Fig 2: TCR microcluster movement is impaired in the absence of Cas-L. (a) Western blot of total cell lysates from CD8+ T cells isolated by negative selection from spleen of Cas-L-null (Cas-L-/-) mice, and from the control Cas-L heterozygous littermates (WT). A monoclonal antibody for Cas-L was used to assess the presence of Cas-L. (b) Cells were fixed in 2% paraformaldehyde 15 min after seeding, and images of synapses formed in both cell types were acquired in both channels. Red=TCR; blue=ICAM-1; yellow=synapse outline. Scale bar=2 µm. (c) Radial profiles of TCR and ICAM-1 fluorescence intensities at the synapses of WT and Cas-L-/- cells (radial sweep averaged from at least 50 cells in each cell type and two independent experiments). Solid and dashed lines represent WT and Cas-L-/- cells, respectively. Red=TCR, blue=ICAM-1. P-value<0.05 (d) Time-lapse images of TCR microcluster formation and translocation at the immunological synapse. Yellow arrowheads highlight individual TCR microcluster position at the synapse at different time points. Freshly isolated CD8+ T cells from spleen of WT or Cas-L-/- mice were seeded on a bilayer embedded with fluorescently labeled antibody to TCR (red) and ICAM-1 (blue), and imaged in a TIRF microscope to follow immunological synapse formation. Simultaneous imaging of four different fields of the bilayer was performed with a × 100 objective, with an acquisition rate of 13 s between frames. Scale bar=5 µm. (e) Average speed of translocation of individual TCR microclusters during synapse formation. Mean±s.e.m. represent two independent experiments. At least five individual microclusters per cell were analyzed in at least three cells. One asterisk indicates P-value?0.05. (f) Change in mean fluorescence intensity of TCR microclusters during synapse formation in WT and Cas-L-/- cells. Mean values of four cells of each type, pooled from two independent experiments. P-value<0.0005. (g) Change in area occupied by TCR microclusters at the synapse of WT and Cas-L-/- cells analyzed in D (mean values of four cells of each type, pooled from two independent experiments).

Fig 3: Working model illustrating the critical steps that lead to cytoskeletal stretch-dependent Cas-L activation and TCR signaling. Based on our observations, we propose a model where the initial steps of TCR ligation and clustering (1–2), assembly of actin nucleating factors (3) and initial f-actin polymerization at TCR microclusters (4) take place independently of Cas-L, and the subsequent steps leading to integrin activation and stabilization of lamellipodia structures (6), and TCR transport (7), are regulated by actin-dependent Cas-L activation at TCR microclusters. In turn, this mechanical activation of Cas-L acts via a potential feedback loop that leads to TCR signal amplification (8–9) thus stabilizing the immunological synapse.

Fig 4: Cas-L-/- T cells show impaired release of Ca2+ from intracellular stores. (a) Time-lapse images of T cells from WT or Cas-L-/- mice pre-incubated with Calcium ion fluorescent dye Fluo-4, and seeded on glass coverslips coated with antibodies against TCR/CD28 and ICAM-1. Changes in free intracellular Ca2+ levels were captured in the 488 nm channel (widefield, Fluo-4 top panel), and images of the contact between a cell and the coverslip were visible through internal reflection microscopy (IRM bottom panel). Ionomycin was added to the medium at ~20 min (+Iono), and EGTA was added at ~30 min. The acquisition rate was 4 s between frames. Scale bar=5 µm. (b) Changes in Ca2+ levels were plotted across time for at least ten individual cells of each cell type from two independent experiments. Fluorescence intensity values were normalized for each individual cell by dividing background-corrected intensity values by the absolute maximum intensity value measured after adding ionomycin (imax2). (c) Levels of free intracellular Ca2+ in Cas-L-/- cells only rise up to 47±9% of their maximum potential (as measured by imax2), whereas in control WT cells Ca2+ levels peak at 87±11% of their maximal potential (P-value?0.005). (d) No significant change in the levels of sustained free intracellular Ca2+ (sustained/imax2) between Cas-L-/- and control WT cells ('n.s.': non-significant; error bars represent s.e.m. from two independent experiments). (e) PLC?1 phosphorylation (pPLC?1, green) is reduced at the synapse of Cas-L-/- T cells. Scale bar=5 µm. (f) Co-localization of phospho-PLC?1 with TCR microclusters is impaired in Cas-L-/- cells. Dot plots display the median (central line)±1.5 × interquartile range (whiskers). One asterisk means P-value?0.05, three asterisks mean P-value?0.0005; data represent at least 30 cells from two independent experiments.

Fig 5: Cas-L phosphorylation is dependent on actin polymerization. (a) Actin disruption affects the radial profiles of phospho-Cas-L (pCasL) intensity at the synapse. T cells treated for 30 min with DMSO, or 60 nm and 200 nm Cytochalasin D (CytoD) were seeded on anti-CD3?/ICAM-1-embedded bilayers, allowed to interact with the bilayers for 2 min, fixed with 2% paraformaldehyde and stained with an antibody specific for phosphotyrosine residue repeats in the substrate domain of Cas-L. The synapses were imaged with TIRFM, the levels of mean fluorescence intensity of pCasL were measured, processed and the average radial profiles and standard error of the mean of at least 30 cells per condition were plotted (data represent two independent experiments). (b) Confocal slices from the synapses of WT and Cas-L-/- cells, and corresponding vertical section profiles. The border between lamella and lamellipodia structures is lost in synapses of Cas-L-/- cells. Scale bars=2 µm. (c) Radial profiles of f-actin in synapses of WT and Cas-L-/- T cells indicated lower f-actin accumulation at the synapses of Cas-L-/- T cells. Cells were seeded on bilayers, fixed at 2 min and stained with fluorescently labeled phalloidin (green). Curves represent the mean±s.e.m. of at least 30 cells from three independent experiments. (d) Phospho-Cas-L levels at the synapse are dependent on Arp2/3 activity. T cells were treated with a small molecular inhibitor of Arp2/3 (CK666), or DMSO as control. Three asterisks: P-value?0.0005. (e) Phospho-Cas-L levels at the synapse are dependent on WASp activity. WASp-/- T cells or WT cells were seeded on bilayers, fixed at 2 min, and stained for phospho-Cas-L. (f) Cas-L and PKC? play opposing roles in regulating synapse stability. T cells from Cas-L-/- mice were seeded on bilayers, and 20 min after seeding a small molecule inhibitor for PKC? (C20) was added to the medium (at 10 µm or 33 µm) and its effect was followed for another 20 min. Graph reflects the change in the number of new synapses formed after C20 treatment relative to the DMSO control. Values represent mean±s.e.m. of at least 30 cells from three independent experiments.