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Cytoskeletal forces during signaling activation in Jurkat T-cells.

Hui KL, Balagopalan L, Samelson LE, Upadhyaya A - Mol. Biol. Cell (2014)

Bottom Line: Although cytoskeletal forces have been implicated in this process, the contribution of different cytoskeletal components and their spatial organization are unknown.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.Our results delineate the cytoskeletal contributions to interfacial forces exerted by T-cells during activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Maryland, College Park, MD 20742.

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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.
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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.

Mentions: We first examined the effect of blebbistatin on traction force generation. Because blue light inhibits blebbistatin, we turned off the 491-nm illumination just before adding 50 μM blebbistatin to spreading Jurkat cells and compared the traction stress, as shown in the “before” and “after” stress maps (Figure 3, a and b) for a representative cell. Qualitatively, we found that the cell edge continued to behave in a dynamic manner upon blebbistatin addition. We measured actin retrograde flow in the presence of blebbistatin using TagRFP-T–actin–labeled cells and found that the flow was largely intact, indicating that myosin IIA does not play a significant role in maintaining actin flow in these cells (Supplemental Figure S2). We also found that myosin activity contributed to the formation of the actin ring (Supplemental Figure S4). We noted that the average stress ratio (0.96) was not significantly different from the control (stress ratio, 0.95; Figure 3e). We further found that 10 μM ML-7 had no effect on the traction stresses (stress ratio, 0.97; Figure 3e). On the other hand, treatment with 100 μM Y-27632 decreased the stresses exerted, as shown in the stress maps (Figure 3, c and d). The cell contact area did not decrease upon drug application, and actin lamellipodial structures were maintained even after the addition of Y-27632 (Figure 3, c and d). However, there was a slight decrease in the dynamics of the cell edge upon Y-27632 addition. The summary data show a modest decrease in stresses (stress ratio, 0.8) as compared with the double-distilled H2O control (Figure 3e).


Cytoskeletal forces during signaling activation in Jurkat T-cells.

Hui KL, Balagopalan L, Samelson LE, Upadhyaya A - Mol. Biol. Cell (2014)

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.
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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.
Mentions: We first examined the effect of blebbistatin on traction force generation. Because blue light inhibits blebbistatin, we turned off the 491-nm illumination just before adding 50 μM blebbistatin to spreading Jurkat cells and compared the traction stress, as shown in the “before” and “after” stress maps (Figure 3, a and b) for a representative cell. Qualitatively, we found that the cell edge continued to behave in a dynamic manner upon blebbistatin addition. We measured actin retrograde flow in the presence of blebbistatin using TagRFP-T–actin–labeled cells and found that the flow was largely intact, indicating that myosin IIA does not play a significant role in maintaining actin flow in these cells (Supplemental Figure S2). We also found that myosin activity contributed to the formation of the actin ring (Supplemental Figure S4). We noted that the average stress ratio (0.96) was not significantly different from the control (stress ratio, 0.95; Figure 3e). We further found that 10 μM ML-7 had no effect on the traction stresses (stress ratio, 0.97; Figure 3e). On the other hand, treatment with 100 μM Y-27632 decreased the stresses exerted, as shown in the stress maps (Figure 3, c and d). The cell contact area did not decrease upon drug application, and actin lamellipodial structures were maintained even after the addition of Y-27632 (Figure 3, c and d). However, there was a slight decrease in the dynamics of the cell edge upon Y-27632 addition. The summary data show a modest decrease in stresses (stress ratio, 0.8) as compared with the double-distilled H2O control (Figure 3e).

Bottom Line: Although cytoskeletal forces have been implicated in this process, the contribution of different cytoskeletal components and their spatial organization are unknown.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.Our results delineate the cytoskeletal contributions to interfacial forces exerted by T-cells during activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Maryland, College Park, MD 20742.

Show MeSH
Related in: MedlinePlus