Cytoskeletal tension actively sustains the migratory T‐cell synaptic contact
2020; Springer Nature; Volume: 39; Issue: 5 Linguagem: Inglês
10.15252/embj.2019102783
ISSN1460-2075
AutoresSudha Kumari, Michael Mak, Yeh‐Chuin Poh, Mira Tohmé, Nicki Watson, Mariane B. Melo, Erin Janssen, Michael L. Dustin, Raif S. Geha, Darrell J. Irvine,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle2 January 2020Open Access Cytoskeletal tension actively sustains the migratory T-cell synaptic contact Sudha Kumari Corresponding Author Sudha Kumari [email protected] orcid.org/0000-0001-7082-2825 Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA Search for more papers by this author Michael Mak Michael Mak Department of Mechanical Engineering, MIT, Cambridge, MA, USA Search for more papers by this author Yeh-Chuin Poh Yeh-Chuin Poh orcid.org/0000-0003-3510-6369 Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Department of Mechanical Engineering, MIT, Cambridge, MA, USA Search for more papers by this author Mira Tohme Mira Tohme Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Nicki Watson Nicki Watson Whitehead Institute of Biomedical Research, Cambridge, MA, USA Search for more papers by this author Mariane Melo Mariane Melo Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA Search for more papers by this author Erin Janssen Erin Janssen Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Michael Dustin Michael Dustin orcid.org/0000-0003-4983-6389 Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK Search for more papers by this author Raif Geha Raif Geha Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Darrell J Irvine Corresponding Author Darrell J Irvine [email protected] orcid.org/0000-0002-8637-1405 Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA Department of Biological Engineering, MIT, Cambridge, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Sudha Kumari Corresponding Author Sudha Kumari [email protected] orcid.org/0000-0001-7082-2825 Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA Search for more papers by this author Michael Mak Michael Mak Department of Mechanical Engineering, MIT, Cambridge, MA, USA Search for more papers by this author Yeh-Chuin Poh Yeh-Chuin Poh orcid.org/0000-0003-3510-6369 Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Department of Mechanical Engineering, MIT, Cambridge, MA, USA Search for more papers by this author Mira Tohme Mira Tohme Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Nicki Watson Nicki Watson Whitehead Institute of Biomedical Research, Cambridge, MA, USA Search for more papers by this author Mariane Melo Mariane Melo Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA Search for more papers by this author Erin Janssen Erin Janssen Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Michael Dustin Michael Dustin orcid.org/0000-0003-4983-6389 Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK Search for more papers by this author Raif Geha Raif Geha Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Darrell J Irvine Corresponding Author Darrell J Irvine [email protected] orcid.org/0000-0002-8637-1405 Koch Institute of Integrative Research, MIT, Cambridge, MA, USA Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA Department of Biological Engineering, MIT, Cambridge, MA, USA Howard Hughes Medical Institute, Chevy Chase, MD, USA Search for more papers by this author Author Information Sudha Kumari *,1,2, Michael Mak3,9, Yeh-Chuin Poh1,3, Mira Tohme4, Nicki Watson5, Mariane Melo1,2, Erin Janssen4, Michael Dustin6, Raif Geha4 and Darrell J Irvine *,1,2,7,8 1Koch Institute of Integrative Research, MIT, Cambridge, MA, USA 2Ragon Institute of Harvard, MIT and MGH, Cambridge, MA, USA 3Department of Mechanical Engineering, MIT, Cambridge, MA, USA 4Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 5Whitehead Institute of Biomedical Research, Cambridge, MA, USA 6Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK 7Department of Biological Engineering, MIT, Cambridge, MA, USA 8Howard Hughes Medical Institute, Chevy Chase, MD, USA 9Present address: Department of Biomedical Engineering, Yale University, New Haven, CT, USA *Corresponding author. Tel: +1 617 253 0656; E-mail: [email protected] *Corresponding author. Tel: +1 617 452 4174; E-mail: [email protected] The EMBO Journal (2020)39:e102783https://doi.org/10.15252/embj.2019102783 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott–Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell–APC synaptic contact. Synopsis When naïve T cells detect cognate antigens on the surface of antigen presenting cells, these highly migratory cells undergo immediate arrest and form radially symmetric cell-cell conjugate interfaces termed immunological synapses. The stability of such synapses is a crucial determinant of T cell activation, but the T cell-intrinsic mechanisms that regulate synaptic lifetime are not clear. This study finds that T cells construct specialized actin architectures within the immunological synapse to elevate cytoskeletal tension, thereby reinforcing synaptic stability. In T cells, lamellar protrusions would constantly attempt to break the synapse. Integrin activation is insufficient to sustain the synapse. Antigen recognition-induced actin foci and associated actomyosin arrangements generate high in-plane cytoskeletal tension that actively restrains synapse breaking. Via nucleation of actin foci, WASP acts as a central regulator of this mechanism. Downregulation of WASP following T cell activation leads to loss of foci-dependent actin architecture resulting into synapse unravelling. T cells that lack WASP are predisposed to synapse symmetry breaking, irrespective of the substrate stiffness. Introduction During an immune response, T cells form specialized junctions with cognate antigen-presenting cells (APCs) termed “immunological synapses” (Negulescu et al, 1996; Grakoui et al, 1999). Such synapses provide a stable platform where a number of T-cell surface receptors including T-cell receptors (TCRs), adhesion, and costimulatory receptors engage with counter-ligands on the APC surface (Negulescu et al, 1996; Miller et al, 2002; Dustin, 2008a). A symmetric, sustained synapse is a hallmark of T-cell activation, both in vitro and in vivo (Miller et al, 2002; Stoll et al, 2002; Mempel et al, 2004; Bajenoff et al, 2006; Germain et al, 2012). How T cells, which are otherwise highly motile (Bousso & Robey, 2003; Miller et al, 2003; Tadokoro et al, 2006), sustain prolonged synaptic contacts with APCs remains poorly characterized. This is a crucial gap in our understanding of T-cell biology since synapse lifetime is a critical determinant of T-cell activation and function (Hugues et al, 2004; Celli et al, 2007; Skokos et al, 2007; Zaretsky et al, 2017). Integrins, the actin cytoskeleton, and calcium signaling are all known to play important roles in the initial formation of an immunological synapse (Dustin et al, 1997; Wei et al, 1999; Fooksman et al, 2010; Comrie & Burkhardt, 2016; Martin-Cofreces & Sanchez-Madrid, 2018), but their roles in the subsequent maintenance and eventual dissolution of the synaptic contact are unclear. For example, integrin activation and actin polymerization are essential processes for T-cell adhesion and synapse formation, but these processes could also terminate the synapse by engaging new adhesions and cellular protrusions, respectively, and thus driving T-cell migration away from the APC. Furthermore, T cells are inherently highly migratory and have pronounced F-actin-dependent lamellar undulations even in the synaptic phase (Sims et al, 2007; Roybal et al, 2013). These lamellar dynamics could promote polarization and lateral movement of the T cell (Mullins, 2010), thereby breaking symmetry of the synapse. F-actin along with the activated integrin lymphocyte function-associated antigen-1 (LFA-1) is organized into a ring-like structure in the early synapse, and this ring-like pattern is thought to promote synaptic junctional stability (Wulfing et al, 1998; Kaizuka et al, 2007; Babich et al, 2012). However, this F-actin/integrin ring undergoes continuous fluctuations and is therefore amenable to symmetry breaking (Sims et al, 2007; Mullins, 2010; Lomakin et al, 2015). In addition, genetic lesions in actin regulatory proteins such as the Wiskott–Aldrich syndrome protein (WASP) do not interfere with T cells adhering to and forming synaptic contacts with APCs, but result in highly unstable synapses (Cannon & Burkhardt, 2004; Sims et al, 2007; Thrasher & Burns, 2010; Calvez et al, 2011; Kumari et al, 2015). In summary, the role of the basic motility apparatus, especially the actin cytoskeleton, in sustaining already formed T cell–APC contacts is unclear and warrants further investigation. Maintenance of the immune synapse is known to be influenced by T cell-intrinsic factors such as the strength of TCR signaling (Henrickson et al, 2008; Bohineust et al, 2018). However, the processes downstream of the cellular migration machinery that enable the transition from an arrested to a motile state are not clear (Hugues et al, 2004; Celli et al, 2007; Skokos et al, 2007; Shulman et al, 2014). One of the ways in which the TCR-associated actin dynamics could enforce synapse symmetry and stability is by regulating mechanical forces within the synapse. Polymerization of actin is known to generate forces (Ridley et al, 2003), and organization of the polymerized F-actin network tunes the magnitude of these forces (Fletcher & Mullins, 2010; Blanchoin et al, 2014). Notably, adhesion forces in T cell–APC conjugates are actin-cytoskeleton-dependent and continue to evolve after initial synapse formation (Hosseini et al, 2009; Lim et al, 2011; Bashour et al, 2014; Hu & Butte, 2016), raising the possibility that a specialized F-actin organization and associated forces may regulate maintenance of the synaptic contact. We hypothesized that examining the actin cytoskeleton and associated forces in T cells during synaptic contact breaking would provide important clues to the mechanical design principles that T cells employ for maintaining the immune synapse. We studied T-cell cytoskeletal organization at different stages of activation using a combination of super-resolution imaging, genetic and pharmacological perturbations, micromechanical measurements, and computational simulations, employing both model APC-mimetic surfaces and physiological APCs. We found that specialized actin microstructures termed actin foci, which form within the interface following antigen encounter, generate and sustain intracellular tension within the T cell at the contact interface, and this tension actively sustains the synapse after its formation. This process relies on continuous nucleation of actin at TCR microclusters by Wiskott–Aldrich syndrome protein (WASP) and interaction of freshly polymerized actin filaments with myosin II. Myosin contractile activity generates and maintains high in-plane tension across the synaptic interface. This high-tension actomyosin network eventually breaks as activated T cells downregulate WASP, leading to immediate relaxation of cytoskeletal tension followed by synaptic unraveling and resumption of motility. Taken together, these results uncover a novel antigen-triggered biomechanical program that regulates synapse symmetry in primary T cells and highlight sub-synaptic actin architectures that sustain their interactions with APCs. Results Synapse maintenance is associated with actin re-organization, but not integrin activation or calcium signaling The actin cytoskeleton, integrins, and intracellular calcium flux all play important roles in mediating initial T-cell motility arrest and formation of a symmetric synapse with antigen-presenting cells. We thus first examined how these factors impact the maintenance of pre-formed synapses. To model synapse formation and eventual disengagement, we seeded mouse primary naïve CD4+ primary T cells (referred to as “T cells” henceforth) onto an anti-CD3/intercellular adhesion molecule-1 (ICAM-1)-coated coverslip (antigen-presenting surface, APS) and allowed synapses to form. We then assessed cellular polarization over time by recording the cells’ morphologic aspect ratio, AR, because the contact interface elongation, reflected as increase in AR, is tightly linked to the motile state of the T cell (Hons et al, 2018; Houmadi et al, 2018; Mayya et al, 2018; Negulescu et al, 1996; Fig 1A, and Appendix Figs S1 and S2). That is, synapses are radially symmetric in their stable state and polarize to break symmetry thereby acquiring an elongated morphology prior to resumption of motility (Movies EV1 and EV2). Figure 1. Synapse polarization involves actin remodeling in mouse naïve CD4+ T cells (“T cells” henceforth) and proceeds despite of integrin augmentation A . General schematic of the assay system used in this study for examining the process of synapse sustenance vs. polarization, post-initial T-cell adhesion, and spreading on the antigen-presenting surface (APS). Cell–APS interface of T cells was imaged for synapse establishment and maturation (5′) and eventual symmetry breaking and polarization (20′). B–D. T-cell interface actin (phalloidin) re-organization during synapse symmetry breaking, imaged using SIM. Quantification of the shape elongation (AR; B, n = 44 for 5′, 39 for 20′), or relative fluorescence signal of the indicated proteins in the SIM images (C, D) normalized to the mean of values at 5′; points represent values for individual cells. Arrowheads in (C) indicate foci. P values are: ***P < 0.0001 for AR, **P = 0.003 for foci, **P = 0.008 for talin, and P = 0.36 for total F-actin using Mann–Whitney two-tailed test between populations of cells within the same experiment. E. Relationship between AR and foci at individual cell level, each point in the scatter plot represents value obtained from a single cell. F–H. Integrin augmentation does not rescue synapse symmetry breaking. Cells were allowed to adhere to the APS for 5′ or 10′ and were then treated with vehicle control, 0.5 mM MnCl2, or 100 nM A286982 for subsequent 10′. Cells were fixed, stained with phalloidin and anti-talin antibody, imaged using SIM (F), and analyzed for AR (G) as well as talin, total F-actin, and actin foci at the synapse (H). In (E–H), n for 5′ = 40, for 20′ = 62, for MnCl2 = 59, for A286982 = 53. The values in the plots represent the intensity values normalized to the mean of 5′ in each set. P values in the graph, n.s. > 0.05; ***P < 0.001; *P = 0.01; **P < 0.009 using Mann–Whitney two-tailed test between populations of cells within the same experiment. Data information: Central values in the graphs represent Mean, and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5 μm. Download figure Download PowerPoint Within seconds, T cells seeded onto an APS spread to form a symmetric contact interface (Appendix Fig S1). Symmetric synapses persisted for ~ 10 min, followed by a transition period when the cells began polarizing and becoming motile (Negulescu et al, 1996; Hons et al, 2018; Houmadi et al, 2018; Mayya et al, 2018), reflected in an increase in AR (Fig 1B, Movies EV1 and EV2). Structured illumination microscopy (SIM) revealed the presence of peripheral actin-rich lamellipodia and punctate actin structures distributed across the synaptic interface (Fig 1C), termed “actin foci” (Kumari et al, 2015) in stable (5 min) synapse. As synapse began to polarize at later times (20 min), actin foci were reduced even though total F-actin levels in the interface remained constant (Fig 1C–E). Note that the foci quantification methodology we used [employing a Gaussian mask generated by a 1.6 × 1.6 μm2 rolling window, as described previously (Kumari et al, 2015)] identified near-complete loss of foci as a ~ 65% reduction in foci intensity, due to residual signal contributed by the lamellipodial F-actin (Fig EV1). As T cells formed synapses with an APS, the key integrin adaptor protein talin accumulated rapidly at the periphery of the interface and talin levels remained constant at later times as cells began polarizing (Fig 1D). Activating integrins using MnCl2 (Dransfield et al, 1992) after a stable synapse was established did not affect subsequent polarization/synapse breaking at 20 min, and inhibiting LFA-1/ICAM interactions using the small molecule inhibitor A286982 (Liu et al, 2000) blocked T-cell polarization, indicating that integrin activation is not necessary to sustain synapse symmetry and may in fact be required for symmetry breaking (Fig 1F and G). Notably, treatment of cells with A286982 was accompanied by a retention of actin foci at 20′, supporting a previous report that activated integrins may reduce F-actin accumulation in foci (Tabdanov et al, 2015). Under all of the treatment conditions, talin recruitment as well as the total F-actin was unchanged (Fig 1H left and middle graphs); however, higher actin foci levels were associated with reduced synapse polarization at later times (Fig 1H, right graph). Elevation of intracellular calcium is a key signal that triggers initial arrest and formation of adhesive contacts between T cells and APCs (Miller et al, 2004). However, similar to inhibition of integrins, blocking calcium signaling using BAPTA (Balagopalan et al, 2018) induced a retention of actin foci and synapse symmetry at 20′ (Appendix Fig S3). Thus paradoxically, integrins and calcium signaling, which are critical for initial T-cell arrest and formation of a symmetric synapse following TCR triggering, are unable to sustain pre-formed synapses. In contrast, actin foci were enriched in the interface of T cells forming symmetric synapses, and reduced or absent as T cells polarized and regained motility under all treatment conditions (Fig 1F–H). Click here to expand this figure. Figure EV1. Image processing scheme utilized to extract and quantify foci on per cell basis. Related to Fig 1 A , B. The images show 2D F-actin intensity as marked by phalloidin staining (top images), or a 3D view of spatial distribution of intensities in the phalloidin images (bottom images). To process the raw images for extracting foci intensities from overall F-actin signal, a Gaussian mask was generated by using a 1.6 × 1.6 μm rolling window, as optimized previously (Kumari et al, 2015). Subtraction of mask image from raw image generated a processed image that could be quantified to measure the average intensity contributed by the foci. Note that while this method reliably identifies the foci in raw images and reduces intensity contribution from the non-foci uniform lamellar area, the peripheral lamellipodial network still contributes a background of ˜ 35% to the total foci intensity, regardless of the presence of profuse foci in arrested synapse, or their visible reduction in the motile phase. Scale bar, 5 μm. Download figure Download PowerPoint T-cell polarization is associated with WASP/actin foci downregulation To directly visualize the dynamics of actin foci as T cells transitioned from the arrested to the motile state, we imaged LifeAct-GFP-expressing T cells using lattice light-sheet microscopy (LLSM). This technique exposes cells to lower phototoxicity than traditional microscopy (Chen et al, 2014), allowing long-term imaging of actin dynamics in naïve T cells which are highly sensitive to photodamage. LLSM imaging revealed that T cells engaged in a stable synapse continuously nucleated and dissipated actin foci across the contact interface, but as the cells polarized to begin migration, pre-existing foci were lost and new foci ceased to form (Fig 2A and B; Movie EV3). Thus, the loss of actin foci accompanies synapse symmetry breaking. Figure 2. Synapse symmetry involves temporal regulation of WASP and WASP-dependent actin kinetics A . Time-lapse imaging of LifeAct-GFP-expressing T cell using lattice light-sheet microscopy (LLSM) during synapse polarization. Shown are snapshots at selected time points (left) and a kymograph of actin foci (denoted by arrows in the first image panel) over time (right) for a representative T cell. B. Quantification of changes in actin foci and cellular aspect ratio in LLSM images during symmetry breaking. The values in the plot are the raw values normalized to their mean in each case. C–E. T cells incubated with APS for the indicated time periods were processed for phalloidin or immune staining (C, D) or for Western blotting (E). Graph in (D) shows relative levels of total and active WASP (WASP-phosphorylated at Y293, pWASP), normalized to the mean levels at 5′; for pWASP n in 5′ = 49, in 20′ = 44; for WASP, n in 5′ = 56, 20′ = 56. P values, P** = 0.0027 for pWASP and P** = 0.0035 for WASP using Mann–Whitney two-tailed test. The scatterplot in (D) shows the relationship between active WASP and foci on a per cell basis. Numbers in the graph in (E) indicate WASP band intensity divided by the actin band intensity, and resulting ratios normalized to the values at the 0′ time point (non-activated cells), from three independent experiments (n = 3). P value * = 0.025, using paired two-tailed t-test. F–H. Actin dynamics in mature synapse of LifeAct-GFP-expressing T cell using TIRFM. The images show the foci (pseudocolored red) extracted from the total synaptic F-actin (F, G), and kymograph shows the time course of foci and lamellar activity over a period of 210 s. Histograms of foci and lamellar protrusion and retraction (fluctuations) dynamics in WT T cell (H); lifetime of individual foci and lamellar events (graph on the bottom right, each dot represents a single foci/lamellar protrusion–retraction event n = 126 for foci and n = 77 for lamellar protrusions). ***P < 0.0001 using Mann–Whitney two-tailed test between populations of cells within the same experiment. I. Kymograph of the lamellar activity of WASP-deficient cells that lack actin foci. Data information: Central values in the graphs represent Mean, and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5 μm. Download figure Download PowerPoint We posited that the loss of actin foci during synapse disengagement may result from downregulation of WASP since actin foci at the synapse are nucleated by WASP (Kumari et al, 2015) and TCR signaling-induced degradation of WASP has been previously reported (Macpherson et al, 2012; Reicher et al, 2012; Watanabe et al, 2013). Consistent with this hypothesis, we detected a sizeable reduction in total as well as active forms of WASP (phospho-Y293) at 20′ post-T-cell activation. WASP downregulation correlated with diminution of actin foci at the T cell–APS synapse as revealed by TIRF microscopy and Western blotting (Fig 2C–E). Similar correlations between WASP downregulation with reduction in actin foci and onset of cellular morphological polarization at 20 min were found when examining T cell–bone marrow-derived dendritic cell (BMDC) synapses using confocal microscopy (Appendix Fig S4). When the stable synapse was imaged at high resolution, highly dynamic lateral protrusions and retractions at the cell periphery (lamellar fluctuations; Roybal et al, 2013) were visible along with continuously nucleated foci (Fig 2F–H, Movie EV4). Comparison of foci and lamellar dynamics showed that foci had a mean half-life ~ 3 times longer than the lamellar actin fluctuations in WT cells (Fig 2H). In WASP−/− cells lacking foci, lamellar fluctuations dominated the overall F-actin dynamics at the synapse (Fig 2I, Movie EV4), as expected. Thus, even during the stable phase of the arrested synapse, actin polymerization drives constant lamellar fluctuations in the periphery that could drive symmetry breaking (Carvalho et al, 2013; Blanchoin et al, 2014), while actin foci are dynamically nucleated across the interface. Synaptic actin foci dynamics and associated traction forces support synapse symmetry Continuous nucleation of actin filaments at the foci can, in principle, generate localized mechanical stresses (Cossart, 2000; Pollard & Borisy, 2003). To assess whether this was the case here, and whether the resulting forces may help sustain synapse symmetry, we quantified foci-dependent forces in the synaptic contact interface using traction force microscopy (TFM, Fig 3A; Engler et al, 2004; Yeung et al, 2005; Poh et al, 2012). Using this approach, we found that WT T cells exerted substantial forces on the substrate (Fig 3B). These forces were greatly diminished in WASP−/− cells that lacked foci (Fig 3B). Furthermore, the net contractile moment (pJ), a metric describing force magnitude as well as distribution across the cell contact interface (Butler et al, 2002; Wang et al, 2002), showed that there were higher opposing forces separated by longer distances in WT synapses (pJ 0.35 ± 0.17) compared to WASP−/− synapses (pJ 0.06 ± 0.033; P value 0.01 between WT and WASP−/−). Figure 3. Foci-associated mechanical forces are linked to synapse symmetry A , B. Actin foci-deficient cells display poor traction forces in their synapse. WT or WASP−/− T cells were incubated on polyacrylamide substrates covalently functionalized with anti-CD3 and ICAM1, and traction force measurements were carried out as described in “Materials and Methods”. The images in the right show traction force maps without (left panels) or with (right panels) force vectors. P value, **P < 0.005 as determined by using Mann–Whitney two-tailed test between populations of cells within the same experiment, n = 9 and 11. C. SIM imaging of WT or WASP−/− T cells activated by APS for 5′. The graphs show quantification of actin foci (derived from phalloidin staining), pCasL, and talin levels in the synapse normalized to the “WT” mean value in each case (Middle graph; in the case of foci, n = 63 for WT and 86 for WASP−/−; in the case of pCasL n = 63 for WT and 78 for WASP−/−; in the case of talin, n = 59 for WT and 73 for WASP−/−; in the case of AR, n = 46 for WT and 42 for WASP−/−); right graph shows AR measurement. ***P < 0.0001; P value for talin *P = 0.07, as determined by using Mann–Whitney two-tailed test between populations of cells within the same experiment. D. Human WAS patient CD4+ T cells–APC conjugates display synapse symmetry defects similar to those observed in mouse WASP−/− CD4+ T cells. CD4+ T cells purified from healthy controls or WAS patients were incubated with superantigen-loaded HUVEC cells for 5′ (APC; see “Materials and Methods”) and imaged using confocal microscopy. Each image represents a maximum intensity projection of the synaptic area. Graph in the middle shows values normalized to the mean value of the “Healthy” case. In the case of “actin” and “actin foci”, n = 87 for healthy and n = 63 for WAS; in the case of pCasL, n = 56 for healthy and n = 21 for WAS; and for “AR” graph, n = 88 for healthy and n = 63 for WAS case. P values; ***P < 0.001; n.s. P = 0.09, as determined by using Mann–Whitney two-tailed test between populations of cells within the same experiment. Data information: Central values in the graphs represent Mean, and error bars represent ± SEM. These experiments were repeated at least twice with similar results. Scale bar, 5 μm. Download figure Download PowerPoint We further confirmed the link between foci and mechanical stresses at the synapse by determining their localization with the phosphorylated form of the mechanosensory protein CasL (pCasL)—a molecular marker of localized cytoskeletal tension. CasL interacts with the actin cytoskeleton and undergoes a conformational change in response to local cytoskeletal tension allowing stretching and phosphorylation of its N-terminal domain (Sawada et al, 2006; Janssen et al, 2016; Santos et al, 2016; Fig EV2). This conformational change
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