Figure 2.

Load-Fail Dynamics Is Dependent on Actin Dynamics but Not Myosin-II Contractility

(A) Confocal fluorescent imaging time-lapse of a 1G4-TCR Jurkat T cell expressing actin-SNAP labeled with SNAP-Cell-505 interacting with an antigen-coated coverslip before and after the addition of 500 nM jasplakinolide. The red arrowheads indicate stationary features in the lamellipodium after treatment. At right is the corresponding kymograph. The red arrowheads show actin retrograde flow before treatment and arrested flow after treatment.

(B) Confocal fluorescent imaging time-lapse of a 1G4-TCR Jurkat T cell expressing actin-SNAP labeled with SNAP-Cell-505 interacting with an antigen-coated coverslip before and after the addition of 100 μM Y27632. The red arrowheads indicate lamellipodial width reduction after treatment. The blue arrowheads indicate the loss of actin arcs following treatment. At right is the corresponding kymograph.

(C) Optical flow analysis of actin flow during activation. The red arrows indicate a vector field, showing the direction of actin flow. Scale bar, 2 μm. At bottom is an extreme closeup of the dashed box at top.

(D) Pseudo-color intensity map of radial and azimuthal actin velocity before and after the addition of 500 nM jasplakinolide. Scale bar, 2 μm. Bottom: corresponding line profiles.

(E) Pseudo-color intensity map of radial and azimuthal actin velocity before and after the addition of 100 μM Y27632. Scale bar, 2 μm. Bottom: corresponding line profiles.

(F) Fold change of radial (top) and azimuthal (bottom) actin velocity within the lamellipodium of a 1G4-TCR Jurkat T cell interacting with an antigen-coated coverslip after the addition of 500 nM jasplakinolide or 100 μM Y27632. Error bars show means and SDs; N = 12 cells per condition.

(G) Left: kymograph showing the dynamics of actin and fluorescent beads before and after the addition of 500 nM jasplakinolide (time point indicated by the large arrowhead). Actin dynamics shows clear retrograde flow pre-treatment and the stabilization of flow immediately afterward. Load-fail events are visible before treatment, as indicated by the small white arrowheads, and the events are dramatically reduced following treatment. Scale bar, 1 μm. Center: representative selection of tracks showing the dynamics of beads pre- and post-addition of 500 nM jasplakinolide. Treatment leads to the complete termination of load-fail events. Right: quantification of load-fail event rate before and after the addition of jasplakinolide. Error bars show means and SDs; N = 4 cells.

(H) Left: kymograph showing the dynamics of actin and fluorescent beads before and after the addition of 100 μM Y27632 (time point indicated by the large arrowhead). Scale bar, 1 μm. Center: representative selection of tracks showing the dynamics of beads pre- and post-addition of 100 μM Y27632. Right: quantification of load-fail event rate before and after the addition of Y27632. Error bars show means and SDs; N = 5 cells.