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. 2015 Feb 15;26(4):685-95.
doi: 10.1091/mbc.E14-03-0830. Epub 2014 Dec 17.

Cytoskeletal forces during signaling activation in Jurkat T-cells

Affiliations

Cytoskeletal forces during signaling activation in Jurkat T-cells

King Lam Hui et al. Mol Biol Cell. .

Abstract

T-cells are critical for the adaptive immune response in the body. The binding of the T-cell receptor (TCR) with antigen on the surface of antigen-presenting cells leads to cell spreading and signaling activation. The underlying mechanism of signaling activation is not completely understood. Although cytoskeletal forces have been implicated in this process, the contribution of different cytoskeletal components and their spatial organization are unknown. Here we use traction force microscopy to measure the forces exerted by Jurkat T-cells during TCR activation. Perturbation experiments reveal that these forces are largely due to actin assembly and dynamics, with myosin contractility contributing to the development of force but not its maintenance. We find that Jurkat T-cells are mechanosensitive, with cytoskeletal forces and signaling dynamics both sensitive to the stiffness of the substrate. Our results delineate the cytoskeletal contributions to interfacial forces exerted by T-cells during activation.

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Figures

FIGURE 1:
FIGURE 1:
Jurkat T-cells are weak generators of traction force. (a) Time-lapse fluorescence images of EGFP-actin expressing Jurkat T-cells spreading on an anti-CD3–coated elastic substrate (of stiffness 1.2 kPa). Scale bar, 10 μm. (b) Time-lapse images of traction stress color maps for the same cell as in a. The colors correspond to magnitudes of stresses as indicated in the color bar. (c) Vector map of traction force vectors showing the direction of exerted traction stresses. Scale bar, 10 μm. (d) Development of total force as a function of time for three example cells. Black trace corresponds to the cell in a. (e) Histogram of total traction force exerted by Jurkat T-cells (N = 95). (f) Comparison of traction stresses generated by cells on substrates coated with stimulatory antibody anti-CD3 and nonstimulatory antibody anti-CD45. (g) Snapshot of an EGFP-actin cell on an elastic substrate (left; scale bar, 10 μm), and a kymograph (right) drawn along the dashed line. The linear streaks illustrate actin retrograde flow in the cell periphery. Scale bar, 5 μm (horizontal), 5 min (vertical). (h) Histogram of retrograde flow speeds of cells spreading on gels in the stiffness range 1–2 kPa (N = 46).
FIGURE 2:
FIGURE 2:
Loss of F-actin dynamics reduces cellular force generation. Fluorescence images of EGFP-actin expressing Jurkat T-cells on an elastic substrate 1 min before (left) and 9 min after (right) application of (a) 1 μM latrunculin-A, (c) 100 μM CK666, (e) 1 μM jasplakinolide, and (g) 0.1% DMSO. Color maps of traction stresses of the same cells before (left) and after (right) addition of (b) 1 μM latrunculin-A, (d) 100 μM CK666, (f) 1 μM jasplakinolide, and (h) 0.1% DMSO. (i) Total traction force as a function of time for a representative cell in each of the conditions described. The dashed line represents the time point at which the drug was added. (j) Comparison of the after-to-before ratios of traction stresses for application of Lat-A (N = 20 cells), CK666 (N = 17 cells), and Jasp (N = 10 cells) with control (DMSO carrier, N = 20 cells). The average stresses in a 3-min time interval just before addition of drug and in the time interval 9–12 min after addition of drugs were used to compute the ratios. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 3:
FIGURE 3:
Effect of myosin II activity on cellular force generation. (a) Fluorescence image of an EGFP-actin Jurkat T-cell on an elastic substrate 1 min before addition of 50 μM blebbistatin. (b) Traction stress color map of the same cell 1 min before (left) and 9 min after (right) addition of blebbistatin. (c) Fluorescence images of an EGFP-actin Jurkat T-cell on an elastic substrate 1 min before (left) and 9 min after (right) application of 100 μM Y-27632. (d) Traction stress color maps of the same cell before and after addition of Y-27632. (e) Comparison of the after-to-before ratios of traction stresses upon addition of blebbistatin (N = 20 cells) and ML7 (N = 17 cells) with control (DMSO carrier) and comparison of traction stress ratios upon addition of Y-27632 (N = 20 cells) with double-distilled H2O control (N = 11 cells). The average stresses in a 3- min time interval just before addition of drug and in the time interval 9–12 min after addition of drug were used to compute the ratios. **p < 0.01. (f, g) Traction stress color maps for example cells (at the indicated time points after stimulation). Drug or vehicle was added at 5 min after stimulation (f, DMSO; g, blebbistatin). (h) Traces of the total force exerted by four example cells with drug addition 5 min after stimulation (vertical dashed line). The total force is normalized to the value exerted at 5 min after stimulation. Red lines indicate vehicle, and blue lines indicate the time of blebbistatin addition. (i) Summary statistics of the stress ratio after drug addition for cells averaged between 9 and 12 min after stimulation. N = 13 for blebbistatin and N = 12 for DMSO (p < 0.05, Wilcoxon's rank sum test). Scale bars, 10 μm.
FIGURE 4:
FIGURE 4:
Substrate stiffness affects traction forces and signaling. (a) Average total force exerted by cells (between 14 and 15 min of spreading initiation) as a function of gel stiffness (N = 500). The data are fit to formula image (red curve) with Fsat = 5 nN, and kcell = 1 nN/μm (corresponding to 1.5 kPa). (b) Top, DIC images of two representative cells spreading on soft (200 Pa) and stiff (10 kPa) gels. Kymographs of edge dynamics for the two cells along the locations indicated by the red lines. (c) Example time traces of Pearson coefficient between cell edge's radial position profile at 15 min and at earlier time points for cells spreading on soft (blue) and stiff (red) gels. (d) Comparison of the percentage of time for which the cell edge profile had correlation coefficient >0.5 compared with the profile at 15 min for softer (<1.5 kPa stiffness) and stiffer (>1.5 kPa stiffness) gels. The difference between the two conditions is significant (t test, p < 0.001) and indicates that cell edges are more dynamic on softer gels. (e) Western blot analysis of tyrosine phosphorylation (pY) levels (of LAT and ZAP70/SLAP76 substrates) at the indicated times on two different gel stiffnesses (∼1 and ∼5 kPa). (f) Densitometry analysis of relative pY levels (for LAT substrate) as a function of time for cells on soft gels (blue curve, ∼1 kPa) and stiff gels (red curve, ∼ 5 kPa). Analysis represents average of five different experiments.

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