Murine Syk^–/–PMNs formed multiple lamellipodia and showed impaired site-directed migration. (A) Confocal microscopy images of polarized murine Syk^+/– and Syk^–/– PMNs. Adhesion and polarization on immobilized fibrinogen was induced by fMLP (100 nM) for 30 minutes at 37°C. Indirect fluorescence staining of Syk using a polyclonal Syk Ab and a secondary Alexa 546–conjugated anti–rabbit IgG Ab (red). F-actin was assessed using Alexa 488–conjugated phalloidin (green). In Syk^+/– cells, F-actin and Syk were enriched at the leading edge (arrow); colocalization is shown in yellow (merge). In contrast, Syk^–/– PMNs formed multiple lamellipoda (arrowheads). Transmission images show the morphology of the corresponding cell (transmission). Scale bar equals 10 μm. (B-C) Analysis of migrating Syk^+/– and Syk^–/– PMNs in Zigmond chambers on immobilized fibrinogen in response to a gradient of 10 μM fMLP. (B) Time-lapse images (1-5) of Syk^+/– and Syk^–/– PMNs were recorded at intervals of 1 minute. Syk^+/– PMNs formed 1 stable lamellipodium and performed site-directed migration over the observed time period (arrow). In contrast, Syk^–/– PMNs showed multiple unstable lamellipodia (arrowheads) and impaired migration. Scale bar equals 10 μm. Supplementary video sequences show migrating Syk^+/– PMNs (Video S4) and Syk^–/– PMNs (Video S5). (C) Polar plot of tracked migration paths of Syk^+/– and Syk^–/– PMNs. Time-lapse images were taken every 15 seconds over a period of 12 minutes. In contrast to control cells, Syk^–/– PMNs showed impaired site directed migration. Scale bar equals 25 μm. Migration tracks from one experiment are shown. Numbers indicate the percentage of cells that ended up within a 180° angle facing the source of the chemoattractant. Results are representative of 3 independent experiments.