Rap1 accumulates at anchor insertions and prevents cell displacement. (a and c) Representative images of U937 cells transfected with wild-type Rap1-GFP (a) or Rap1-N17–GFP (dnRap1-GFP; c) and Lifeact-RFP. Cells were adhered to VCAM-1 under static conditions (2 min) and then exposed to a fluid flow (2 dynes/cm2). Flow induced accumulation of Rap1 and F-actin in the region of anchor insertion (open arrowheads). (b) Quantification of Rap1-GFP accumulation at the level of anchor insertion. The ratio of upstream versus downstream Rap1-GFP in the vicinity of the cortical actin cytoskeleton was determined for each cell before and during fluid flow (n = 15 cells; two IEs; *, P < 0.0001). MFI, mean fluorescence intensity. (c–e) In cells transfected with dnRap1-GFP, fluid flow did not induce anchor formation and upstream accumulation of F-actin or dnRap1-GFP. Cells gradually pulled away from their initial adhesion site. The dashed yellow lines in c provide a spatial reference between images. (d) Displacement of Rap1-GFP– versus dnRap1-GFP–transfected U937 cell 45 s after exposure to fluid flow (2 dynes/cm2; n = 10 per group; two IEs; *, P < 0.0001). (e) Displacement over time of representative U937 cells transfected with Rap1-GFP or dnRap1-GFP. Arrows indicate the direction of fluid flow. Error bars show means ± SD. Bars, 10 µm.
