Fluid flow triggers the formation of upstream F-actin–rich anchors. (a and e–h) Live-cell imaging of Lifeact-GFP–transfected U937 cells. Cells suspended in an Mn²⁺-containing buffer were adhered under static conditions to a VCAM-1–coated surface of a flow chamber and then exposed to fluid flow (2 dynes/cm² for 2 min). (a) Images of a representative cell at the indicated levels from the adhesion surface before and after exposure to fluid flow. Upstream anchors (solid arrowheads) and anchor insertion sites (open arrowheads) are located within several micrometers of the adhesion surface. The circles represent the cell periphery at the midpoint of the cell as determined by superimposing DIC images. (b) Representative SEM images of perfusion-fixed untransfected U937 cells and monocytes (Mono) exposed to fluid flow. Upstream anchors (solid arrowheads) are visualized clearly when chucks were rotated to obtain a side view. (c) Quantification of anchor dimensions in SEM images (n = 32 and 36; three IEs). Data were normalized to the mean cell height or width (8.03 ± 0.82 and 8.55 ± 0.46 µm for U937 cells, n = 10; and 4.94 ± 0.54 and 5.33 ± 0.57 µm for monocytes, n = 15). (d) SEM showing anchor formation in human monocytes infused into VCAM-1–coated chambers (0.5 dynes/cm² for 30 s and then 2 dynes/cm² for 60 s). Upstream anchors are indicated by solid arrowheads. (e and f) Sequential images reveal the kinetics of F-actin accumulation and anchor formation. The time relative to the introduction of a fluid shear force (2 dynes/cm²) is indicated. (e) Top and side views of collapsed z stacks show F-actin–rich anchors (arrowheads) at 15 s. The GFP signal is enhanced in the inset. (f) Images obtained just above the adhesion surface reveal that anchors develop from projections that contacted the adhesion surface under static conditions (solid arrowheads). The accumulation of F-actin in the cortical actin cytoskeleton (open arrowheads) is evident within 1 s after exposure to fluid flow. See Video 6. (g and h) Lifeact-GFP fluorescence intensity analysis across cells. (g) Representative images of a Lifeact-GFP–transfected U937 cell before and after exposure to fluid flow and corresponding GFP fluorescence profiles within yellow lines. Images were obtained at a level at which anchors insert into the cell body. The horizontal dashed red lines correspond to the GFP intensity in the downstream cortical actin cytoskeleton. The graph shows upstream to downstream GFP mean fluorescence intensity (MFI) ratios of 10–15 randomly selected cells (four IEs; *, P < 0.001). (h) Analysis of GFP intensity at different levels from the adhesion surface as shown in the schematic. In each optical slice from a z stack, fluorescence profiles within the yellow lines are shown. Dashed lines at peaks correspond to the downstream (yellow) and upstream (red) cortical actin cytoskeleton and anchors upstream of the cell body (white). The numbers at the top of images refer to the height of each image above the adhesion surface. The upstream to downstream cortical actin mean fluorescence intensity ratios are plotted. The maximal difference in upstream versus downstream cortical actin fluorescence ratio is observed at a level at which anchors insert into the cortical actin cytoskeleton. This representative experiment demonstrates how optical slices from a z stack were selected for analysis in g. Also refer to Fig. S3. Arrows indicate the direction of fluid flow. Error bars show means ± SD.