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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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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).
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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).

Mentions: To measure the forces exerted by T-cells, we performed traction force microscopy, which allows the measurement of spatially resolved traction stresses (Dembo and Wang, 1999). Jurkat T-cells expressing enhanced green fluorescent protein (EGFP)–actin were allowed to spread on polyacrylamide gels coated with anti-CD3 antibody and embedded with fluorescent beads on the top surface as fiduciary markers. We imaged the spreading dynamics of cells starting from the earliest time points before the cell established contact with the substrate and continued imaging for at least 15 min. Typically, cells were completely spread before 15 min, as shown in the EGFP-actin images (Figure 1a) of a typical cell spreading on a gel of stiffness 1.7 kPa (which approximates the stiffness of APCs; Rosenbluth et al., 2006). To measure the traction stresses exerted by cells, we tracked the fluorescent beads using particle image velocimetry (PIV). The first frame of the live-cell image sequence, before the cell exerted traction on the surface, was used as the “zero displacement” or reference image. Unconstrained Fourier transform traction cytometry (FTTC; Butler et al., 2002) was used to calculate the traction stress map from the measured bead displacements at different times (Figure 1b). The average traction stresses exerted by cells were in the range of 5–10 Pa, whereas the peak traction stresses exerted were in the range of 10–30 Pa, in the same range as stresses exerted by neuronal growth cones (Betz et al., 2011; Koch et al., 2012). By contrast, rapidly migrating keratocytes and strongly adherent fibroblasts are known to exert traction stresses in the range of 100 Pa to several kilopascals (Lee et al., 1994; Dembo and Wang, 1999).


Cytoskeletal forces during signaling activation in Jurkat T-cells.

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

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).
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Related In: Results  -  Collection

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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).
Mentions: To measure the forces exerted by T-cells, we performed traction force microscopy, which allows the measurement of spatially resolved traction stresses (Dembo and Wang, 1999). Jurkat T-cells expressing enhanced green fluorescent protein (EGFP)–actin were allowed to spread on polyacrylamide gels coated with anti-CD3 antibody and embedded with fluorescent beads on the top surface as fiduciary markers. We imaged the spreading dynamics of cells starting from the earliest time points before the cell established contact with the substrate and continued imaging for at least 15 min. Typically, cells were completely spread before 15 min, as shown in the EGFP-actin images (Figure 1a) of a typical cell spreading on a gel of stiffness 1.7 kPa (which approximates the stiffness of APCs; Rosenbluth et al., 2006). To measure the traction stresses exerted by cells, we tracked the fluorescent beads using particle image velocimetry (PIV). The first frame of the live-cell image sequence, before the cell exerted traction on the surface, was used as the “zero displacement” or reference image. Unconstrained Fourier transform traction cytometry (FTTC; Butler et al., 2002) was used to calculate the traction stress map from the measured bead displacements at different times (Figure 1b). The average traction stresses exerted by cells were in the range of 5–10 Pa, whereas the peak traction stresses exerted were in the range of 10–30 Pa, in the same range as stresses exerted by neuronal growth cones (Betz et al., 2011; Koch et al., 2012). By contrast, rapidly migrating keratocytes and strongly adherent fibroblasts are known to exert traction stresses in the range of 100 Pa to several kilopascals (Lee et al., 1994; Dembo and Wang, 1999).

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