Figure 7.

Vinculin and talin couple integrins to actin. (A) Jurkat T cells were stably transfected with shRNA vectors targeting talin (shT), vinculin (shV), and α-actinin 4 (shA4). Untransfected cells (UT) and cells expressing the empty shRNA vector (EV) were used as controls. Expression of each protein was tested by Western blotting with the indicated antibodies; the antibody against α-actinin recognizes both the 1 and 4 isoforms. Panels are regions from one blot, adjusted individually to enhance brightness and contrast, using only linear tools. The complete blot is available in Figure S2 in Supplementary Material. (B) Cells generated as in (A) were stimulated on coverslips coated with OKT3 and VCAM-1, and actin flow rates in the LP region were quantified. Mean ± SD were calculated from measurements made in 10–40 cells for each condition, ***p < 0.001 compared with EV control. (C–E) Untransfected (C), talin-suppressed (D), and vinculin-suppressed (E) Jurkat T cells were stimulated on coverslips patterned with VCAM-1 surrounded with OKT3 for 15 min, fixed, and labeled as indicated. (F) Untransfected Jurkat T cells were treated with Y-27632 and Jasplakinolide to arrest actin flow, and stimulated on coverslips patterned with VCAM-1 surrounded with OKT3. Scale bar = 10 µm.