translate signals transduced by chemokine triggered Gi-proteins and PIP2 have

only recently begun to unfold [39] (Fig. 1). Apart from activating talin-integrin associations, PIP2 activates additional focal adhesion proteins, potentially important for leukocyte integrin adhesiveness, recruits many regulators of actin polymerization including the Cdc42 GTPase and maintains membrane integrity [40–42]. Thus, de novo generation of PIP2 following chemokine triggering of PIP2 generating enzymes and its hydrolysis by leukocyte PLCs, can serve as sequential checkpoints in initial arrest and subsequent integrin-mediated adhesion strengthening of leukocytes adhered to target endothelial sites. Recent data suggest that rapid activation of lymphocyte LFA-1 by talin1 involves chemokine triggered Gi stimulated RhoA and Rac1 GTPases, phospholipase D (PLD) and the PIP2 generating enzyme, PIP5Kγ (PIPK1γ90). This PIPK avidly binds talin and is thought to be recruited by a talin homodimer to the vicinity of its target integrin where, by locally raising PIP2 levels, it relieves autoinhibitory interactions between the talin head and rod domain [26], and thereby drives talin head association with target integrins [39] (Fig. 1). Apart from inducing PIP2, chemokine activated Rho and its downstream mDIA1 [43] may also generate new actin filaments near talin to optimize the buildup of tension

Fig. 1 – Sequential activities of chemokine signals in integrin-mediated arrest, adhesion strengthening, spreading and crawling to sites of diapedesis. The diagram depicts postulated signaling modules of the LFA-1 integrin in adherent and crawling T lymphocytes. Other integrins may use different pathways which partially overlap with these modules. Steps 1A–C. Talin activation close to the target integrin is a rate limiting step in integrin activation. This event requires a second integrin tail binding adaptor, Kindlin-3. Talin activation is triggered by PIP2 locally generated by talin-associated PIP5Kγ stimulated by a nearby Gi-coupled chemokine receptor. Bi-directional integrin activation involves a force-facilitated co-occupancy of the integrin heterodimer by extracellular and cytoplasmic ligands. These steps may be sensitive to both the strength of the chemokine signal as well as the distance between the signaling GPCR and the target integrin. F, force. Step 2. Integrin microclustering is driven by proximal ligand-occupied chemokine-stimulated integrins within focal dots. The focal dots may serve as nucleating assemblies for the GTPases listed in steps 3 and 4. Steps 3 and 4. Microvillar collapse and lammelipodia formation promote lymphocyte crawling. Vertical protrusions (invasive filopodia) triggered by chemokine activated Cdc42 and possibly RhoA are each surrounded by rings of activated integrins. A list of potential lymphocyte GEFs and their main GTPase targets thought to coordinate these various chemokine triggered actin remodeling processes is shown. Both shared and unique GEFs are likely involved in parallel processes orchestrated by other types of leukocytes. For simplicity, the functions of chemokine regulated PKCs and PI3Ks are not shown.

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by the integrin-ligand complex necessary for full integrin activation. Another mode of chemokine triggered talin activation could involve talin rod phosphorylation [26]. Interestingly, although both talin and PIP5Kγ also co-regulate chemokine stimulation of the VLA-4 integrin, PLD is not involved in chemokine triggered VLA-4 activation [30]. Instead of triggering PLD, chemokine signals seem to activate lymphocyte VLA-4 by activating the Zap-70 tyrosine kinase, which rapidly phosphorylates the Rho GTPase guanine exchange factor (GEF), VAV1. Rather than activating its target GTPases, RhoA and Rac1, the phosphorylated VAV1 appears to activate a nearby VLA-4 by releasing a pool of active talin1 which has been sequestered by its pre-phosphorylated VAV1 isoform [30]. At the same time, the chemokine activated phosphorylated VAV1 can initiate a Rac activation program for lymphocyte spreading and motility. Additional talin1 molecules may be co-recruited to the vicinity of adjacent integrin molecules by the Rap-1 effector, RIAM [44], although the role of this adaptor may be cell-type and chemokine-type specific [45]. Rap-1 is one of the major chemokinestimulated GTPases involved in rapid integrin-mediated activation in both leukocytes and platelets [46–48] (see below).

Chemokine triggered integrin microclustering and macroclustering Lymphocyte spreading and crawling on endothelial cells taking place under shear flow are mediated by scattered microclusters of LFA-1 occupied by ICAM-1, which rapidly form and dissipate underneath the entire lymphocyte body [49]. Chemokine activated VLA-4 molecules also rearrange in scattered microclusters around their endothelial ligand, VCAM-1. In the absence of ligands, however, LFA-1 and VLA-4 are readily mobilized to the leading and trailing edges of chemokine-stimulated lymphocytes, respectively, where they remain in macroclusters [50]. Chemokine activated ICAM-1occupied LFA-1 microclusters are localized, on the other hand, to the contact area of the lymphocyte and the endothelial surface it adheres to. These focal contacts average between 30 and 40 per lymphocyte, are enriched with talin [49] and vinculin (Shulman and Alon, unpublished) and appear instrumental for millipede-like shearresistant crawling of lymphocytes on endothelial surfaces [49]. Thus, ventrally scattered ligand-occupied integrin microclusters, rather than macroclusters, [51] are the main functional units of lymphocyte motility over endothelial barriers. These ventral focal dots are molecularly distinct from the classical stress fiber enriched focal adhesions, which orchestrate the adhesion and motility of adherent mesenchymal cells [52]. Although supported by high affinity bonds, these dots must be disassembled within seconds to allow rapid leukocyte locomotion over and through vascular barriers, while maintaining high leukocyte resistance to detachment by disruptive shear forces [49]. Indeed, T cells with LFA-1 artificially locked into high affinity states, although resistant to detachment, cannot crawl and fail to transmigrate across endothelial barriers [53].

The PLC-Rap-1 axis in chemokine-dependent control of integrin adhesiveness Chemokine-stimulated Rap-1 is thought to play a key role in the rapid and reversible modulation of integrin affinity and

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microclustering underlying chemokine triggered lymphocyte adhesion and crawling. Chemokine triggered PLCs appear to be key upstream regulators of these Rap-1 activities in lymphocytes as well as in other hematopoietic cells, like neutrophils [47]. General blockade of T cell PLC activities indeed interferes with both VLA-4 and LFA-1 activation by chemokines [54] (Sagiv and Alon, unpublished), although formal proof of a role of a particular chemokine-stimulated PLC isoform in this activation is yet to be obtained [55]. Since both Gi-proteins and Gq can trigger various PLC activities, an intriguing possibility is that a subset of chemokine-occupied GPCRs activate Gq heterotrimeric proteins [56] acting along with or downstream of Gi-proteins. PLCs are believed to trigger Rap-1 activities via the Ca2+ and DAG dependent Rap-1 GEF, Cal-DAGGEF-I (CDG-I), an emerging regulator of leukocyte and platelet integrins [57]. Notably, the second main target of PLC activity, DAG dependent PKCs are not involved in either β2 or β1 integrin activation by rapid chemokine signals [3,30,58,59]. CDG-I, on the other hand, is required for rapid integrin activation by leukotriene B4 (LTB4) and platelet activating factor (PAF) in neutrophils [57] as well as for sub-second CXCL2 triggered activation of β2 integrin adhesiveness on murine bone marrow derived leukocytes (Pasvolsky, Alon, unpublished). CDG-I and Rap-1 are also required for optimal chemokine-stimulated conformational activation of LFA-1 in T cells [54]. Notably, CDG-I and Rap-1 play more critical roles in chemokine-stimulated LFA-1 adhesiveness and crawling following arrest on endothelial cells, than in initial LFA-1 mediated arrest [49,54], presumably due to sequential roles of the PIP2 generating GTPases (e.g., Rho and Rac) and of the PIP2 consuming GTPases (i.e. Rap-1) in integrin activation (Fig. 1). Interestingly, chemokine triggering of T cell PLC and CDG-I does not seem to require free cytosolic Ca2+, since chelation of Ca2+ has no effect on LFA-1 activation by chemokines [3,58,59]. In contrast, chemokine triggering of monocyte VLA-4 is blocked by chelation, but the role of CDG-I in this activation pathway has not been determined [60]. In addition to activating CDG-I and Rap-1, chemokine-stimulated PLCs may elevate cytoslic free Ca2+, and activate calmodulin dependent proteases such as calpains. These enzymes are thought to release integrins from actin constraints, increase their mobility and regulate both integrin microclustering and macroclustering [61]. Thus, different types of lymphocytes as well as monocytes and neutrophils may utilize different chemokine-stimulated Ca2+ dependent programs for either triggering proteolytic machineries nearby target integrins or activating the CDG-I-Rap-1 integrin axis [60]. In addition to recruiting and activating talin via its effector RIAM, chemokine activated Rap-1 can also recruit to the vicinity of particular integrins a multifunctional adaptor molecule, RAPL (regulator of adhesion and cell polarization enriched in lymphoid tissues). This Rap-1 effector is involved in chemokine triggered lymphocyte polarity, the transport of intracellular LFA-1 containing vesicles to the leading edge of motile lymphocytes and the generation of high avidity LFA-1 macroclusters critical for a variety of LFA-1-dependent adhesion strengthening processes [48,62,63]. Together with Rac1, RAPL appears to regulate polarized LFA-1 adhesiveness in the leading edge of crawling lymphocytes, although its earlier roles in the turnover of the ventral LFA-1-ICAM-1 focal dots are still unclear.

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The rapid turnover of focal adhesive contacts in crawling lymphocytes It is increasingly evident that crawling lymphocytes and other leukocytes can use a diverse set of chemokine controlled adhesion and de-adhesion machineries to migrate over and through endothelial barriers, while resisting detachment by the shear forces they experience near blood vessel walls. Chemokine regulated GTPases, via a delicate balance of their numerous GEFs and GTPase activating proteins (GAPs), control this adhesion-deadhesion cycling necessary for leukocyte motility and extravasation [64]. The transient nature of chemokine-driven GTP loading by and activation of RhoA, Rac1 and Rap-1 is probably the major factor in the transient nature of integrin affinity regulation by chemokine signals [58,65]. In addition, chemokine activated Cdc42 may also actively downregulate integrin affinity by competition with RhoA and Rac1 activation [39]. The chemokine-stimulated calpains mentioned in the previous section [61] may also regulate the turnover of integrin focal contacts by releasing talin and vinculin from these focal contacts. Calpains may also induce de novo associations of integrin subsets with either positive or negative integrin regulatory adaptors such as α-actinin and filamin A [66,67].

Chemokine triggered integrin macroclustering at the leading edge In addition to their tight regulation by chemokine triggered GTPases, integrin macroclustering may be controlled by chemokine-stimulated lipid and protein kinases also involved in regulation of cell polarity. Integrin macroclustering is thought to promote leukocyte-endothelial associations especially at endothelial compartments that express low density integrin ligands, which fail to support leukocyte adhesions unless numerous integrin molecules can be mobilized into large multivalent patches [58]. Key players in these processes are chemokine-stimulated PI3K and PKCζ, an atypical DAG-independent PKC isoform, both postulated to regulate Rac GTPase activation at the cell front [68,69]. PI3K and PKCζ promote LFA-1 mobility to and macroclustering at the leading edge of chemokine-stimulated T cells and neutrophils [58,70–72]. These events also require a specific RhoA domain [71] whereas microclustering of high affinity LFA-1ICAM-1 bonds within ventral focal dots is PI3K independent and does not require this RhoA domain (Shulman, Pasvolsky, Laudanna and Alon, unpublished). Notably, chemokine-promoted VLA-4 adhesiveness in T cells also does not involve these chemokinestimulated activities [59]. Instead, VLA-4 activation by chemokines is regulated by DAG dependent PKCs [54], possibly at a late stage of adhesion strengthening mediated by VLA-4-VCAM-1 interactions [30,59] (Manevich and Alon, unpublished).

Chemokine triggered collapse of leukocyte microvilli: a checkpoint of lateral motility Concomitantly with their localized action on conformational integrin activation and clustering, chemokine signals trigger potent Rac1-mediated actin remodeling events that lead to the

collapse of leukocyte microvilli [73] and the establishment of an anterior-posterior axis. DOCK2, a hematopoietic GEF of Rac1 and 2 was found to control this microvillar collapse in murine T cells interacting with various chemokine-bearing substrates [74]. DOCK2-mediated microvillar collapse, together with sequential Rac activation events mediated by a second Rac GEF, Tiam1, are critical for lamellipodia formation, for lateral T lymphocyte locomotion on integrin ligands, as well as for integrin-independent chemokine-mediated lymphocyte motility in vitro and in vivo [74–76]. Rac driven microvillar collapse, cell spreading and locomotion appear to be regulated independently of the earlier Rac1-mediated chemokine triggering of integrin affinity and microclustering underlying lymphocyte arrest and adhesion strengthening [39]. Interestingly, although the contact area between the chemokine-stimulated leukocyte and its endothelial target is enlarged by and cell spreading [73], this is not a limiting factor in the ability of leukocytes to develop adhesion strengthening in response to chemokine signals. Nevertheless, murine DOCK2 is involved in integrin activation by chemokines in B lymphocytes [77]. Thus, in T cells, chemokine triggering of Rac-1 and talin1 seems to involve a different set of Rac GEFs than in B cells. Furthermore, transient silencing of DOCK2 in human lymphocytes interferes with chemokine-mediated integrin activation [78], suggesting that DOCK2 and Rac contribution to integrin activation by chemokine signals vary with the leukocyte type and the mode of DOCK2 silencing. Indeed chemokine stimulation of Rac1 and of other Rac family members depend also on a third Rac GEF, Vav1 [79].

Triggering of ventral invasive filopodia by chemokine and integrin cues during lymphocyte TEM Both lymphocytes and neutrophils use invasive filopodia to protrude into endothelial cells during their crossing between and through these cells [49,80–82]. Once reaching paracellular endothelial junctions, lymphocytes and possibly other leukocytes use these protrusions to probe subendothelial chemokines [83,84]. At the same time, leukocyte β2 integrin members continuously guide the leading edge of the transmigrating leukocyte to serially engage cognate ligands at the basolateral endothelial surface [49]. Invasive filopodia are sent out by crawling leukocytes into the apical endothelial membrane independently of their positioning at endothelial junctions, much before the leukocytes begin to transmigrate [85] and may therefore guide both the classical paracellular migration route as well as transcellular and pericellular routes of leukocyte TEM [49,82]. Studies on primary resting lymphocytes have suggested that invasive filopodia are rarely generated underneath crawling lymphocytes in the absence of shear force application or when chemokine signals are limiting. Since under these conditions lymphocyte TEM is rare, the frequency of invasive filopodia tightly correlates with successful TEM [49,81]. Nevertheless, invasive filopodia are never observed on the leading edge of lymphocytes that have already crossed the endothelium. Electron microscopy (EM)-aided immunostaining analysis of invasive filopodia suggests that the scattered focal contacts observed at the lymphocyte contact with the apical endothelial surface are in fact submicron LFA-1 rings that surround invasive

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filopodia at their base [49]. These submicron rings and their corresponding ICAM-1 rings [82] may serve as outside-in signaling moieties as well as nucleating assemblies for subsequent cytoskeletal remolding by chemokine triggered Rho and Rap-1 GTPase activities on both the leukocyte and the endothelial sides [86]. These integrin rings may preferentially form within specialized “endothelial nanoplatforms” enriched with ICAM-1, VCAM-1, and their tetraspanin partners [87]. Initially, these filopodia and their integrin rings function individually, but during later stages of TEM, they seem to organize in larger assemblies described as macro-rings or transmigratory cups [88–90]. Notably, invasive filopodia generated by chemokine-stimulated lymphocytes do not form when lymphocyte Cdc42 is sequestered from the plasma membrane [49]. Lack of the Cdc42 effector, WASP, also perturbs filopodia formation [82]. WASP, together with the Rho effector, mDIA1 [43], may drive the formation of these lymphocyte protrusions via triggering the phosphorylation of ERM actin linker adaptors [49]. WASP is also involved in earlier integrin clustering events [91]. Notably, it is still unclear if Cdc42 and WASP are activated solely by chemokine signals [92] or also by integrin outside-in signaling [93]. It is also unclear which of the numerous Cdc42 GEFs is key for translating chemokine signals to Cdc42 activation and function during leukocyte TEM. A novel DOCK180 related GEF for Cdc42, DOCK11, was found to be expressed predominantly in lymphocytes [94], and is an attractive candidate. In addition, chemokine activated Rap-1 has been postulated to activate Cdc42 [76]. Consistent with the highly specialized role of Cdc42 in the function of invasive filopodia during lymphocyte TEM, DOCK2/ Tiam1 dependent Rac-1 activation by chemokine signals is not required for lymphocyte crossing through endothelial barriers [74,76]. Interestingly, subsequent lateral locomotion of the extravasating lymphocyte re-uses DOCK2 for chemokine triggering of Rac-1 [74]. Thus, distinct sets of GEFs and GTPases regulated by sublumenal endothelial chemokines appear to mediate the terminal steps of leukocyte crossing of vascular barriers and probing of the basement membrane for additional crossing cues [86]. The terminal steps of lymphocyte TEM also require adequate chemokine triggering of RhoA and its serine-threonine kinase effector, ROCK, at the lymphocyte's uropod [95]. This Rho effector could be crucial for myosin-mediated contractility of the uropod and detachment of integrin bonds within this compartment [96] especially during the terminal steps of leukocyte TEM. LFA-1 recycling between the uropod and the leading edge has been also postulated to regulate motility over ICAM-1 bearing surfaces [97], yet the contribution of this process to chemokine-driven crawling and TEM of primary lymphocytes and other leukocytes is still unclear. LFA-1, as well as other leukocyte integrins, can transmit outside-in signals to the different Rho GTPases discussed above, even in the absence of chemokine signals [98]. These costimulatory signals require intact Src activities both in lymphoid and myeloid leukocytes, and seem to involve integrin crosstalk with members of the VAV Rho GEF family [82,99]. Indeed, subsets of invasive filopodia initiated by LFA-1 ligation by endothelial ICAM-1 display podosome-like features and involve Src dependent activation of Cdc42 and WASP [82]. Nevertheless, chemokine signals activate this pathway independently of Src [49,100], highlighting the interesting possibility that the regulation of

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invasive filopodia by chemokine signaling and integrin signaling involves distinct subsets of Rho GEFs [101].

Importance of chemokine presentation for leukocyte activation at distinct vascular synapses Soluble chemokines produced by endothelial and stromal cells and secreted to the circulation are immediately diluted, quenched by erythrocyte intereceptors [102] or proteolytically degraded [103]. However, even when compared in vitro in the absence of these suppressive mechanisms, surface-presented chemokines are far more efficient than their soluble counterparts in activating integrin adhesiveness to both endothelial cells and to isolated endothelial ligands [3]. Soluble chemokines induce leukocyte microvillar collapse and cell spreading, mobilize integrins to macroclusters, induce leukocyte protrusions, and trigger additional promigratory actin remodeling machineries [92]; nevertheless, surface immobilized chemokines may be indispensible for the accurate coupling of talin1, Kindlin-3 and their activating GTPases to the highly dynamic signaling events triggered by endothelial integrin ligands during leukocyte adhesion to and motility on endothelial surfaces under disruptive shear forces [49]. Surface immobilized chemokines may also stabilize associations of their cognate leukocytes GPCRs with lipid rafts [104], preferential sites of integrin microclustering [105] and GTPase activation [106]. Almost all known vascular chemokines bind in vitro to heparin sulfate GAGs presented on various endothelial scaffold proteins [107,108] Heparin sulfates also function in chemokine transcytosis and presentation on endothelial microvilli [109]. Nevertheless, genetic evidence is still missing to support the importance of GAG presentation of endothelial chemokines for the multiple proadhesive and promigratory functions required for leukocyte TEM. Thus, the physiological roles of specific heparan sulfate scaffolds in the presentation and integrin activating functions of vascular chemokines are still obscure [108]. How apical and subluminal pools of chemokines support the high directionality of leukocyte TEM is also unknown. Likewise, the functions of endothelial stored inflammatory chemokines in leukocyte adhesion and TEM remain to be addressed [110].

Conclusions and open questions Chemokine signals are the most efficient endothelial-displayed triggers of leukocyte integrins, the key adhesion molecules that mediate leukocyte motility on and through endothelial barriers. We propose that chemokine regulated focal integrin microclusters and their associated invasive filopodia comprise the quantal units of leukocyte adhesion, crawling and transendothelial migration. We have discussed the central role of these chemokinestimulated integrin assemblies and their dynamic generation at the leukocyte-endothelial interface by chemokine triggered RhoA, Rap1, Cdc42 and Rac1 GTPase activities. Despite of our increasing understanding of the integrin conformational switches facilitated by chemokine signals within these adhesive units, many open questions remain unresolved. A major challenge is to further elucidate how and within what membranal microdomains specific chemokine receptors and their various scaffolds [111,112]

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engage target GTPases and their numerous upstream and downstream effectors during the various integrin activation events discussed above. Another intriguing issue is whether endothelial chemokines and other endothelial chemoattractants indeed exclusively use Gi/o proteins to activate integrins and actin remodeling machineries on responding leukocytes or can subsets of these cytokines also trigger Gq and G12/13 proteins to fine tune the control of leukocyte polarization and motility on and across endothelial barriers. Recent evidence also suggests that Gs proteins may downregulate integrin adhesiveness and induce de-adhesive events in a cAMP dependent manner [113], yet, the identity of the endothelial cytokines which may contribute to these regulatory activities is still unknown. Another open question is how integrin affinity and avidity are downregulated to provide the fast turnover of integrin adhesions involved in leukocyte crawling and TEM. Different integrins in different cellular environments may use distinct modalities to downregulate their adhesive states. These modalities include shutting down of integrin activating chemokine-stimulated GTPases, localized proteolysis of cytoskeletal adaptors initially anchoring ligand-occupied integrins to the cortical cytoskeleton, and dephosphorylation of specific adaptors that facilitate talin associations with integrin tails at their phosphorylated states. It is also unclear how myeloid cells can integrate integrin activating signals from endothelial selectins [114] and immunoreceptors [115] to either prime leukocyte integrins for subsequent chemokine signals or to amplify integrin outside-in signaling [2]. In this regard, it is unclear how and where specific integrin outside-in signals collaborate with chemokine signals during specific stages of leukocyte TEM. Another intriguing question is if the invasive filopodia sent by crossing leukocytes into endothelial junctions and endothelial cell bodies can function as ultra-sensitive sensors of endothelial-associated chemokines. Another standing question is the interplay between endothelial and stromal produced chemokines that guides the leukocyte underneath the endothelial barrier it has just crossed [81], and facilitates leukocyte detachment from the vascular wall and its subsequent penetration through the basement membrane. The molecular variation of these programs in various types of hematopoietic and mesenchymal stem cells as well as in malignantly transformed hematopoietic cells is very large. Future studies are therefore likely to identify novel regulatory molecules as well as additional levels of communication between known integrin and GPCR adaptors and their actin remodeling partners. While some key integrin and actin remodeling GTPases are universally used by many motile cells, their mode of operation in one type of leukocyte cannot be extrapolated to another type of leukocyte nor to other types of extravasating cells circulating in blood vessels. The dissection of these high diversified systems, while challenging, may be clinically beneficial for targeting highly selective anti-migratory therapies to singular subsets of circulating cells.

Acknowledgments We thank Dr. S. Schwarzbaum for the editorial assistance, and Mrs. Channa Vega for the assistance in scheme preparation. R. Alon is Incumbent of The Linda Jacobs Chair in Immune and Stem Cell

Research. R.A. is supported by the Israel Science Foundation, by the German Israeli Foundation and by the FAMRI Foundation, U.S.A.

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