Cytoskeletal cross-talk in the control of T cell antigen receptor signaling

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1 FEBS Letters 584 (2010) journal homepage: Review Cytoskeletal cross-talk in the control of T cell antigen receptor signaling Rémi Lasserre 1, Andrés Alcover Institut Pasteur, Department of Immunology, Lymphocyte Cell Biology Unit, 28, Rue du Dr Roux, F Paris, France CNRS URA 1961, 28, Rue du Dr Roux, F Paris, France article info abstract Article history: Received 19 July 2010 Revised 19 August 2010 Accepted 1 September 2010 Available online 7 September 2010 Edited by Israel Pecht Keywords: T cell receptor Immunological synapse Actin Microtubules Ezrin Ezrin radixin moesin T cell antigen receptor signaling is triggered and controlled in specialized cellular interfaces formed between T cells and antigen-presenting cells named immunological synapses. Both microtubules and actin cytoskeleton rearrange at the immunological synapse in response to T cell receptor triggering, ensuring in turn the accuracy of intracellular signaling. Recent reports show that the crosstalk between the cortical actin cytoskeleton and microtubule networks is key for structuring the immunological synapse and for controlling T cell receptor signaling. Immunological synapse architecture and the interaction between the signaling machinery and various cytoskeletal elements are therefore crucial for the fine-tuning of T cell signaling. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1. Introduction T cells recognize antigens as molecular fragments (i.e. peptides) associated to major histocompatibility complex (MHC) molecules expressed on the surface of antigen-presenting cells (APC). T cell antigen receptors (TCR) bind peptide-mhc complexes, through their immunoglobulin-like variable domains, triggering T cell activation. The TCR does not contain enzymatic activities, but recruit several protein tyrosine kinases, including the src-family kinase Lck and the syk-family kinase ZAP-70 that perform initial phosphorylation events [1]. Soon after antigen recognition and in response to initial TCR signaling, the T cell polarizes toward the APC, generating an organized cell cell interface called the immunological synapse, which ensures the communication between both cells. Immunological synapses can be formed between helper or cytotoxic T cells and various types of APCs (dendritic cells, B cells or macrophages), or target cells (virus-infected or transformed cells). Two broad functions are assigned to immunological synapses, first to trigger and Abbreviations: APC, antigen-presenting cell; ERM, ezrin radixin moesin; H- DAC6, histone deacetylase 6; MTOC, microtubule-organizing center; MHC, major histocompatibility complex; TCR, T cell antigen receptor Corresponding author. Address: Unité de Biologie Cellulaire des Lymphocytes, Institut Pasteur, 28, rue Docteur Roux, Paris Cedex 15, France. Fax: addresses: remi.lasserre@pasteur.fr (R. Lasserre), andres.alcover@ pasteur.fr (A. Alcover). 1 Fax: control TCR signal transduction; second, to restrict the action of T cell effector functions, like the secretion of cytokines or cytotoxic granules, to interacting APCs or target cells. The structure, function and outcome of each type of immunological synapse may be different leading to T cell proliferation and differentiation, T cell help of B cells, or killing of target cells. A number of cell surface receptors, adhesion molecules, and signaling effectors gather at the immunological synapse ensuring the triggering and the control of T cell activation. Concomitantly, T cell activation induces the reorganization of both microtubules and actin cytoskeleton. Cytoskeleton rearrangements at the synapse are in turn crucial for sustaining and tuning T cell activation and for the achievement of T cell effector functions. The regulation of cytoskeleton reorganization by TCR signaling, and conversely, the control of TCR signaling by the cytoskeleton have been areas of active investigation. However, whether there exists a coordinate action of the actin-based cytoskeleton with microtubules for the control of T cell signaling remained so far poorly elucidated. We will discuss here how interactions between the cortical actin cytoskeleton and microtubule networks are crucial for ensuring the immune synapse architecture and the finetuning of TCR signal transduction. 2. Cytoskeleton reorganization at the immunological synapse The rapid development during the last decade of live cell microscopy imaging, as well as of experimental systems using /$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 4846 R. Lasserre, A. Alcover / FEBS Letters 584 (2010) planar surfaces as surrogate APCs, has allowed a much better spatial and temporal resolution and has provided compelling evidence that the T cell APC contact site was a specialized subcellular area in which TCR signaling was translated into dynamic rearrangements of actin and microtubule cytoskeleton that in turn were required to control TCR signaling Actin cytoskeleton TCR signaling regulates actin dynamics at the immunological synapse [2] involving an array of signaling effectors, such as the signaling adaptors Nck and SLP-76 [3], the guanine exchange factor Vav for the Rho GTPases Rac and Cdc42 [4], and the large GTPase Dynamin2 [5]. Moreover, several actin nucleation-promoting factors are involved, including the Wiskott-Aldrich syndrome protein (WASp), the WASp-interacting protein (WIP), WAVE2, the cortactin homologue HS1, etc. that regulate actin polymerization through the Arp2/3 complex (reviewed in [6]). In turn, the actin cytoskeleton may control TCR signaling at two stages: first, by regulating the steady state diffusion of surface receptors at the plasma membrane, and second, by regulating the activation-dependent dynamics of signaling complexes at the synapse [7,8]. Thus, it was recently shown that the cortical actin cytoskeleton controls, via the membrane-cytoskeleton linker ezrin, B cell receptor dynamics at the plasma membrane. Disrupting the actin cytoskeleton enhanced B cell receptor diffusion and led to a concomitant cell signaling like calcium flux and Erk activation. These data suggest that the cortical actin cytoskeleton may prevent receptor triggering, maintaining immune cells in an inactive state below an activation threshold [9]. It is at present unknown whether the TCR may behave in a similar manner in non-activated cells. In agreement with a negative regulatory role of actin on TCR activation, inhibiting actin polymerization with cytochalasin D led to enhanced Calcium fluxes in T cells stimulated with an anti-cd3 antibody [10,11]. The actin cytoskeleton may also regulate TCR signaling by controlling the dynamics of signaling complexes at the immunological synapse [8]. Indeed, following T cell interaction with an APC, molecular complexes containing TCR and signaling molecules nucleate into microclusters that form at the periphery of the immune synapse and then move centripetally towards central areas in which signal extinction occurs [12] (see Chapter 3). Dynamic actin polymerization control the nucleation and centripetal movement of signaling microclusters [8,13], which sequentially contribute to sustain and extinguish TCR signaling [12,14]. In this line, myosin IIA inhibition impairs signaling microcluster movement and T cell activation [15], indicating an implication of this actin-based motor in immune synapse regulation. Therefore, actin cytoskeleton dynamics are key to set up the immunological synapse and control TCR signaling Microtubule cytoskeleton The microtubule cytoskeleton is reorganized during the formation of the immunological synapse. The T cell microtubule-organizing center (MTOC) reorients and contacts the plasma membrane at the APC contact site [16,17] and microtubule arrays dynamically irradiate from the MTOC to the periphery of the synapse [18,19]. Microtubule network reorganization at the synapse requires TCR signaling and conditions immunological synapse structure and functions. The effectors involved in MTOC reorientation in response to TCR stimulation are not fully defined. Immunoreceptor tyrosine-based activation motifs (ITAM) in TCR subunits, the src family protein tyrosine kinases Lck and Fyn [20,21] and the SLP-76-associated scaffold protein ADAP [22] appear necessary, whereas some controversy exists on the involvement of Cdc42 Rho GTPase [23,24]. In addition, post-translational modifications of tubulin mediated by histone deacetylase 6 (HDAC6) are also involved [25]. Interestingly, the actin nucleating formins, Diaphanous-1 and Formin-like- 1, control MTOC relocalization to the immunological synapse rather than actin dynamics at the synapse [24]. The molecular mechanism involved in microtubule anchoring to the synapse periphery and in microtubule tension facilitating the approach of the MTOC to the contact site is still an open question. An intricate ensemble of protein complexes, together with molecular motors may be necessary. For instance, the PDZ domain-containing scaffold protein Dlg1 and the membrane-microfilament linker ezrin are involved in microtubule network organization and MTOC positioning at the synapse [19]. Moreover, the microtubule-based molecular motor dynein is necessary for MTOC translocation to the synapse [26]. An additional candidate might be IQGAP, an effector of Rac and Cdc42 that links microtubules with the actin cytoskeleton [27], and relocates to the periphery of the immune synapse [17], although a more direct prove for IQGAP involvement in microtubule organization in the synapse is still needed. Finally, microtubule stability at the immunological synapse may be regulated by HDAC6, a cytosolic deacetylase that acts on tubulin [25]. Microtubules are in turn necessary for immunological synapse formation and function. Thus, microtubules drive vesicular traffic that facilitates molecular targeting to the synapse, as shown for TCR subunits [28,29], the tyrosine kinase Lck [30,31], the adaptor LAT [32,33], or the GTPase Rap1 [34]. Altogether may regulate TCR signaling at the synapse and TCR-induced T cell adhesion through integrins. MTOC could also interact via paxillin with other signaling molecules, like the tyrosine kinase PYK-2, positioning them close to the synapse to perform their function [35,36]. Microtubules drive the dynamics of signaling microclusters at the immune synapse, tuning TCR signaling [19]. Impairing HDAC6, or dynein dynactin complexes, inhibited molecular clustering at the synapse and T cell activation [25,26], further indicating a role for microtubules in synapse formation and function. However, these proteins might also perform other functions non-directly related with microtubule organization that could influence T cell activation [37]. Finally, microtubules are key for T cell effector functions, like polarized secretion of helper cytokines (reviewed in [38]). 3. Spatio-temporal organization of the TCR signaling machinery In order to trigger and control receptor signal transduction, signaling effectors need to be coordinated in time and space. This coordination may be different depending on the cell type, the receptor involved and the outcome of the activation process. TCR engagement by antigen-mhc on the APC surface, or by activatory planar surfaces, is rapidly followed by the generation of signaling complexes that nucleate into dynamic microclusters visible at the light microscopy level. Signaling microclusters are composed of TCR subunits and various signaling effectors, including the adaptors SLP-76 and LAT, and the ZAP-70 protein tyrosine kinase. Signaling microclusters nucleate at the periphery of the immunological synapse, in which signaling is initiated and sustained and then move to the center of the synapse in which the activation signal is extinguished [12,39 42]. The centripetal movement of signaling microclusters depends on the integrity of the actin and microtubule cytoskeleton [8,19,39] and appears to condition the intensity and duration of TCR signaling [13,14,19]. Although initially colocalized at the periphery of the synapse, some signaling molecules separate from TCR subunits during microcluster centripetal movement. Thus, whereas the TCR ends up coalescing in the center of the synapse, SLP-76, LAT and

3 R. Lasserre, A. Alcover / FEBS Letters 584 (2010) ZAP-70 microclusters disappear before reaching the center [40]. Whether the association of signaling microclusters with subcellular structures changes along their route from the periphery to the center of the synapse is still unclear. It is possible that initial signaling complexes form first at the plasma membrane and may then join endocytic vesicles that run parallel to the plasma membrane. TCR signaling might continue to take place in endosomal vesicles, as described for other receptors [43]. Dynamic interaction between plasma membrane signaling complexes and intracellular vesicles carrying signaling molecules like LAT seem also to take place and participate in the signaling process [44]. Where molecular separation takes place (i.e., plasma membrane versus endocytic vesicles), and how the sorting between TCR subunits and SLP-76, LAT and ZAP-70 signaling molecules occurs is still an open question. Interestingly, ubiquitin and sorting proteins of the ESCRT complex appear involved in some of those sorting pathways that target signaling complexes to sites of signal extinction [12,41,42,45]. Various non-exclusive mechanisms have been proposed to account for TCR signal downregulation at the immunological synapse. These include dephosphorylation of critical tyrosine residues by the CD45 phosphatase [12]; phosphorylation of serine residues that allow binding of negative regulatory proteins [46], which in turn may favor signaling complex disassembly; finally, sorting the different components of signaling complexes to vesicular compartments from which they might be either recycled back to the plasma membrane [28], or be degraded [12,41,42,45,47,48]. 4. Interplay between the actin and microtubule cytoskeleton in the control of TCR signaling T cells continuously move around in lymphoid organs sensing the environment and integrating signals received from APCs [49]. When a certain threshold of signaling is attained during contacts with APCs displaying cognate antigen, T cells slow down their movement, adhere more strongly to the APC, polarize and form immunological synapses. Dustin proposed that highly dynamic T cell APC interactions, which he named immunological kinapses, might provide stimulatory signals that could be integrated by T cells during their movement. T cell stimulation in kinapses would be different than the more sustained stimulation provided by immunological synapses and would lead to different outcomes (i.e., anergy, different level of cytokine production, proliferation etc.). In lymphoid organs a T cell would pass from a kinapse to a synapse state, and vice versa, depending on the signals provided by APCs (MHC-antigen, chemokines, cytokines, etc.) [50,51]. The signaling molecules that ensure the cytoskeleton interplay in T cells and regulate the transition between migratory T cells and T cells forming synapses are poorly known, but they are likely conserved in other cells types and among various cellular processes. In T cells forming kinapses, which behave as migrating cells, the coordinate action of actin and microtubule dynamics likely ensure the membrane expansion and contraction that occur at the front and the rear of the cell, as described for other cells [52]. Then, a stronger TCR signaling would provide a stop signal, driven in part by enhanced integrin adhesion, and the transition from kinapse to synapse. This transition, is regulated by a balance between the actin regulator WASp and the protein kinase C (PKC)h [53], and needs to modulate the activity of the actin-based motor myosin IIA [54]. PKCbI was also shown to regulate microtubule network organization and migratory T cell shape [55], and could cooperate with PKCh to balance TCR and integrin signaling that modulate T cell locomotion and T cell dynamics during APC interactions. During kinapse to synapse transition T cells adopt more symmetrical shapes [53] with radial arrays of microtubules [19] and peripheral actin dynamics [13]. This likely involves a multidirectional coordinated action of the cortical actin cytoskeleton and microtubules in order to create stability and symmetry [19]. The periphery of the immunological synapse resembles the front lamellipodium of migrating cells. It is therefore likely that the interplay between actin and microtubules described in lamellipodia could also take place at the periphery of the immunological synapse conditioning its shape and stability. The Rho family GTPases are likely key for this cytoskeleton interplay. For instance, in fibroblasts, microtubule growth promotes Rac1 activity, which in turn drives actin polymerization and lamellipodium formation [56]. Conversely, the Rho GTPase regulates via the formin mdia the formation and orientation of stable microtubules in fibroblasts [57], which together with Cdc42 and the motor Dynein drive MTOC reorientation [58]. Interestingly, lymphocytes may utilize these molecules differently depending on whether they are migratory or forming synapses. Thus, formins, but not Cdc42, were involved in MTOC reorientation to the immune synapse [24], although mdia regulated actin cytoskeleton rearrangements driving cell shape and motility in migrating T cells [59]. Finally, Rac may also promote microtubule stabilization by increasing detyrosinylated tubulin, or inhibiting microtubule destabilizing proteins [52]. Myosin IIA may also represent an additional link between the actin cytoskeleton and microtubules. In other cell types, myosin IIA silencing increased microtubule stability in membrane protrusions [60]. It was also shown that microtubule movement requires a balanced action of myosin II and the microtubule-mediated motor dynein [61]. The interplay between cortical actin and microtubules is crucial for synapse stability and symmetry. The cell cortex organizer ezrin and its partner Dlg1 are key for this interplay [19]. Their silencing alters microtubule network organization at the synapse, as well as synapse shape and symmetry. Ezrin is a membrane-microfilament linker involved in cell cortex organization in a wide variety of cells, including T lymphocytes [6,62]. Dlg1 is a PDZ domain-containing scaffold protein, which is part of a conserved complex of cell polarity regulators controlled by Cdc42. These include adenomatous polyposis coli, Scribble, Lgl and the protein kinase C n. Their role in cell polarization and microtubule localization during cell migration has been largely studied [63]. These proteins are also involved to various extents in T cell polarization during T cell migration and immune synapse formation [64]. Microtubule network reorganization during cell migration and immunological synapse formation may also help the targeting of endosomal vesicles containing the LFA-1 integrin and the GTPase Rap1, which, together with RapL and the actin-binding protein talin, may drive the localization and activation of integrins at the front versus the rear of migrating cells, and at the periphery of the immunological synapse, thus regulating T cell APC adhesion during kinapse and synapse formation (reviewed in [34]). The nucleation of signaling microclusters at the immunological synapse relies on a functional actin cytoskeleton [8,13], whereas microtubules seem to be dispensable for this early activation phase [19]. In contrast, both actin dynamics and an organized microtubule network are necessary for microcluster centripetal movement [13,19]. This indicates that both cytoskeleton systems cooperate to regulate microcluster dynamics. On one hand, retrograde actin flow occurring at the lamellipodium-like membrane extensions favors the centripetal movement of signaling microclusters [13]. The actin-based motor protein myosin IIA was also proposed to be required for microcluster movement and synapse stabilization [15]. On the other hand, signaling microclusters at the synapse localize along microtubules and their movements depend on microtubule integrity and organization at the synapse [19,39]. Interestingly, altering signaling microcluster dynamics with physical barriers [14], by strengthening cell adhesion [13], or by altering the

4 4848 R. Lasserre, A. Alcover / FEBS Letters 584 (2010) interaction between cortical actin and microtubules [19] leads to enhanced TCR signaling. Therefore, the interplay between the actinomyosin cytoskeleton and the microtubule network regulates immunological synapse architecture and TCR signaling. The molecular actors involved started to be uncovered. A working model for some of the steps involving this interplay is shown in Fig. 1. T cell spreading and immunological synapse architecture depends on tight interactions between the cortical actin cytoskeleton and microtubules via ezrin and Dlg1 (Fig. 1 top and lower panels). Once cells spread, signaling complexes nucleate at the plasma membrane in the synapse periphery forming microclusters that start moving centripetally. This may be driven by retrograde actin flows, actin-based motors and microtubules, although the relative involvement of these various elements is not known. Microclusters align with microtubules from the edge of the synapse in the zone of active actin dynamics. Microclusters might be then taken by endocytic vesicles, which would be transported on microtubules by still undefined microtubule-based motors (Fig. 1, central panel). TCR signal transduction could continue in endocytic compartments, as shown for other receptors. Microcluster associated to endosomes would keep on moving close and parallel to the plasma membrane. Some signaling molecules may either detach from the endocytic vesicles becoming again cytosolic proteins (i.e., ZAP-70 and SLP-76), or be targeted to other vesicular compartments by ubiquitin-dependent sorting processes (i.e., LAT and TCR). Sorting may be different for LAT and TCR, which could end up in distinct compartments. This would lead to the extinction of TCR signaling through the dephosphorylation of critical tyrosine residues, or through the degradation in lysosomal compartments. Part of the signaling molecules sorted in intracellular compartments could be engaged in a recycling pathway that would allow them to be ready for another round of TCR engagement. 5. Conclusions and perspectives Various lines of evidence indicated a key role for both the actin and the microtubule cytoskeleton in the formation and function of immunological synapses. However, most of the studies published so far dealt with one or the other cytoskeletal element separately, and little knowledge existed on the interplay between actin and microtubules in the regulation of TCR signaling at the synapse. As already known for cell migration or cell division processes, the dynamic interaction between both cytoskeletal systems is also likely to be crucial for the various functions of immunological synapses. Consistently, recent evidences support the importance of a cross-talk between the cortical actin cytoskeleton and microtubules in setting the architecture of immunological synapses and in ensuring its function in TCR signaling regulation. New molecular actors in the immune synapse will need to be unveiled to better understand this complex molecular interplay. It is however likely that some of these molecules will be also involved in other cellular processes, but might be controlled in the synapse by a set of specific signaling proteins. Likewise, microtubule-based molecular T Cell Stimulatory surface Signal down-regulation area 1 Stimulatory surface Microtubules network organization MTOC - Dlg1? Ezrin F-actin microtubule : signaling microcluster : retrograde actin flow : signaling microcluster containing vesicle : exosomal vesicle Fig. 1. Interplay between the actin cytoskeleton and microtubules in the control of TCR signaling. (Top panel) Schematic representation of a T cell spread on a stimulatory planar surface. (Central panel) Schematic representation of the lamellipodium-like membrane extension characteristic of the immunological synapse. Microtubules irradiate from the MTOC and rich the periphery of the immunological synapse in an ordered manner. At the edge of the lamellipodium, microclusters nucleate and start moving centripetally driven by retrograde actin flows together with microtubules (1). Alternatively, microtubules may drive transport of signaling molecules only after being incorporated in endocytic vesicles (2). In both cases microcluster components would be driven to sites of signal extinction in the centrosomal area. Finally an outward transport on microtubules of exocytic vesicles containing signaling molecules may also exist to bring recycled or newly synthesized molecules towards the sites of microcluster nucleation at the synapse periphery (3). (Low panel) Schematic representation of the interaction between microtubules and the cortical actin cytoskeleton via the membrane-microfilament linker ezrin and its partner the PDZ domain-containing scaffold protein Dlg1, which in turn could indirectly link microtubules. These interactions are key for synapse architecture and function.

5 R. Lasserre, A. Alcover / FEBS Letters 584 (2010) motors will be likely revealed as key players in signaling microcluster dynamics and therefore in the regulation of TCR signaling. Acknowledgements We thank Dr. M.I. Thoulouze and Dr. V. Di Bartolo for stimulating discussions. The research of our laboratory is supported by Grants from Association pour la Recherche sur le Cancer (ARC), Ligue Contre le Cancer, Agence National de Recherche (ANR), Agence National de Recherche sur la SIDA (ANRS), Institut Pasteur and CNRS. RL has been supported by a Bourse Roux from Institut Pasteur and by the ANR. References [1] Acuto, O., Bartolo, V.D. and Michel, F. (2008) Tailoring T-cell receptor signals by proximal negative feedback mechanisms. Nat. Rev. Immunol. 8, [2] Bunnell, S.C., Kapoor, V., Trible, R.P., Zhang, W. and Samelson, L.E. (2001) Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal adaptor LAT. 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