Cytoskeletal forces during signaling activation in Jurkat T-cells.
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.
Affiliation: Department of Physics, University of Maryland, College Park, MD 20742.Show MeSH
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Mentions: We first focused on the role of actin polymerization and depolymerization dynamics on cellular traction forces with latrunculin-A (Lat-A) to inhibit polymerization of actin, jasplakinolide (Jasp) to stabilize preexisting actin filaments, and CK-666 to inhibit the activity of Arp2/3, an actin-nucleating protein. Inhibition of actin polymerization by Lat-A resulted in the disruption of preexisting lamellipodia and actin-rich structures visible in the cell surface contact zone (Figure 2a), with a reduction in traction (Figure 2b). Application of Lat-A 5 min after initiation of spreading resulted in similar disruption of actin and loss of traction (unpublished data). Initial TCR signaling upon stimulation has been shown to result in immediate recruitment of signaling proteins that subsequently lead to Arp2/3 activation (Billadeau et al., 2007; Burkhardt et al., 2008; Beemiller and Krummel, 2010; Dustin and Groves, 2012). Hence the Arp2/3 complex might also be important in mechanically linking TCR to the F-actin cytoskeleton and therefore involved in the force generation process. Inhibition of Arp2/3 activation by addition of CK-666, an inhibitor that locks the Arp2/3 complex in an inactive conformation (Nolen et al., 2009), led to the retraction of lamellipodia and termination of edge dynamics (Figure 2c). This is in accordance with several previous observations that the Arp2/3 complex is essential for maintaining lamellipodial structure (Gomez et al., 2007). Consistent with our expectations, addition of CK-666 led to a reduction in the traction forces, as seen from the before-and-after traction maps (Figure 2d). CK-666 application also led to a reduction in F-actin intensity and retrograde flow but not a complete inhibition of actin flow (Supplemental Figure S1). Stabilization of F-actin upon addition of Jasp reduces the retrograde flow of actin in cells (Babich et al., 2012). Addition of 1 μM Jasp resulted in the reduction of traction forces in most cells, as shown in the traction maps before and after inhibitor addition (Figure 2, e and f). However, the effect of Jasp was somewhat variable, with some cells showing little effect of Jasp on actin flow and edge dynamics. These cells typically did not show a decrease in traction. As a control, addition of dimethyl sulfoxide (DMSO) carrier alone did not significantly affect the actin structures in the cell or the traction forces generated (Figure 2, g and h, and Supplemental Figure S1).
Affiliation: Department of Physics, University of Maryland, College Park, MD 20742.