Multiple Pathways Leading from the T-Cell Antigen Receptor to the Actin Cytoskeleton Network ⇑ Barak Reicher, Mira Barda-Saad

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FEBS Letters 584 (2010) 4858–4864

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Review Multiple pathways leading from the T-cell antigen receptor to the actin cytoskeleton network ⇑ Barak Reicher, Mira Barda-Saad

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel article info abstract

Article history: Dynamic rearrangements of the actin cytoskeleton, following T-cell antigen receptor (TCR) engage- Received 26 July 2010 ment, provide the structural matrix and ﬂexibility to enable intracellular signal transduction, cellu- Revised 10 August 2010 lar and subcellular remodeling, and driving effector functions. Recently developed cutting-edge Accepted 1 September 2010 imaging technologies have facilitated the study of TCR signaling and its role in actin-dependent pro- Available online 7 September 2010 cesses. In this review, we describe how TCR signaling cascades induce the activation of actin Edited by Israel Pecht regulatory proteins and the formation of actin networks, and how actin dynamics is important for T-cell homeostasis, activation, migration, and other effector functions. 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ó TCR Actin polymerization Lymphocyte activation Signaling complex WASp

1. Introduction dously [3,4]. Alongside RNAi, animal models and characterization of diseases in which the cytoskeleton regulators are defective, Upon encountering an antigen-presenting cell (APC), T cells rap- our increased understanding of the events following T-cell activa- idly reorient their cellular organelles to the contact area, which is tion was made possible largely by the use of new advanced micros- termed the immunological synapse (IS), where they undergo com- copy techniques; these techniques enabled the spatial and plex structural and cytoskeletal changes. In order to carry out their temporal resolution of signaling molecules, implicated in cytoskel- effector functions, T cells must interact with peptide major histo- etal network dynamics and regulation during T-cell activation. In compatability complex (pMHC) on an APC or a target cell. The pri- this review, we will present the diverse signaling complexes form- mary antigenic stimulation ultimately results in cell cycle ing within the IS and the mechanisms by which these complexes progression and clonal expansion. T cells leave the lymphoid tissue govern the formation of cytoskeletal structures that are critical and search the periphery for infected or transformed cells carrying for T cell functions. their cognate antigen. In order to interact with their target cells, T cells circulate between lymphatic vessels, blood and tissues. Dur- 2. T-Cell antigen receptor (TCR) signaling and actin cytoskeleton ing circulation, T cells undergo marked changes in size and shape. These changes enable their binding to and extravasation through When encountering an APC or a target cell, TCR ligation with the endothelium into the surrounding tissue. Once the T cell de- the cognate pMHC results in the activation of the T-cell-speciﬁc tects its target cell, it again reorients its cellular machinery toward Fyn and Lck Src-kinases, which in turn phosphorylate immunore- the contact zone; then, a co-stimulation event triggers the polar- ceptor tyrosine-based activation motifs (ITAMs) within the TCR- ized secretion of cytolytic granules and/or cytokines, mediating associated CD3-chains. Phosphorylated ITAMs serve as docking the T cell’s effector function [1,2]. Over the past several years, sites for the Syk-kinase zeta chain-associated protein of 70 kDa our understanding of the molecular mechanisms regulating dy- (ZAP70). Activated ZAP70 phosphorylates many members of the namic actin cytoskeletal rearrangements has expanded tremen- membrane-proximal activation complex, including the linker for the activation of T cells (LAT). As a transmembrane adapter protein, LAT provides a link between upstream signaling of Lck/ZAP70 to ⇑ Corresponding author. Address: The Mina and Everard Goodman Faculty of Life downstream signaling events including calcium ﬂux, phosphati- Sciences, Bar-Ilan University, Bldg. 204, Rm. 210, Ramat-Gan 52900, Israel. Fax: +972 3 7384058. dylinositol turnover and Ras activation. The adapter protein E-mail address: [email protected] (M. Barda-Saad). Grb2-like adapter downstream of Shc (Gads) binds phosphorylated

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.09.002 B. Reicher, M. Barda-Saad / FEBS Letters 584 (2010) 4858–4864 4859

LAT and recruits the scaffold protein SLP-76 to the activation com- tein (EYFP), we studied the dynamics of multiple proteins includ- plex. Upon phosphorylation by ZAP70, SLP-76 binds the adaptor ing Nck upon TCR engagement (Fig. 2). We learned that the protein, Nck, and the guanine nucleotide exchange factor (GEF), initial contact was generated by lamellipodia formation. VAV. While Nck recruits the actin nucleation factor Wiskott–Al- Recently a direct association between Nck and VAV was identi- drich Syndrome Protein (WASp) to the TCR site, VAV in turn acti- fied and this complex was shown to regulate actin polymerization, vates the Rho family GTPases, Cdc42 and Rac1. Activated Cdc42 in the absence of SLP-76 [7]. and Rac1 facilitate the regulation of actin filament formation Actin polymerization is initiated by a process termed nucleation through the nucleation promoting factors (NPFs) such as actin-re- [8]. There are currently three recognized classes of actin nucleation lated proteins 2/3 (Arp2/3) and WASp family members (Fig. 1) factors that can induce the polymerization of actin filaments: the [5,6]. Indeed by using time-lapse imaging of T cells expressing a Arp2/3 complex which is activated by WASp family members, chimera of signaling protein and enhanced yellow fluorescent pro- the formins, and the spire proteins [8].

Fig. 1. The molecular signaling cascades that link TCR to cytoskeleton remodeling. T-cell activation is initiated by APCs containing stimulatory MHC-peptide complexes. Phosphorylation of the TCR is mediated by the Src family protein tyrosine kinases. ZAP-70 is recruited to the phosphorylated ITAMs and is being activated. LAT and other signaling proteins are tyrosine phosphorylated by ZAP-70. LAT contains nine tyrosine residues which act as docking sites for adapter proteins such as Grb2 and Gads. SLP-76 is recruited by the LAT-nucleated complex and serves as a docking site for other signaling molecules, including Nck, VAV, ITK and ADAP. Nck binds WASp which is involved in actin-dependent processes such as lamellipodia formation and endocytosis. Independent interactions of WIP with the TCR signaling complex occur through binding to ZAP-70 and CrkL. WIP and WASp function coordinately with Arp2/3 complex and induce actin polymerization. WIP stabilizes WASp and is also likely to be involved in WASp- independent actin polymerization cascade. WAVE2 exists in a pentameric hetero-complex with other proteins including Abi1/2, HEM1 (NAP1), PIR121 (SRA1) and HSPC300. The WAVE complex components are interdependent, stabilizing one another against degradation. The recruitment of WAVE2 to the IS is poorly understood, to date. Recent evidence indicates that WAVE2 complex regulates TCR-stimulated integrin activation and involves Ca2+ entrance through the CRAC channel. Phosphorylated HS1 stabilizes newly generated actin ﬁlaments while Formins play a role in MTOC polarization and T-cell cytolytic function. 4860 B. Reicher, M. Barda-Saad / FEBS Letters 584 (2010) 4858–4864

Fig. 2. The use of molecular imaging to follow the dynamics of Nck by confocal microscopy. Live Jurkat T cells expressing YFP-Nck were plated over coverslips and observed dynamically using spinning wheel confocal microscopy. Cells were dropped over a stimulatory coverslip coated with monoclonal anti-CD3. Four-image, 0.5 lm deep Z-stacks were collected every 10 s.

The central actin regulator Arp2/3 requires for its activity inter- spreading, chemokine-induced migration, T reg formation and actions with two proteins expressed in immune cells and were the induction of autoimmune diseases such as colitis. found to be important for cytoskeletal reorganization, WASp and WASp and N-WASp differ structurally from the other members the WASp family-verprolin homologous protein 2 (WAVE2). of the family in their N-terminal WH1 (EVH1) domain, followed by the unique elements, a basic region and a GTPase-binding domain 3. Requirement of actin nucleation factors for T-cell effector (GBD). In resting cells, WASp is mainly present in an autoinhibited functions form induced by the interaction of its VCA domain with a hydrophobic patch in the GBD, thereby masking the binding sites for 3.1. The WASp family of proteins the Arp2/3 complex. The Rho family GTPase, Cdc42, activated by the Rho GTPase exchange factor (GEF), VAV, binds to the WASp One of the diseases in which the cytoskeletal regulation is GBD site. This binding, together with phosphorylation of WASp defective is the Wiskott–Aldrich syndrome (WAS). WAS is an X- on the Y291 site by protein tyrosine kinase (PTK), induces a dra- linked recessive disease characterized by immunodeficiency, matic conformational change. The hydrophobic core is disrupted, thrombocytopenia and eczema. Untreated, most patients die by releasing the VCA domain, enabling its interaction with the Arp2/ 10 years of age due to recurrent infections, bleeding or autoim- 3 complex, thereby promoting actin polymerization. mune disease. Genetic deficiency of WASp was identified as the TCR ligation leads to the activation of protein tyrosine kinases causative defect in WAS. Hematopoietic cells in which WASp is including the Src-kinase Fyn, IL2-inducible T Cell Kinase (ITK), or mutated and/or absent show abnormally smooth cell membranes, Hck, and the subsequent activation of the Rho family GTPase, decreased proliferation, reduced spreading and diminished actin Cdc42. Interaction of GTP-bound Cdc42 with the WASp GBD is polymerization [9,10]. WASp is expressed by all hematopoietic cell thought to release the C terminus from auto-inhibition, allowing lineages and precursor cells including immune cells T, B and natu- binding by the Arp2/3 complex and actin nucleation [10]. However, ral killer (NK) lymphocytes. In addition to WASp, the WASp family data from early studies indicated that WASp can also be activated of proteins consists of three additional proteins: neuronal (N)- independently of Cdc42 [12,13] suggesting additional mechanisms WASp, WAVE 1–3 and the recently discovered Wiskott–Aldrich for allosteric release from the autoinhibitory state. These suggested syndrome protein and SCAR homologue (WASH) [11]. These addi- mechanisms of WASp activation involve Nck and phosphatidylino- tional proteins are widely expressed in almost all tissues. The sitol 4,5 bisphosphate (PI(4,5)P(2)), as well as the dimerization of WASp family of proteins shares a similar structure, a conserved WASp [14]. Phosphorylation of WASp on Y291 (the homologue C-terminal verprolin homology/cofilin homology/acidic region of murine Y293) by PTK in addition to enhancing the sensitivity (VCA) domain enabling each protein to associate with actin mono- of WASp to the small G-protein, Cdc42, was shown to be an essen- mers and to bind and induce actin-nucleating activity of the Arp2/3 tial physiological regulator of WASp activity in T cells [15]. WASp- complex and a proline-rich domain (PRD), which facilitates inter- deficient mice expressing a phospho-abolishing WASp mutant actions with several Src-homology 3 (SH3) domain-containing pro- (Y293F) develop many of the same immune defects and abnormal- teins. Interaction with these proteins is pivotal for NPF activation, ities as occurring in WASp-deficient mice and exhibit marked regulation and trafficking [9]. Although the members of the WASp reduction of actin polymerization, in vivo and in vitro, further family share the same VCA region that implicates them in the demonstrating the importance of Y293/Y291 phosphorylation for Arp2/3-dependent branched actin nucleation activity, they differ normal WASp function. In the same model, mice expressing a in their N-terminus. Each NPF’s distinct N-terminus defines its phospho-mimicking WASp mutant (Y293E) exhibit increased actin localization, regulation and interactions with other proteins, giving polymerization; however, in these mice, WASp is unstable, under- the protein its unique function, as will be discussed later. going proteosomal-dependent degradation [16]. Other studies show that phosphorylation of WASp is unnecessary for its activa- 3.2. Structure, activation and function of WASp and N-WASp tion, but works cooperatively with Cdc42 to bring active WASp to sites required for F-actin-based structures, such as podosomes, WASp is a 502 a.a cytosolic protein, expressed exclusively in membrane structures enabling cell protrusion and invasion [17]. hematopoietic cells and, similar to the other WASp family proteins, does not exhibit intrinsic catalytic activity, but functions as a scaf- 3.3. WASp WH1 domain and the WASp-interacting protein (WIP) fold, directing Arp2/3-dependent actin nucleation. WASp was suggested to be involved in multiple cell functions In immune cells, most of the WASp is in a constitutive complex including the formation of membrane structures e.g., lamelliopodia with WIP, which wraps its C-terminal domain around the WASp and fillopodia, cell migration, phagocytosis, and proliferation. In- WH1 domain. The vast majority of missense mutations in WAS deed, TCR signaling, actin reorganization, TCR internalization, and are located in the WH1 domain of WASp, demonstrating the migration were found defective in T cells from WAS patients and importance of WIP–WASp interaction to the function of immune in waspÀ/À mice. cells. Studies show that WIP has four major cellular functions in WASp initially appeared to be dispensable for T-cell develop- regulating actin dynamics: As a chaperone of WASp, WIP is essen- ment; however, the identification of the more generally expressed tial for WASp stability [16,18,19], regulates WASp activation, and homologue, N-WASp revealed that WASp and N-WASp share recruits WASp to areas of active actin assembly [18] following redundant roles in thymocyte development, receptor-mediated TCR activation. WIP also participates in the reorganization of the B. Reicher, M. Barda-Saad / FEBS Letters 584 (2010) 4858–4864 4861 actin-based cytoskeleton and stabilizes actin filaments in a WASp- WASH is a part of a protein complex. This 550 kDa complex con- independent manner. However, the independent role of WIP in tains 4 uncharacterized proteins: KIAA1033, KIAA0196, KIAA0592 actin polymerization and its involvement in cellular motility of im- and ‘coiled-coil domain containing 53’, along with WASH and the mune cells remain mostly unknown to date. 2 subunits of the capping protein (CP) – CP a and CP b. Similar to the WAVE2 complex, WASH complex members are interdependent 3.4. WAVE – a central regulator of actin polymerization and maintain WASH stability [28], suggesting a similar mode of regulation. Studies in Drosophila revealed WASH’s ability to crosslink/ Although WASp was established as essential for the activation bundle linear F-actin as an effector of Rho1. Interestingly, binding of of multiple immune cell types in response to receptor ligation the Arp2/3 complex was found to inhibit WASH-dependent linear i.e., T and NK lymphocytes, some data demonstrate that T cells actin bundling, promoting branched actin nucleation, suggesting from WASp-deficient mice are still able to form conjugates and Arp2/3 as a molecular switch between WASH-dependent linear polymerize actin at the IS under certain experimental conditions. and branched actin polymerization. Unlike WASp, Drosophila This suggested the presence of additional actin-regulating mole- WASH does not appear to be autoinhibited, but either constitutively cules in T cells. One candidate, the Rac1 effector molecule, WAVE2, active or trans-inhibited [29]. Imaging studies on T cells showed was identified as a central regulator of actin polymerization in T that WASH exhibits a distinct punctuate localization and unlike cells [20]. WAVE2 is one of the three WAVE isoforms and is pre- WASp and WAVE2, which are generally localized to the membrane dominantly expressed in hematopoietic cells. WAVE1 and 3 are ex- cortex, WASH puncta appeared only to cluster centrally upon TCR- pressed mainly in central nervous system (CNS) cells. Unlike induced spreading. Although the molecular mechanisms regulating WASp, which exists in an autoinhibited conformation that is re- WASH and its activation and recruitment to sites of actin polymer- lieved by GTP-Cdc42 binding, WAVE2 is not autoinhibited and is ization are still unknown, it appears to play an important role in regulated by Rac1-mediated recruitment to areas of cellular activa- endosomal sorting and exhibit unique abilities to polymerize actin tion [1]. WAVE2 exists in a pentameric hetero-complex with other and bind microtubules. This potential ability of WASH to interact proteins including Abi1 and 2, HEM1 (NAP1), PIR121 (SRA1) and with both actin and microtubules and its involvement in the endo- HSPC300. The WAVE complex components are interdependent, somal sorting pathway make it a promising subject of further stabilizing one another against degradation [20]. The link between investigations. TCR-mediated Rac1 activation and WAVE2 complex recruitment to the membrane is yet to be determined. However, in macrophages 3.6. HS1 serves as an actin stabilization factor and non-hematopoietic cell lines, two possible links have been established: the adaptor protein IRSp53, which binds both Rac1 Another Arp2/3-related NPF in T cells is the hematopoietic-spe- and WAVE2, and a direct interaction between NAP1, SRA1 and cific cortactin homologue (HS1). TCR ligation results in the IL-2- Rac1 - both lead to the recruitment of the complex to the mem- inducible, ITK-dependent tyrosine phosphorylation of HS1. This brane and subsequent F-actin polymerization [21]. Suppression phosphorylation recruits HS1 to the membrane and provides dock- of Abi1/2 or HEM-1 also results in actin defects, in agreement with ing sites for several SH2 domain-containing proteins, including WAVE2 depletion [22]. Abi1/2 also mediates an interaction be- PLCc1 and VAV1 [30]. In this manner, HS1 stabilizes VAV1 at areas tween Abl kinase and WAVE2, possibly resulting in phosphoryla- of F-actin polymerization at the IS. Once recruited to the IS, HS1 tion at Y150, which increases the actin polymerization activity of has the capacity to stimulate Arp2/3-induced actin assembly as WAVE2. On the other hand, suppression of c-Abl markedly impairs well as to stabilize filament branch-points [1]. Consistent with this WAVE2 recruitment to the T cell–APC contact zone, further high- notion, HS1-depleted Jurkat T cells exhibit defective TCR-stimu- lighting the importance of WAVE2 phosphorylation [23]. Most related F-actin structures and decreased stability of F-actin at the cent studies suggest that the WAVE complex is constitutively IS in vivo. Additionally, the same F-actin structural defects were inactive and requires simultaneous interactions with prenylated observed in T cells from hs1À/À mice. HS1 deficiency also results Rac-GTP and acidic phospholipids, as well as a specific state of in diminished TCR-induced proliferation and calcium store release phosphorylation to promote full activation; activation is depen- [30,31]. The ability of HS1 to induce the formation and to stabilize dent upon an allosteric change in the complex, rather than simple F-actin at the IS, along with its adaptor properties, can facilitate recruitment or dissociation of the subunits [24]. However, eluci- coordination of actin polymerization. Thus, for example, HS1 dating the exact mechanism by which WAVE2 is activated will re- recruitment to the IS can activate WASp and WAVE2 through the quire further study. The use of advanced microscopy methods such stabilization of VAV1, leading to the activation of Cdc42 and Rac1 as fluorescence resonance energy transfer (FRET) to individually GTPases. probe each of the complex members and to follow their dynamics and interactions in vivo may make this challenge achievable. 4. The Arp2/3 complex – the motive force of branched actin 3.5. WASH – a novel molecular link between the actin cytoskeleton polymerization and endosomal sorting The best characterized and first actin nucleator to be identified The most recently identified WASp family NPF is the WASp and is Arp2/3, a 220 kDa complex of seven stably associated polypep- SCAR homologue (WASH). WASH is widely expressed in mamma- tides. The complex includes a core of Arp2 and 3, plus five addi- lian tissues and cell lines both in the membranes and in the cyto- tional subunits, ArpC1–ArpC5. Arp2/3 nucleation activity solic fractions. Like the other WASp family members, WASH produces branched F-actin networks by initiating new actin fila- facilitates Arp2/3-dependent branched actin nucleation through ments on the sides of preexisting filaments. These dendritic actin its VCA domains. The N-terminal WASH homology domain 1 networks, created by Arp2/3, commonly form the structure of (WAHD1) facilitates WASH cellular distribution and its interaction lamellipodium. Arp2 and Arp3 themselves bear structural similar- with other proteins, while the tubulin-binding region (TBR) enables ity to an actin dimer and act as a template for the formation of the its association with microtubules in a Rho GTPase-regulated man- branch-point of F-actin. Alone, the Arp2/3 complex is an inefficient ner. Mammalian WASH localizes to early and recycling endosomes, nucleator, requiring the binding of actin filaments to increase its where WASH-mediated Arp2/3 activity regulates retromer-depen- activity and couple it with nucleation and branching. The engage- dent endosomal sorting [25–27]. Like the other WASp family NPFs, ment of this complex by NPFs, i.e., WASp or WAVE2, further 4862 B. Reicher, M. Barda-Saad / FEBS Letters 584 (2010) 4858–4864 enhances Arp2/3 nucleation activity by the binding of the C-termi- the pSMAC and then to the cSMAC, where these receptors are tar- nal domains of WASp to the Arp2/3 complex, leading to conforma- geted for degradation [36]. This coordinated movement of TCRs tional changes of the latter and delivery of actin monomers to the and their recycling may provide the physical basis for controlled barbed ends of Arp2/3 in the primed complex [8]. Studies that em- T cell function by IS stabilization, signaling sustention and eventu- ployed both scanning electron microscopy (SEM) and fluorescence ally signaling termination. microscopy demonstrated that in T cells, Arp2/3 depletion leads to the formation of filopodial protrusions rather than lamellipodia, 6.2. The IS and actin cytoskeleton following TCR ligation or APC encounter [32]. Arp2/3 depletion also results in decreased T cell-APC conjugate formation and dimin- Prior to IS formation, actin polymerization at the leading edge ished integrin activation and TCR internalization [32]. These results and cytoskeletal contraction at the lymphocyte uropod mediate ra- further underscore the importance of the Arp2/3 complex for T cell pid migration during scanning. Upon TCR engagement with a effector functions. In addition, the formation of filopodial actin- pMHC, T-cell mobility is arrested and radial actin polymerization based structures suggests the existence of Arp2/3-independent ac- creates a lamellipodia over the APC. This is coupled with move- tin nucleators. Such alternative factors including mammalian ment of F-actin toward the center of the IS, forming the cSMAC. Diaphanous-1 (mDia1) have been implicated in similar mecha- The formation of the IS and its stabilization by F-actin filaments al- nisms in T cells as well as in neutrophils, including regulation of low T cell to scan the APC surface for antigen as well as to generate cell motility, chemotaxis, polarization and interactions with WASp the mechanical force required to trigger the TCR [35]. The forma- [33]. tion and movement of TCR-signaling complexes during T-cell activation are dependent on F-actin dynamics. The involvement of 5. Formins promote linear actin polymerization and MTOC additional actin regulatory proteins, such as WAVE2, formins, recruitment HS1 and PKC-h in IS dynamics [20,30–32,37], strongly suggests a complex and orchestrated cytoskeletal mechanism, involving sev- Unlike Arp2/3, all other known actin nucleators produce un- eral signaling pathways for IS modulation. However, although it branched filaments. The best characterized of these are the for- was demonstrated that the formation, movement and stability of mins, which represent a large family of proteins that are TCR signaling complexes are dependent on F-actin, the direct link activated by Rho GTPases to nucleate actin filaments in an Arp2/ between F-actin and TCR signaling clusters is under intensive 3-independent manner [26]. Two formins are known to be ex- investigation. Aspects related to the roles of actin machinery in pressed in T cells, Formin-like-1 (FMNL1) and mDia1 [32]. mDia1 the segregation of the IS domains and the fate of signaling assem- is distinguished by the presence of an FH2 (formin homology 2) do- blies are poorly understood. Also, the correlation between actin main, which binds the barbed ends of actin filaments and promotes regulatory proteins to specific actin structures and their biological elongation of actin filaments. Similar to WASp family proteins, in outcome, as well as their potential overlapping, are just starting to naive cells, mDia1 is rendered inactive by autoinhibitory intramo- be explored. lecular interactions and activated by the association to Rho GTPas- es [2]. In addition to their actin polymerization activity, formins 6.3. Endocytosis regulate MTOC polarization in T cells in a Rac1-dependent manner [32]. The use of mDia1-deficient mice demonstrated a critical role TCR engagement with a pMHC and the subsequent formation for mDia1 in modulating T cell effector functions. These mice have of the cSMAC lead to downregulation of the TCR [36,38]. TCR fewer lymphocytes and their T cells display defects in adhesion, downregulation can be the consequence of a combination of migration, actin polymerization and impaired proliferation upon events including reduced recycling back to the membrane, in- TCR activation [34]. Elucidating the possible connection of formins creased internalization and degradation. It is well established to other actin polymerization pathways, such as WASp, can shed that the TCR is endocytosed via clatherin-dependent pathways light on the complexity of the regulation and dynamics of the actin [39]. In addition to TCR endocytosis, data from imaging studies cytoskeleton network. show the endocytosis of signaling complexes containing the adaptor molecules upstream to the actin polymerization machinery: LAT, Gads and SLP-76 [40,41]. Therefore, endocytosis might 6. Regulation of the actin cytoskeleton network is critical for T be a possible mechanism for both TCR signaling attenuation and cell effector functions quick redistribution of signaling complexes. As a major regulator of T-cell activation, the actin cytoskeleton is likely to be involved Actin polymerization plays a pivotal role in multiple and crucial in T-cell endocytic pathways. Early studies indicated the involve- aspects of the immune response including the formation of the IS, ment of WASp in endocytosis by showing an impairment of antigen recognition, signal transduction, T-cell proliferation, ligand binding-induced endocytosis of the TCR in WASp-deficient migration, adhesion and invasion through tissues. Once the TCR T cells [42]. Following TCR activation, the endocytic adaptor pro- binds foreign antigens on APCs, multiple proteins redistribute to tein intersectin 2 binds both WASp and Cdc42, thereby inducing form the IS. WASp activation and its translocation to endocytic vesicles [43]. WASp was also found to mediate CD28 co-receptor internaliza- 6.1. Formation and stabilization of the IS tion in an activation-dependent manner, by an interaction with sorting nexin 9 (SNX9), which is involved in endocytosis and The IS contains compartmentalized clusters of TCRs, additional vesicular trafficking [44]. These findings suggest a link between membrane receptors, and cytosolic signaling effectors [2]. Data the actin cytoskeleton and the endocytic regulation of T-cell from imaging studies show that the IS is organized into three con- activation. centric supramolecular activation clusters (SMACs): a TCR:pMHC- containing central SMAC (cSMAC), a surrounding peripheral ring 6.4. Integrin activation of adhesion molecules (pSMAC) and a distal rim of CD45-enriched membrane (dSMAC) [2,25,35]. The IS is suggested to modulate TCR Adhesive interactions which are critical for T-cell trafficking and signaling by regulating the trafficking of TCR-signaling clusters ini- antigen recognition are facilitated by integrins, a large family of ab tially formed by TCR:pMHC ligation, from the dSMAC through to heterodimeric cell surface proteins that promote cell to cell B. Reicher, M. Barda-Saad / FEBS Letters 584 (2010) 4858–4864 4863 interactions and interactions of cells with extracellular matrix pro- [2] Zhang, J., Dong, B. and Siminovitch, K.A. (2009) Contributions of Wiskott– teins [45] Key T-cell integrins include the leukocyte function-asso- Aldrich syndrome family cytoskeletal regulatory adapters to immune regulation. Immunol. 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