T cell antigen receptor signaling and immunological synapse ...

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  • 1. ARTICLES T cell antigen receptor signaling and immunological synapse stability require myosin IIA Tal Ilani1, Gaia Vasiliver-Shamis2, Santosh Vardhana2, Anthony Bretscher1,3 & Michael L Dustin2,3 Immunological synapses are initiated by signaling in discrete T cell antigen receptor microclusters and are important for the differentiation and effector functions of T cells. Synapse formation involves the orchestrated movement of microclusters toward © 2009 Nature America, Inc. All rights reserved. the center of the contact area with the antigen-presenting cell. Microcluster movement is associated with centripetal actin flow, but the function of motor proteins is unknown. Here we show that myosin IIA was necessary for complete assembly and movement of T cell antigen receptor microclusters. In the absence of myosin IIA or its ATPase activity, T cell signaling was interrupted ‘downstream’ of the kinase Lck and the synapse was destabilized. Thus, T cell antigen receptor signaling and the subsequent formation of immunological synapses are active processes dependent on myosin IIA. The specific and long-lasting interface between a T cell and an antigen- proposed on the basis of size-dependent segregation of proteins presenting cell (APC), called the ‘immunological synapse’, is critical coupled with receptor-ligand interaction kinetics and membrane for the afferent and efferent limbs of the adaptive immune response1,2. dynamics16. T cell synapses have been shown to have a centripetal The supramolecular organization of the immunological synapse was version of retrograde actin flow2,17, a process that propels growth described more than a decade ago3–5, yet the mechanisms leading to cones of neurons and other motile cells18. A close examination of the its formation and persistence are unknown. No function for motor centripetal movement of TCR microclusters shows that it is F-actin proteins in signaling and synapse formation by cells of the immune dependent and that they move at about half of the speed of the system has been established6,7. underlying actin cytoskeleton (140 nm/s versus 320 nm/s, respectively) The first step in synapse formation is engagement of the T cell and can change course to move around barriers2,17. It has been antigen receptor (TCR) with the appropriate major histocompatibility proposed that intermittent coupling between the retrograde actin complex–antigenic peptide complex, which leads to actin-dependent flow and the microclusters may drive centripetal movement, but the microcluster formation and recruitment of signaling components function of ‘motors’ in this process is not known. to form a signalosome within seconds8–10. The TCR signalosome Members of the nonmuscle myosin II subfamily are critical to many includes tyrosine-phosphorylated forms of the kinase Lck (A001394), cellular functions, including cell polarization, migration, adhesion and the kinase Zap70 (A002396) and the membrane adaptor Lat cytokinesis19. Members of the myosin II family are composed of a (A001392) and excludes the transmembrane phosphatase CD45 heavy-chain dimer; each heavy chain is associated with two myosin (refs. 8,9,11–13). The contact area expands by integrin-mediated light chains (MLCs). Nonmuscle myosin II is activated by phosphor- spreading as TCR microclusters continue to form at the outer ylation of the MLCs to induce assembly into bipolar filaments and edge11,13. Over a period of minutes, the microclusters move to the contraction after interaction with actin filaments19,20. Three genes center of the contact area, where they fuse into larger clusters and encode mammalian nonmuscle myosin II heavy chains, producing the become part of the nonmotile central supramolecular activation following three isoforms: MyH9 (A004003), MyH10 and MyH14 cluster (cSMAC)13. As there is less tyrosine phosphorylation in the (refs. 21,22). Of those three isoforms, only MyH9 is dominant in cSMAC, it has been suggested to be the site of inactivation of old T cells6,23. MyH9 pairs with regulatory MLCs to form a complex we clusters, whereas new microclusters form at the periphery9,13,14. The refer to here by its common name, myosin IIA. T cell crawling and the formation and movement of new TCR microcluster–based signalo- movement of beads attached to the surface of T cells have been somes toward the cSMAC sustains signaling13. shown to require myosin IIA–mediated contractility6,24. In both of The driving force for protein rearrangement in the immunological those studies, the immunological synapse seemed to form in synapse is unknown, although actomyosin-driven contraction has the absence of myosin IIA activity or in cells depleted of myosin been proposed to drive TCR movement15. An alternative has been IIA by small interfering RNA (siRNA). Myosin IIA was recruited 1Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York, USA. 2Molecular Pathogenesis Program, Helen L. and Martin S. Kimmel Center for Biology and Medicine of the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA. 3These authors contributed equally to this work. Correspondence should be addressed to A.B. (apb5@cornell.edu) or M.L.D. (michael.dustin@med.nyu.edu). Received 11 December 2008; accepted 5 March 2009; published online 6 April 2009; doi:10.1038/ni.1723 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1
  • 2. ARTICLES a 0s 6s 12 s 18 s 24 s 72 s b TCR Myosin c Control Control siRNA TCR (% of control) 120 100 80 60 40 20 Blebb 0 Total cSMAC Figure 1 Effect of the inhibition or depletion of myosin IIA on the centripetal motion of TCR microclusters. (a) TIRF microscopy of microclusters in control Jurkat T cells (top) or cells pretreated with blebbistatin (Blebb; bottom) added to a planar lipid bilayer containing Alexa Fluor 568–labeled anti-TCR and ICAM-1 and imaged during the initial minutes of synapse formation. Yellow plus symbols indicate initial microcluster localization; red circles indicate microcluster location; red lines indicate tracks of individual clusters. (b) Microscopy of primary human CD4+ cells unaffected by siRNA treatment (left; n ¼ 1 cell per image) or treated with MYH9-specific siRNA (arrows; n ¼ 1 cell per image), then added for 20 min to a planar lipid bilayer containing Alexa Fluor 568–labeled anti-TCR (red) and ICAM-1, and then fixed and stained for myosin IIA (green). Scale bars (a,b), 5 mm. (c) Total TCRs and TCRs at the center of the contact area (cSMAC) in control and myosin IIA–depleted (siRNA) cells (n ¼ 30 cells per condition). Data are representative of seven (a) or three (b) experiments with 26 samples per condition or three experiments (c error bars, s.e.m.). © 2009 Nature America, Inc. All rights reserved. to the synapse6, but its activation and function in signaling and Thus, the microclusters continued to move at 40% the speed of synapse formation were not firmly established. control Jurkat T cells but with over fourfold more meandering and Here we show that the actin-based ‘molecular motor’ myosin IIA is only 10% of the displacement of control Jurkat T cells (treated with an essential participant in the formation and persistence of immuno- DMSO). In mature synapses with formed cSMACs, blebbistatin logical synapses and TCR signaling. Myosin IIA was rapidly activated treatment did not disrupt the cSMAC, but the peripheral TCR after TCR engagement, and its activity was essential for the centripetal microclusters ceased directed movement shortly after addition of the movement of TCR microclusters. Additionally, both immunological drug (Supplementary Movie 7 online). These results suggest that synapse stability and signaling ‘downstream’ of the TCR required myosin IIA activity is required for the centripetal movement of TCR intact myosin IIA. microclusters but not for microcluster formation. To further assess the involvement of myosin IIA in the transloca- RESULTS tion of TCR microclusters, we targeted MyH9 by siRNA. Jurkat TCR microcluster movement requires myosin IIA T cells did not recover sufficiently from nucleofection of control As translocation of TCR microclusters is an essential part of the siRNA to form mature synapses (data not shown). As myosin II is formation of the immunological synapse, we first assessed whether required for cytokinesis19, siRNA vectors that require growth and myosin IIA was required for this motion. TCR microclusters can be selection would also not be usable. Therefore, we ‘knocked down’ tracked with a supported planar bilayer system and total internal MyH9 in primary activated human CD4+ T cells, which recover well reflection fluorescence (TIRF) microscopy11,13. We used TIRF micro- from nucleofection. The best knockdown efficiency achieved in the scopy to image the motion of TCR microclusters in Jurkat T cells primary T cells was 35%, as assessed by immunoblot analysis (data on supported planar bilayers containing laterally mobile Alexa Fluor not shown). However, immunofluorescence analysis showed that 568–labeled antibody to TCR (anti-TCR; OKT3) and intercellular this was due to nearly complete knockdown of MyH9 in one third of adhesion molecule 1 (ICAM-1)17. In agreement with published cells (data not shown). We analyzed microcluster tracking on planar studies17, TCR microclusters in Jurkat T cells moved centripetally bilayers of all cells in several microscopic fields while indexing the with an average velocity (± s.e.m.) of 0.15 ± 0.05 mm/s (P o 0.0001; x-y coordinates of the fields, then fixed the cells and stained for Fig. 1a and Supplementary Movie 1 online) to generate the cSMAC. intracellular MyH9, which allowed us to identify the cells in which The average displacement of a microcluster from its point of forma- MyH9 was ‘knocked down’ in the previously tracked and indexed tion to the cSMAC was 2.6 ± 0.8 mm (P o 0.0001), and the cells. Primary T cells depleted of MyH9 failed to form the typical meandering index, calculated as displacement divided by track length, condensed cSMAC and instead had small, scattered TCR micro- was 0.83 ± 0.09 (P o 0.0001); both are consistent with published clusters (Fig. 1b). TCR microclusters in cells treated with control values17. To assess the involvement of myosin IIA activity in micro- siRNA had an average centripetal velocity of 0.12 ± 0.034 mm/s, with cluster translocation, we first treated the Jurkat T cells with blebbis- an average displacement of 2.2 ± 0.53 mm and a meandering index of tatin, a well established specific inhibitor of myosin II ATPase 0.85 ± 0.07 (P o 0.0001 for all measurements). TCR microclusters activity25. Jurkat T cells pretreated for 10 min with blebbistatin in MyH9-deficient cells had a speed of 0.062 ± 22 mm/s, a displace- (50 mM) formed microclusters but showed less directed microcluster ment of 0.26 ± 0.11 mm and a meandering index of 0.25 ± 0.11 movement, with an average speed of 0.06 ± 0.02 mm/s, a displacement (P o 0.0001 for all measurements; Supplementary Fig. 1). Notably, of 0.25 ± 0.13 mm and a meandering index of 0.17 ± 0.09 (P o 0.0001 there was significantly less TCR accumulation at the cSMAC for all measurements; Fig. 1a and Supplementary Fig. 1 and Supple- (P o 0.0001) but only slightly, nonsignificantly less total TCR mentary Movie 2 online). We detected equivalent blockade of in the entire contact area in cells depleted of MyH9 (Fig. 1c). microcluster centripetal motion with ML-7 an inhibitor of myosin These results obtained by siRNA knockdown of MyH9 expression light-chain kinase (MLCK; Supplementary Movie 3 online). We reproduced the results obtained by inhibition of myosin II activity noted similar effects on the inhibition of microcluster movement with blebbistatin and ML7. Thus, myosin IIA activity is required for when we inhibited myosin IIA activity in primary human CD4+ T cells the translocation of TCR microclusters to form a cSMAC but not for by treatment with blebbistatin (Supplementary Movies 4–6 online). the formation of TCR microclusters. 2 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY
  • 3. ARTICLES Figure 2 Phosphorylation and redistribution a c TCR Myosin HC Merge of myosin IIA during activation of T cells. Stim (min): 0 0.5 1 3 5 10 30 (a) Abundance of phosphorylated MLC (p-MLC) p-MLC and total MLC (MLC) in total T cell lysates at various times during stimulation (Stim) by soluble Coomassie anti-TCR (OKT3). (b) Microscopy of resting Jurkat TCR p-MLC Merge T cells fixed and stained for F-actin (green) and MLC myosin IIA heavy chain (HC; red). (c) Microscopy of Jurkat T cells stimulated for 1 min with OKT3, then fixed and stained for TCR, myosin heavy chain and phosphorylated MLC. Cells showing b F-actin Myosin HC Merge colocalization: 83%, TCR and myosin IIA heavy Myosin HC p-MLC Merge chain; 92%, TCR and phosphorylated MLC; 90%, myosin IIA heavy chain and phosphorylated MLC. Scale bars (b,c), 5 mm. Data are representative of three experiments (n ¼ 30 cells per condition). Myosin IIA is activated during T cell stimulation Immunological synapse stability requires myosin IIA Our initial results indicated that myosin IIA participates in the To understand the consequences of myosin IIA activity for the © 2009 Nature America, Inc. All rights reserved. formation of the immunological synapse. Activation of myosin IIA immunological synapse, we determined the effect of pretreating through phosphorylation of MLCs during the formation of the Jurkat T cell with 50 mM blebbistatin on synapse formation with immunological synapse has not been evaluated so far. We therefore ‘superantigen’-loaded Raji B cells. Unexpectedly, we found that examined the phosphorylation status of MLCs in Jurkat T cells inhibition of myosin II activity did not inhibit the concentration of stimulated either with soluble OKT3, which activates the TCR only, TCR, ezrin, F-actin or myosin IIA itself at the contact site between the or with ‘superantigen’ presented by Raji B cells as APCs, which engages two cells (Fig. 3b). As we could not use siRNA in the Jurkat model, we the TCR and multiple adhesion and costimulatory molecules. MLCs used both ML7 and an additional inhibitor, Y27632, which inhibits were not detectably phosphorylated in resting Jurkat T cells, but within Rho-associated kinase (ROCK). Both ROCK and MLCK phosphory- 30 s of stimulation by soluble OKT3, they became phosphorylated and lated and activated MLCs, and both ML7 and Y27632 inhibited this phosphorylation was sustained for at least 30 min (Fig. 2a). In phosphorylation of MLCs during T cell stimulation with soluble resting Jurkat T cells, myosin IIA was uniformly distributed in the anti-TCR (Supplementary Fig. 3 online). Conjugates between ‘super- cytoplasm, whereas after stimulation with soluble OKT3, myosin IIA antigen’-loaded Raji B cells and Jurkat T cells pretreated with either of and its phosphorylated MLCs rapidly became enriched at the area of those drugs had apparently normal accumulation of myosin IIA TCR clusters at the plasma membrane (Fig. 2b,c). (Supplementary Fig. 4 online). We obtained similar results with In synapses formed between Jurkat T cells and ‘superantigen’- primary human CD4+ cells pretreated with blebbistatin and incubated loaded Raji B cells, we detected the typical accumulation of TCR, for 5 min with Raji B cells (Supplementary Fig. 2). These data F-actin and ezrin at the contact site as described before26,27. In a two- confirm and extend earlier indications that the first attachment step of cell system, either the T cell or B cell could contribute to this protein immunological synapse formation does not require myosin IIA redistribution, yet the results obtained with immune synapses between activity6. These results were also consistent with the ability of Jurkat and Raji B cells were identical to results obtained with Jurkat blebbistatin-treated or myosin IIA–depleted cells to form immature T cells stimulated with soluble OKT3 and were indicative of a immunological synapses on planar bilayers containing TCR micro- seemingly normal immune synapse (Figs. 2 and 3). The synapse clusters and ICAM-1 (Fig. 1). was also highly enriched for myosin IIA, with a distribution very Although the conjugates formed after inhibition of myosin IIA similar to that of the TCR (Fig. 3a). We obtained similar results with seemed normal, we found that fewer total conjugates formed with primary human CD4+ T cells (Supplementary Fig. 2 online). The T cells pretreated with 50 mM blebbistatin than with control cells recruitment of activated myosin IIA to the immunological synapse is treated with DMSO (Fig. 3c). Conjugate formation was not further consistent with the observed function of myosin IIA in the movement decreased by pretreatment with 100 mM blebbistatin (data not shown), of TCR microclusters and cSMAC formation. which suggested that the residual conjugate formation was not simply Figure 3 Effect of the inhibition of myosin IIA activity on the formation of the immunological a TCR (89%) Ezrin (86%) F-actin (93%) Myosin HC (83%) c synapse. (a,b) Microscopy of Jurkat T cells pretreated for 10 min with DMSO (a) or 50 mM DMSO blebbistatin (b), incubated for 5 min with SEE 50 superantigen–loaded B cells (prestained with conjugates (%) 40 CMTPX; red), then fixed and stained for TCR, T cells in ezrin, F-actin or myosin II heavy chain (green). 30 Numbers above images indicate percent cells b TCR (92%) Ezrin (79%) F-actin (90%) Myosin HC (81%) 20 with similar protein distribution (n ¼ 30 cells per 10 condition). Scale bars, 5 mm. (c) Conjugate 0 b SO Blebb eb formation by cells treated as described in a,b M Bl D (n ¼ 50 cells per condition). Data are represen- tative of three experiments (error bars (c), s.d.). NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 3
  • 4. ARTICLES Figure 4 Effect of the inhibition of myosin IIA activity on the stability of the a Before blebb After blebb immunological synapse. (a) DIC imaging of conjugate formation and stability T T b 100 of Jurkat T cells pretreated (pretreat) with DMSO or blebbistatin and added DMSO to SEE superantigen–loaded B cells immobilized in dishes with coverslip Synapses (%) (pretreat) 80 inserts, followed by the formation of immunological synapses (top two rows), B B T T 60 or of Jurkat T cells without pretreatment added to the B cells described 40 above, followed by the addition of blebbistatin 1 min or 12 min after synapse T 20 formation (bottom two rows), assessed before (left) and 1–2 min after (right) T T 0 treatment with blebbistatin. T, T cell; B, B cell; white arrows, immunological Blebb b SO synapses; black arrows, loss of synapse. (b) Synapses in the cells in a eb (pretreat) M Bl D present 2 min after the addition of blebbistatin. (c) Duration of synapses in B B the cells in a after the addition of blebbistatin at various times after synapse B c formation (horizontal axis). Data are representative of five experiments (a), B 170 or five experiments with 35 cells per condition (b,c; error bars, s.d.). Synapse duration (s) Blebb 150 T (1 min) 130 110 90 We next assessed whether synapse breakdown resulted from 70 perturbation of the typical accumulation of the adhesion proteins 50 B B 30 LFA-1 and ICAM-1 at the peripheral SMAC5 after inhibition of Blebb 0 1 2 3 4 5 10 12 (12 min) Blebb addition (min) myosin IIA activity with blebbistatin. Pretreatment with blebbistatin T led to a more peripheral distribution of these interactions, consistent © 2009 Nature America, Inc. All rights reserved. T with impaired transport toward the center. However, we did not detect a difference in the intensity of these interactions relative to that of an effect of partial inhibition of myosin IIA activity. To explore the control cells treated with DMSO (Supplementary Fig. 5 online). basis for the lower conjugate number, we examined the effect of Thus, instability of the immunological synapse after the inhibition of blebbistatin addition before and after conjugate formation. We first myosin IIA activity was not due to initial failure of LFA-1 activation. immobilized ‘superantigen’-loaded Raji B cells in dishes with coverslip inserts and then added Jurkat T cells. We monitored conjugate Ca2+ signaling requires myosin IIA activity formation and stability by differential interference contrast (DIC) One of the earliest and most easily monitored signaling events after microscopy, adding blebbistatin at various times relative to conjugate T cell activation is a rapid increase in cytoplasmic Ca2+ (ref. 28). formation (Fig. 4a). The addition of blebbistatin resulted in lower A published study has shown that treatment with butanedione stability of formed conjugates so that only about 20% remained 2 min monoxide, a less specific inhibitor of myosin II than blebbistatin, in after drug addition (Fig. 4b). Jurkat T cells treated with blebbistatin activated primary CD4+ T cells leads to a less sustained increase in formed unstable synapses that only lasted for 102 ± 14 s (Fig. 4c), Ca2+ after stimulation and a partial blockade of the movement of whereas control T cells formed stable synapses that persisted for over membrane proteins to the synapse24. To explore if synapse instability 20 min (data not shown). The addition of blebbistatin at various times correlated with loss of Ca2+ signaling, we preloaded Jurkat T cells with after conjugate formation resulted in instability and detachment with- the fluorescent, cytoplasmic, Ca2+-sensitive indicator dye Fluo-LoJo in 1–2 min of drug addition, with an average time of 109 s (Fig. 4c). and assessed the effect of blebbistatin on cytoplasmic Ca2+ in response As blebbistatin resulted in the same instability regardless of the time of to ‘superantigen’-loaded Raji B cells. Although control Jurkat T cells addition after synapse formation, we concluded that myosin IIA acti- maintained higher cytoplasmic Ca2+ concentrations (Fig. 5a,b), the vity is needed to maintain the stability of both early and mature addition of blebbistatin (50 mM) to an existing immunological synapse synapses. We obtained similar results by inhibiting myosin IIA acti- led to a rapid decrease in Ca2+ concentrations within 1 min (Fig. 5a,b vation with 10 mM ML7 (Supplementary Movie 8 online) and with primary human CD4+ cells (Supplementary Movies 9,10 online). a b Time (s) DMSO Blebb 110 100 Figure 5 Effect of the inhibition of myosin IIA activity on intracellular Ca2+ 0 DMSO (1) Intensity (%) B 90 concentrations. (a) Microscopy of Jurkat T cells incubated with Fluo-LoJo, B DMSO (2) then mixed with SEE superantigen–loaded B cells and allowed to form 80 DMSO (3) Blebb (1) immunological synapses, followed by the addition of DMSO or blebbistatin. 70 Blebb (2) Scale bars, 5 mm. (b) Change in intensity over time of Fluo-LoJo in the cells 9 60 Blebb (3) B in a treated with DMSO (control) or blebbistatin at time 0 (n ¼ 3 cells B 50 per condition), presented relative to the average sustained signal in 0 15 30 45 ‘superantigen’-activated cells, set as 100%. Numbers in parentheses (key) Time (s) identify different samples. (c) Emission ratios of Jurkat T cells incubated 18 c with Fura-2AM, then added to a planar lipid bilayer containing anti-TCR and B B 0.7 ICAM-1 for 15 min before imaging, assessed every 15 s by fluorescence 0.6 Fura-2 (340/380) microscopy and presented as the ratio of absorbance at 340 nm to 0.5 DMSO absorbance at 340 nm (340/380). Gray shaded area indicates the addition 27 0.4 Blebb of DMSO or blebbistatin 15 min after the addition of cells to bilayer (except B Blebb B 0.3 (pretreat) for pretreated cells, which did not receive additional blebbistatin). The low 0.2 and high calcium ratios corresponding to cells in EGTA with Mg2+ and 0.1 without Ca2+ or ionomycin were also determined (data not shown). Data are 0 33 representative of three experiments (a,b) or two independent experiments (c; B 1 2 3 4 5 6 B n ¼ 17 cells per condition). Time (min) 4 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY
  • 5. ARTICLES Figure 6 Effect of the inhibition of myosin IIA activity on TCR microclusters. a Control (a) Microscopy of Jurkat T cells stimulated with Alexa Fluor 488–conjugated 0 min anti-CD3 without additional treatment (Control), after pretreatment for 10 min with 50 mM blebbistatin (Blebb pretreat) or treatment with 50 mM Blebb pretreat Blebb after TCR blebbistatin after TCR stimulation (Blebb after TCR), then imaged immediately (0 min) or at 1 and 5 min after stimulation. Scale bars, 5 mm. Data are representative of five experiments with two cells per condition per 1 min time point. (b) Quantitative analysis of the results in a. Data are representative of five experiments (error bars, s.e.m.; n ¼ 35 clusters per bar). (c) Immunoblot analysis of Src phosphorylated at tyrosine 416 (p-Src), Zap70 phosphorylated at tyrosine 319 (p-Zap70) and Lat phosphorylated at 5 min tyrosine 191 (p-Lat), in Jurkat T cells left untreated (a-TCR À) or treated for 2 min with anti-CD3 (a-TCR +; OKT3) with (+) or without (À) blebbistatin pretreatment. Bottom, actin protein abundance (loading control). Results b 700 1 min c are representative of three independent experiments. 600 5 min α-TCR: – + + TCR cluster (nm) Blebb: – – + 500 p-Src 400 with between 10 mg/ml and 500 mg/ml of antibody (Supplementary 300 p-Lat Fig. 8 online). This result challenges the possibility of insufficient 200 TCR engagement as a mechanism to account for the decrease in 100 p-Zap70 Ca2+ signaling. 0 Actin © 2009 Nature America, Inc. All rights reserved. Blebb: None Before After stim stim TCR signaling requires myosin IIA activity Our results suggested that myosin IIA might be important for the and Supplementary Fig. 6b online). We obtained similar results with function of the TCR signalosome. The simplest way to activate the ML-7 (Supplementary Movie 11 and Supplementary Fig. 7 online). formation of TCR signalosomes is based on the addition of soluble We detected a similar decrease in Ca2+ concentrations in primary anti-CD3e to Jurkat T cells31, shown above to activate MLC phos- human CD4+ cells after inhibition of myosin IIA (Supplementary phorylation. We incubated Jurkat T cells with fluorescence-tagged Movies 12–14 online). For a more quantitative measurement of anti-CD3e and monitored TCR distribution and biochemical indica- cytoplasmic Ca2+ changes, we loaded Jurkat T cells with the ratio- tors of TCR signalosome assembly (phosphorylation of Lck, Zap70 metric Ca2+-indicator dye Fura-2AM and calculated emission ratios. and Lat). Control Jurkat T cells initially showed a uniform surface The addition of 50 mM blebbistatin to cells with preformed synapses fluorescence that aggregated into microclusters with a diameter of diminished cytoplasmic Ca2+ concentrations to baseline within less 280 ± 70 nm by 1 min, followed by coalescence into larger clusters of than 2 min, whereas control cells maintained high Ca2+ concentra- 456 ± 88 nm after 5 min of stimulation (Fig. 6a,b). When we tions (Fig. 5c). Pretreatment with 50 mM blebbistatin blocked the pretreated Jurkat T cells for 5 min with 50 mM blebbistatin and TCR-induced increase in Ca2+ altogether (Fig. 5c). To rule out the then stimulated the cells for 1 min with labeled anti-TCR, the TCR possibility that emission-intensity changes resulted from autofluores- clusters were slightly smaller, with a diameter of 217 ± 63 nm. cence of blebbistatin, we preloaded T cells with Fluo-LoJo and added However, progression in cluster size in the blebbistatin-treated cells blebbistatin without any TCR stimulation. Blebbistatin fluorescence was minimal, reaching a diameter of 247 ± 66 nm after 5 min of was negligible in our assays (Supplementary Fig. 6). Moreover, we stimulation (Fig. 6a,b). We next explored the effect on microclusters found that the addition of 50 mM blebbistatin to the cells, followed by when we added blebbistatin at 1 or 5 min after stimulation. In both illumination, had no toxic effect (data not shown). Notably, in all cases, 5 min after the addition of blebbistatin, the cluster was smaller, these experiments, the decrease in cytoplasmic Ca2+ concentration with a diameter of 217 ± 64 nm or 258 ± 59 nm for 1 min or 5 min, preceded the detachment of the immunological synapse, which respectively (Fig. 6a,b). These results collectively show that TCR indicated that myosin IIA activity is necessary for sustained Ca2+ microclusters about 217 nm in diameter can form in the absence of signaling ‘downstream’ of the TCR in the immunological synapse myosin IIA activity, yet their coalescence into larger clusters and their independently of any effects on adhesion. maintenance in larger clusters require myosin IIA activity. We The serial-triggering model holds that one major histocompatibility obtained similar results with primary human CD4+ cells (Supple- complex–bound antigenic peptide engages a large number of TCRs in mentary Fig. 2). When we treated Jurkat T cells for 2 min with anti- successive rounds, contacting about 50–200 receptors per antigenic CD3e and then analyzed phosphorylated signalosome components by peptide29. This model is compatible with the demonstration that ten direct immunoblot of lysates, we found that phosphorylation of Src complexes of peptide and major histocompatibility complex in the kinases, which probably included phosphorylated Lck, was similar T cell–APC interface can sustain signaling long enough to generate with or without blebbistatin pretreatment (Fig. 6c). In contrast, interleukin 2 (ref. 30). If myosin IIA is needed only to promote an phosphorylation of Zap70 and Lat was substantially lower after active process of serial triggering, then increasing the number of TCRs blebbistatin pretreatment (Fig. 6c). We obtained similar results with triggered in parallel might overcome the requirement for myosin IIA primary CD4+ T cells (Supplementary Fig. 2). We also assessed activity. To test that possibility, we explored whether more activating whether Jurkat T cells pretreated with blebbistatin increased their anti-TCR could overcome the effect of blebbistatin on Ca2+ signaling. Ca2+ in response to stimulation with soluble anti-CD3e. T cells We preloaded Jurkat T cells with Fluo-LoJo and then stimulated the preloaded with Fluo-LoJo and stimulated with soluble anti-TCR cells with increasing concentrations of anti-TCR. Once the cytoplas- underwent a robust Ca2+ response, whereas cells pretreated with mic Ca2+ concentrations had risen, we added 50 mM blebbistatin and blebbistatin failed to increase Ca2+ concentrations in response monitored Ca2+ concentrations for an additional 1 min. The decrease to stimulation (Fig. 5c and Supplementary Fig. 8). These results in cytoplasmic Ca2+ concentration was independent of the concentra- indicate quantitative defects in TCR microcluster size and defective tion of activating antibody, with a similar decrease in cells stimulated signalosome function in a synapse-free assay. NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 5
  • 6. ARTICLES a CD3 Src (pY416) Merge b CD3 Src (pY416) Merge Control 120 DMSO intensity (%) Src (pY416) 120 siRNA 100 intensity (%) Src (pY416) 100 80 80 60 60 40 40 20 20 0 0 Myosin IIA Control Myosin IIA Blebb SO b siRNA siRNA eb siRNA M Bl D CD3 Zap70 (pY319) Merge CD3 Zap70 (pY319) Merge Zap70 (pY319) Control 120 intensity (%) DMSO 120 Zap70 (pY319) siRNA 100 intensity (%) 100 80 80 60 60 40 40 20 20 0 0 Myosin IIA Control Myosin IIA Blebb SO b siRNA siRNA eb M siRNA Bl D CD3 Lat (pY191) Merge CD3 LatT (pY191) Merge © 2009 Nature America, Inc. All rights reserved. Control 120 DMSO 120 intensity (%) Lat (pY191) siRNA 100 intensity (%) Lat (pY191) 100 80 80 60 60 40 40 20 20 0 0 Myosin IIA Control Myosin IIA Blebb SO b siRNA eb siRNA siRNA M Bl D Figure 7 Effect of the inhibition or depletion of myosin IIA on signaling in T cells. (a) Localization of TCR microclusters and phosphorylated Src, Zap70 and Lat (left) in Jurkat T cells (with or without blebbistatin pretreatment; left margin) added for 25 min to a planar lipid bilayer containing Alexa Fluor 568–labeled anti-TCR and ICAM-1, then fixed and stained with antibody to Src phosphorylated at tyrosine 416 (Src(pY416)), Zap70 phosphorylated at tyrosine 319 (Zap70(pY319)) or Lat phosphorylated at tyrosine 191 (Lat(pY191)). Right, quantification of protein phosphorylation. Data are representative of three experiments (n ¼ 15 cells per bar; error bars, s.d.). (b) Localization of TCR microclusters and phosphorylated Src, Zap70 and Lat (left) in primary human CD4+ cells treated with control or MYH9-specific siRNA and added for 25 min to a planar lipid bilayer containing Alexa Fluor 568–labeled anti-TCR and ICAM-1, then fixed and stained as described in a. Myosin IIA–depleted cells were identified by the lack of central TCR clustering (as in Fig. 6b). Right, quantification of protein phosphorylation. Data are representative of two experiments (n ¼ 15 cells per bar; error bars, s.d.). TCR signalosome function can also be evaluated in a synapse-based of the immunological synapse, and synapse persistence. Published system with supported planar bilayers presenting OKT3 (ref. 17). work has shown that the F-actin cytoskeleton is required for all of T cells interacting with a planar bilayer containing OKT3 and ICAM-1 these processes17,32 and that TCR engagement induces actin polymer- for 5 min had a central condensed TCR cluster surrounded by ization through recruitment of the adaptor protein Nck and Wiskott- peripheral microclusters containing TCRs, as well as phosphorylated Aldrich syndrome protein to the TCR microclusters33. Our study has Zap70 and Lat (Fig. 7), similar to published studies9. When we added shown that after T cell engagement, myosin IIA was activated by Jurkat T cells pretreated with 50 mM blebbistatin to the bilayers, phosphorylation of MLCs and its activity was necessary for proper followed by staining with a specific antibody to each phosphorylated assembly of the signalosome. Inhibition of myosin IIA activity with protein, we found that phosphorylated Src was localized together with the highly specific myosin II inhibitor blebbistatin or depletion of TCR microclusters, but the abundance of phosphorylated Zap70 and myosin IIA expression with specific siRNA resulted in a complete Lat, as measured by fluorescence intensity, was significantly lower, by halting of directed motion of microclusters, prevented the formation 80% each (P o 0.0001; Fig. 7). We also extended this analysis to of the cSMAC and prevented amplification of TCR signals after Lck primary CD4+ T cells treated with control and MyH9-specific siRNA activation. Whether myosin IIA activity was inhibited pharmacologi- during activation; this resulted in nearly complete knockdown of cally, in which case myosin IIA was still recruited to the synapse, or if myosin IIA in one third of the cells. We found that knockdown its expression was diminished by siRNA, in which case it was of myosin IIA diminished phosphorylation of Src by only 25% profoundly depleted from the synapse, the formation of initial small (P o 0.0001) but lowered phosphorylation of Zap70 at tyrosine 319 TCR microclusters remained intact. However, these clusters did not by 80% (P o 0.0001) and phosphorylation of Lat by 70% increase in size, did not fully signal and did not undergo directed (P o 0.0001). These data demonstrate, by both pharmacological and translocation. Thus, we have defined distinct F-actin-dependent and reverse-genetic approaches, that myosin IIA is required for amplifica- actomyosin-dependent phases of T cell activation and immunological tion of TCR signaling between the Lck and Zap70 activation steps. synapse formation. The potential involvement of myosin II in the formation of DISCUSSION immunological synapses has been reported before. One study showed Here we have provided evidence that myosin IIA is central to synapse that movement of ICAM-1-coated beads on T cells after activation by assembly and signaling, being necessary for TCR signaling, the a B cell is inhibited by butanedione monoxime with concurrent centripetal motion and fusion of microclusters during the formation decrease in Ca2+ signaling, although the B cell–T cell conjugates 6 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY
  • 7. ARTICLES remain stable24. The authors hypothesized that myosin II–mediated This is the first report to our knowledge to link myosin II activity to transport is delivering components to the immunological synapse that signaling through an immunoreceptor. In examining the ‘downstream’ are needed for sustained signaling. Another study showed that myosin signaling pathway, we found that phosphorylation of the Src family IIA is necessary for T cell motility and uropod maintenance and kinases was unimpaired by either inhibition or depletion of myosin postulated that inhibition of myosin IIA filament formation is IIA, whereas ‘downstream’ signaling, including phosphorylation of required for the T cell ‘stop signal’ after antigen encounter6. These Zap70 and Lat and an increase in cytosolic Ca2+, were much more authors also reported that immunological synapse formation seemed dependent on myosin IIA activity. The truncation in signaling ‘down- unaffected by pretreatment with blebbistatin. That result is in agree- stream’ of Lck was not due to defects in adhesion, as inhibition of ment with our finding that immunological synapses formed with myosin IIA activity in Jurkat T cells stimulated with soluble OKT3 also blebbistatin-treated T cells were initially similar to synapses with resulted in less phosphorylation of Zap70 and Lat and a decrease in control cells. The T cell blasts used in the earlier study6 have high intracellular Ca2+ concentrations to baseline. Our data support a two- constitutive LFA-1 activity, so myosin IIA–dependent signaling is not step model in which initial conjugate formation involving the forma- required for conjugate formation. We focused on two systems here, tion of TCR microclusters, recruitment of myosin IIA and activation Jurkat T cells and primary human T cells, in which basal LFA-1 of Lck are all independent of myosin IIA activity, whereas amplifica- activity is low and ‘inside-out’ signaling through the TCR is required tion of signaling and microcluster movement are dependent on for conjugate formation34. In retrospect, evidence of spreading and myosin IIA activity. Our work here and published work indicates contraction in the immunological synapse formation process was that there is careful ‘tuning’ of myosin IIA activity during T cell visible in earlier studies5,9 and has been explicitly described for activation, with negative regulation through inhibition of the forma- B cell synapse formation without indicating involvement of myosin tion of thick filaments6 and positive regulation through phospho- © 2009 Nature America, Inc. All rights reserved. II (ref. 35). Contractile oscillations have been reported at the outer rylation of MLCs, which leads to maintenance of the cortical tension edge of the immunological synapse formed by T cells32. Contractile needed for TCR signaling and synapse stabilization. oscillations require myosin IIA in fibroblasts. Our results suggest that this is also probably true in lymphocytes36. METHODS Myosin II–based cortical movement has been documented in Cells and antibodies. Jurkat T cells and Raji B cells were from American Type several other situations. Myosin II is necessary for cortical tension Culture Collection. Human peripheral blood lymphocytes were isolated from citrate-anticoagulated whole blood by dextran sedimentation (Blood Centers and functions in the contractile ring during cytokinesis37,38. Several of America–hemerica), followed by density separation over Ficoll-Hypaque studies have suggested that an imbalance in cortical tension contri- (Sigma). The resulting mononuclear cells were washed in PBS and were further butes to cytokinesis, with cortical loosening at the cell poles and purified by nylon wool and plastic adherence as described48. Human peripheral enhanced tension at the cell equator, leading to equatorial movement, CD4+ blasts were prepared as described49. Anti-ezrin and anti–myosin II heavy assembly and contraction of the contractile ring39. In a related chain have been described50. Affinity-purified polyclonal antibodies to MLC mechanism, anterior-posterior polarity in the one-cell nematode phosphorylated at serine 19 (3671), Src phosphorylated at tyrosine 416 (used to embryo is established by myosin II–mediated cortical contraction, measure phosphorylated Lck, the most abundant Src member in T cells; 2101), which moves granules and fate determinants toward the future Zap 70 phosphorylated at tyrosine 319 (2701), and Lat phosphorylated at tyro- anterior pole40. It is possible that a related myosin II–dependent sine 191 (3584) were from Cell Signaling. OKT3 mouse anti–human CD3 was cortical tension may move TCR microclusters toward the center of the purified from an OKT3 hybridoma cell line (14-0037; eBioscience). Rhodamine- phalloidin, Alexa Fluor 568–phalloidin, Alexa Fluor 488–conjugated donkey immunological synapse. Such cortical tension seemed to be required anti-mouse (21202) and Alexa Fluor 568–conjugated goat anti-rabbit (11011) for TCR signalosome function even in the absence of a synapse, on the and goat anti-mouse (11004) were from Invitrogen. Horseradish peroxidase– basis of results obtained with soluble OKT3. The reported particle-size conjugated goat anti-rabbit (W4011) was from Promega. All procedures were requirements for T cell stimulation may arise from the need for approved by the Health and Safety Committee of Cornell University. myosin IIA–mediated tension across an interface or crosslinked Immunofluorescence. Cells were plated on poly-L-lysine-coated glass slides, protein network41,42. Myosin IIA–mediated cortical tension may be were fixed for 30 min at 25 1C with 3.7% (wt/vol) formaldehyde, were made required for the rearrangement of cytoskeletally associated ‘protein permeable by treatment for 2 min with 0.1% (vol/vol) Triton X-100 in PBS and islands’ into functional signalosomes43. then were rinsed three times in PBS. Cells were then incubated for 1 h with 5% The activation of myosin II by phosphorylation of its MLCs can (vol/vol) BSA in PBS, then were incubated for 1 h with primary antibody in 5% be mediated by several different kinases, including the calcium- (vol/vol) BSA in PBS, washed in PBS and incubated for 1 h with the calmodulin–dependent MLCK44, ROCK and protein kinase C45. appropriate secondary antibody (or phalloidin) in 5% (vol/vol) BSA in PBS. Shortly after stimulation of T cells, Vav1, a Rho guanine-exchange After additional washes, 5 ml Vectashield (Vector Labs) was added to cells and factor, is recruited to TCR microclusters through interaction with the slides were covered with coverslips. Cells were viewed with a Nikon Eclipse adaptor protein SLP-76, which is then followed by the recruitment of TE2000-U (100Â objective; numerical aperture, 1.4) with the Perkin Elmer the GTP-binding protein Cdc42 and ROCK46,47. T cell stimulation also UltraVIEW LCI spinning-disk confocal imaging system and a Hamamatsu 12-bit C4742-95 digital charge-coupled device camera. results in more cytoplasmic Ca2+, which is known to activate MLCK44. We have shown that treatment with either the ROCK inhibitor Y27632 Immunoblot analysis. Jurkat and primary T cells were lysed and were resolved or the MLCK inhibitor ML-7 inhibited phosphorylation of MLCs after by SDS-PAGE, followed by transfer to polyvinylidene difluoride membranes T cell stimulation. Thus, both kinases take part in activating myosin II (Immobilon-P; Millipore) with a semidry transfer system (Integrated Separa- even when the TCR is triggered by OKT3. As myosin IIA activity was tion Systems). After 1 h of blocking in 5% (wt/vol) milk in Tris-buffered saline necessary to maintain higher Ca2+ concentrations, a plausible model is with Tween, membranes were incubated for 1 h with primary antibody, then that Rho-GTP–activated ROCK initially phosphorylates MLCs, then were washed and were incubated for 1 h with the appropriate horseradish peroxidase–conjugated secondary antibody. Blots were developed by the Ca2+ concentrations increase, which maintains light-chain phosphoryl- enhanced chemiluminescence system (Amersham). ation through persistent activation of MLCK. Thus, one crucial func- tion of myosin IIA activity is to maintain signaling that then feeds back Cell stimulation and conjugate formation. Jurkat and primary human T cells to maintain higher Ca2+ concentrations and active myosin IIA. were activated for various times with the antibody OKT3 (10 mg/ml). For NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 7
  • 8. ARTICLES stimulation with B cells, Raji B cells were labeled with the fluorescent dye 3. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional CellTracker Red (CMTPX; Molecular Probes) and were loaded with the segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998). ‘superantigen’ staphylococcus enterotoxin E (SEE; 2 mg/ml; Toxin Technology). 4. Dustin, M.L. et al. A novel adaptor protein orchestrates receptor patterning and Equal numbers of T cells and B cells were incubated together. cytoskeletal polarity in T-cell contacts. Cell 94, 667–677 (1998). 5. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell Conjugate stability and DIC microscopy. Raji B cells were loaded with SEE activation. Science 285, 221–227 (1999). superantigen and then were immobilized in dishes containing coverslip inserts 6. Jacobelli, J., Chmura, S.A., Buxton, D.B., Davis, M.M. & Krummel, M.F. A single class (MatTek) and viewed on an Axiovert 100 TV microscope (Carl Zeiss) equipped II myosin modulates T cell motility and stopping, but not synapse formation. Nat. Immunol. 5, 531–538 (2004). with a charge-coupled device (C4742-95-12ERG; Hamamatsu) with a DIC 7. Combs, J. et al. Recruitment of dynein to the Jurkat immunological synapse. Proc. Natl. prism and Openlab 4.0 software (Improvision). After initial B cell imaging, Acad. Sci. USA 103, 14883–14888 (2006). Jurkat T cells or primary human T cells were added to plates and cells were 8. Bunnell, S.C. et al. T cell receptor ligation induces the formation of dynamically allowed to form conjugates. Blebbistatin (50 mM), ML7 (10 mM) or DMSO was regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002). 9. Campi, G., Varma, R. & Dustin, M.L. Actin and agonist MHC-peptide complex- added at various times and conjugates were continuously imaged. Movies were dependent T cell receptor microclusters as scaffolds for signaling. J. Exp. Med. 202, analyzed with ImageJ software. 1031–1036 (2005). 10. Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a Ca2+ assays. For nonratiometric assays, Jurkat T cells and primary human photoactivatable agonist. Immunity 27, 76–88 (2007). T cells were loaded with 1 mM Fluo-LoJo (TefLabs), then were added to 11. Yokosuka, T. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat. Immunol. 6, 1253–1262 SEE-loaded Raji B cells and allowed to form synapses or were stimulated with (2005). OKT3. Blebbistatin, ML7 or DMSO was added at various times and fluores- 12. Douglass, A.D. & Vale, R.D. Single-molecule microscopy reveals plasma membrane cence intensity was measured with the spinning-disk confocal imaging system. microdomains created by protein-protein networks that exclude or trap signaling For ratiometric analysis, Jurkat T cells were loaded with 2.5 mM Fura-2AM molecules in T cells. Cell 121, 937–950 (2005). 13. Varma, R., Campi, G., Yokosuka, T., Saito, T. & Dustin, M.L. T cell receptor-proximal (acetoxymethyl ester; Molecular Probes) as described13. © 2009 Nature America, Inc. All rights reserved. signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006). Bilayer assembly and TIRF microscopy. Glass-supported bilayers of dioleoyl 14. Lee, K.H. et al. The immunological synapse balances T cell receptor signaling and phosphatidylcholine incorporating 0.01% (wt/vol) 1,2-dioleoyl-sn-glycero-3- degradation. 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Note: Supplementary information is available on the Nature Immunology website. 26. Blanchard, N. et al. Strong and durable TCR clustering at the T/dendritic cell immune synapse is not required for NFAT activation and IFN-g production in human CD4+ ACKNOWLEDGMENTS T cells. J. Immunol. 173, 3062–3072 (2004). We thank D. Garbett for help with data analysis with Volocity and for 27. Ilani, T., Khanna, C., Zhou, M., Veenstra, T.D. & Bretscher, A. Immune synapse comments, and D.W. Pruyne for help in setting up DIC microscopy. Supported formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin. by the European Molecular Biology Organization (T.I.) and the US National J. Cell Biol. 179, 733–746 (2007). 28. Weiss, A., Imboden, J., Shoback, D. & Stobo, J. Role of T3 surface molecules in human Institutes of Health (GM36652 to A.B.; and AI44931 and Nanomedicine T-cell activation: T3-dependent activation results in an increase in cytoplasmic free Development Center EY16586 to M.L.D.). calcium. Proc. Natl. Acad. Sci. USA 81, 4169–4173 (1984). 29. Valitutti, S., Muller, S., Cella, M., Padovan, E. & Lanzavecchia, A. Serial triggering of AUTHOR CONTRIBUTIONS many T-cell receptors by a few peptide-MHC complexes. Nature 375, 148–151 The laboratories of A.B. and M.L.D. did independent work on the involvement of (1995). myosin IIA in the formation of immunological synapses and continued the work 30. Krogsgaard, M. et al. Agonist/endogenous peptide-MHC heterodimers drive T cell activation and sensitivity. Nature 434, 238–243 (2005). collaboratively focusing on studies in the human system initiated by T.I. and A.B.; 31. Janeway, C.A. Jr. & Bottomly, K. Responses of T cells to ligands for the T-cell receptor. 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