Fig 1.
Strong but not weak ligand induces organized, radially-symmetric actomyosin arcs.
(A) Representative 3D-SIM images of OT1 T cells fixed 7 min after surface attachment and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2Kb plus CD80, G4:H-2Kb plus CD80, CD80 only, or streptavidin (SA) only. The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (B) Examples for FibrilTool analysis. 7–8 similar-sized trapezoidal ROIs covering the pSMAC portion of the IS were drawn to measure the anisotropy of actin arcs. (C) Average anisotropies of actin arcs in the pSMAC region of OT1 T cells activated on surfaces coated with OVA, G4, or CD80-only. (D) Total anisotropies of actin arcs from all ROIs measured in OT1 T cells activated on surfaces coated with OVA, G4, and CD80-only. (E) Histogram of total anisotropies from all ROIs measured on OVA- and G4-coated surfaces (p < 0.00001). Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. ***, **** indicate p < 0.001, 0.0001.
Fig 2.
The ATPase activity of myosin 2 promotes actomyosin arc formation in a ligand-dependent manner.
(A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-myosin 2A antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2Kb plus CD80 (A) or G4:H-2Kb plus CD80 (B). The pSMAC regions are labeled with yellow or white brackets. Scale bar, 5 μm. (C) Frequency of arc morphologies in OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Arcs were scored as either Organized (i.e. present and concentric), Disorganized (i.e. present but either pointing inwards or entangled), or No arcs (i.e. not present). (D) Average anisotropies of actin arcs in the pSMAC region of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. (E) Histogram of total anisotropies from all ROIs measured on OVA-coated surfaces with or without pnBB treatment (p < 0.0001). (F) Same as (E) but using G4-coated surfaces (p = 0.44). Mean ± SEM. **** indicates p < 0.0001.
Fig 3.
Actomyosin arcs promote tension at the IS in a ligand-dependent manner.
(A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach to the activating surface for 7 min, and then fixed and stained with phalloidin (red) and anti-pCasL antibody (green). The merged images are shown in the bottom row. The cells were activated on glass surfaces coated with either OVA:H-2Kb plus CD80 (A) or G4:H-2Kb plus CD80 (B). Scale bar, 5 μm. (C) Mean intensities of pCasL at the IS from 3D-SIM images of OT1 cells pretreated with either DMSO or pnBB in DMSO and activated on OVA- or G4-coated surfaces. Mean ± SEM. An unequal variance T-test (Welch's T-test) was used. * and ** indicate p < 0.05 and < 0.01, respectively.
Fig 4.
Actomyosin arcs facilitate the accumulation of TCR MCs at the IS center in a ligand-dependent and contractility-dependent manner.
(A and B) Representative 3D-SIM images of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO, allowed to attach for 7 min to planar lipid bilayers containing ICAM-1 and either OVA:H-2Kb (A) or G4:H-2Kb (B) bound via fluorescent SA (to indirectly report the position of TCR MCs in the T cell), and then fixed and stained with phalloidin (red). The distribution of SA (green) and the merged images are shown in the middle and bottom rows, respectively. Scale bar, 5 μm. (C) Averaged, normalized radial integral intensity profiles of fluorescent SA in bilayers containing OVA:H-2Kb (red) or G4:H-2Kb (blue) (p <0.001). (D) Averaged, normalized radial integral intensity profiles of fluorescent SA in bilayers containing OVA:H-2Kb using OT1 T cells pretreated with either DMSO (red) or pnBB in DMSO (black) (p < 0.0001). (E) Averaged, normalized radial integral intensity profiles of fluorescent SA in bilayers containing G4:H-2Kb using OT1 T cells pretreated with either DMSO (blue) or pnBB in DMSO (black) (p < 0.0001). For (C), (D) and (E), the mean value is represented by the bold line, while the SEM is represented by the surrounding shaded area.
Fig 5.
Myosin 2 facilitates proximal TCR signaling in a ligand-dependent manner.
(A) Representative example of AMNIS Imagestream images used to quantitate signaling molecule phosphorylation at the IS of OT1 T cells conjugated with EL4 target cells by employing a combination masking strategy (see Methods for details). Shown from left to right are the bright-field (BF) image with the portion of the combination mask that corresponds to the IS (blue), the signal for GFP-F-Tractin (green) in the OT1 T cell, the signal for farnesylated RFP (pseudocolored orange) in the EL4 target cell, the signals for the nucleus (purple) in both cells, and the portion of the combination mask that corresponds to the IS (blue) overlaid with the signal for pLAT at the IS (red). Scale bar, 10 μm. (B, C, and D) MFIs for pSrc (B), pZap70 (C), and pLAT (D) at the IS of OT1 T cells that had been pretreated with either DMSO or pnBB in DMSO and allowed to form conjugates for 10 min with EL4 target cells loaded with either OVA:H-2Kb or G4:H-2Kb prior to fixation and staining. Each circle represents a single OT1: EL4 conjugate. (E, F, G) Normalized MFIs from three or more experiments performed exactly as in (B), (C) and (D), except that these normalized MFIs were background corrected by subtracting the MFI for the null peptide control. (H, I, and J) Shown are the differences in mean MFI values presented in (H), (I) and (J) between OVA:H-2Kb-engaged and G4:H-2Kb-engaged T cells. The value obtained, referred to as “ΔMFI OVA vs G4” on the y axis, represents a measure of the contribution made by myosin 2 contractility to ligand discrimination. Mean ± SEM (N ≥ 3). *, **, *** indicate p < 0.05, 0.01, 0.001.
Fig 6.
Myosin 2 facilitates T cell adhesion in a ligand dependent manner.
(A) Shown are scatter plot profiles from conjugation assays obtained using flow cytometry (see Methods for details). OT1 T cells pretreated with either DMSO or pnBB in DMSO were incubated for 30 min with EL4 target cells loaded with either OVA peptide, G4 peptide, or null peptide. The cell population in the anti-CD8/H-2Kb double-positive staining channel (gated as blue square) corresponds to OT1: EL4 conjugates. (B) Conjugation frequency (note that these values were background corrected by subtracting the percent conjugation obtained using the null peptide control). Mean ± SEM (N ≥ 3). * indicates p < 0.05. We note that steric hindrance from the bulky antibodies employed in this assay could result in a reduction in conjugation frequency.