Rac, but not Cdc42, is critical for preventing cell displacement. Transfected U937 cells were adhered to VCAM-1 under static conditions (2 min) and exposed to fluid flow (2 dynes/cm2 for 2 min). Images at the level of anchor insertion were selected from confocal z stacks obtained before and after initiation of flow. (a) Representative cells transfected with PAK-PBD-YFP and their fluorescence intensity profiles within the indicated red lines. Each red dashed line provides a reference for PAK-PBD-YFP fluorescence intensity determined at the downstream edge of a cell and facilitates comparison to the fluorescence intensity at the upstream edge. (b) Quantification of PAK-PBD-YFP accumulation in upstream versus downstream regions (n = 10; three IEs; *, P < 0.0001). MFI, mean fluorescence intensity. (c) dnRac-GFP–transfected cells deformed upon exposure to flow. Lifeact-RFP and dnRac-GFP were colocalized in upstream adhesion contacts (arrowheads). (d) Displacement of U937 cells transfected with PAK-PBD-YFP, dnRac-GFP, and dnCdc42-GFP after exposure to fluid flow (2 dynes/cm2 for 60 s; means ± SD; n = 10 cells per group; two IEs; *, P < 0.001). (e) Displacement over time of representative transfected U937 cells. Arrows indicate the direction of fluid flow. Bars, 10 µm.
