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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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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  (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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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 (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.

Mentions: Many types of cells that interact with soft materials have the ability to sense the stiffness of their mechanical environment and respond to it by exerting larger forces on stiffer substrates (Janmey and McCulloch, 2007). Whether Jurkat cells respond similarly to substrate stiffness is not known. Recent experiments suggest that physical forces, such as those generated by the actin cytoskeleton, may be important for T-cell signaling. To examine whether Jurkat cells are sensitive to substrate stiffness, we fabricated polyacrylamide gels with varying concentrations of cross-linker to change the elastic modulus of the gels. We used gels that ranged in stiffness from 200 Pa to ∼6 kPa embedded with beads and imaged for traction force measurements as before. For comparison of forces between gels of different stiffness, we calculated the average traction stress exerted by stably spread cells between minutes 14 and 15 after spreading initiation. We found that the total force exerted by cells increased for soft substrates and rapidly saturated for stiffer substrates, as shown in Figure 4a. Similar results were obtained for earlier time points (unpublished data). Our observations suggest that Jurkat T-cells have the ability to sense the substrate stiffness and modulate the internally generated cytoskeletal forces as a function of substrate stiffness.


Cytoskeletal forces during signaling activation in Jurkat T-cells.

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

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  (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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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 (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.
Mentions: Many types of cells that interact with soft materials have the ability to sense the stiffness of their mechanical environment and respond to it by exerting larger forces on stiffer substrates (Janmey and McCulloch, 2007). Whether Jurkat cells respond similarly to substrate stiffness is not known. Recent experiments suggest that physical forces, such as those generated by the actin cytoskeleton, may be important for T-cell signaling. To examine whether Jurkat cells are sensitive to substrate stiffness, we fabricated polyacrylamide gels with varying concentrations of cross-linker to change the elastic modulus of the gels. We used gels that ranged in stiffness from 200 Pa to ∼6 kPa embedded with beads and imaged for traction force measurements as before. For comparison of forces between gels of different stiffness, we calculated the average traction stress exerted by stably spread cells between minutes 14 and 15 after spreading initiation. We found that the total force exerted by cells increased for soft substrates and rapidly saturated for stiffer substrates, as shown in Figure 4a. Similar results were obtained for earlier time points (unpublished data). Our observations suggest that Jurkat T-cells have the ability to sense the substrate stiffness and modulate the internally generated cytoskeletal forces as a function of substrate stiffness.

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