Actin polymerization and coordination at the leading edge of dendritic cells. (A) DCs were injected between a glass coverslip and a layer of agarose containing 2.5 μg/mL CCL19. This experimental setup served for studying the chemokinetic response and actin dynamics of DCs at 37°C, 5% CO₂ (in panels B-E). (B) Initial radial spreading, symmetry breaking, and subsequent polarization of DCs was recorded by time-lapse videomicroscopy. Morphology outlines of DCs were obtained by morphometric analysis (MetaMorph). A time sequence of a representative WT and Cdc42^−/− DC is shown. Time (minutes:seconds). Scale bar represents 50 μm. Objective: LD A-Plan 20×/0.30 Ph1 (Zeiss). (C,D) WT (C) and Cdc42^−/− (D) DCs were transfected with the actin marker lifeact:GFP and actin dynamics observed by TIRF microscopy over time. (Top panels) A time sequence of a representative entire cell is shown. Time (minutes:seconds). Scale bar represents 10 μm. Objective: Plan-FLUAR 100×/1.45 oil (Zeiss). (Bottom panels) Leading edge regions indicated by the white boxes were analyzed for phases of protrusion and/or retraction. After background flattening (MetaMorph), a time-lapse montage over 91 seconds (2 seconds/frame) presents the dynamics at the leading edge. (E) Quantification of the actin polymerization rate at the leading edge by kymograph analysis. Cells were derived from 2 independent BM DC cultures (of both WT and Cdc42^−/− mice). Single cells (dots) were analyzed (line: mean, t test, P = .069, WT: n = 19, Cdc42^−/−: n = 24).