Figure 3. Stress-induced Rac activation is independent of Src activity.

(A) Time-lapse images of a representative smooth muscle cell transfected with the Rac biosensor and pretreated with a specific Src inhibitor, PP1 (10 µM for 1 hr), before stress application. Florescence resonance energy transfer (FRET) changes were observed within 0.3 s after stress application as shown in the inset with enlarged cell periphery. White arrow indicates magnetic bead movement direction when a stress of 17.5 Pa is applied. Scale bar  = 10 µm. (B) Average data showing rapid Rac activation by stress even though Src was inhibited by PP1. (n = 6 cells, mean +/− s.e.). (C) Time-lapse emission ratio images of the Rac biosensor in response to 17.5 Pa stress in a representative Src/Yes/Fyn triple-knockout (SYF−/−) MEF cell. The inset with the enlarged area of the cell periphery shows rapid activation of Rac. White arrow indicates RGD coated magnetic bead movement direction. Scale bar  = 10 µm. (D) Probe specificity of Rac activation. Normalized YPet/CFP emission ratio shows that Rac activation is induced in SYF−/− mouse MEFs by mechanical stress applied via RGD-coated magnetic beads that bind specifically to integrin receptors (n = 6 cells). Although the PDGF-induced Rac activation is known to be downstream of Src [4], knocking-out Src did not prevent Rac activation by stress. However, application of the same magnitude of stress at 17.5 Pa with beads coated with transferrin (TRF-bead) or poly-L-lysine (PLL-bead) did not activate Rac (TRF-bead, n = 4 cells; PLL-bead, n = 4 cells). Mean +/− s.e.