CD81 regulates integrin-dependent adhesion and retrograde actin flow at the cell front. Confocal microscopical analysis of actin cytoskeleton dynamics at the cell front of migrating LPS-stimulated wild-type (A-D) and CD81^−/− BM-DCs (E-H) in a CCL19 gradient (600 ng/mL). To vizualize actin dynamics, cells were transfected with Lifeact-EGFP. Because of low adherence of mature BM-DCs, the cells were squeezed between an agarose gel and a fibronectin-coated surface, which represents a 3-dimensional environment. Actin dynamics and chemotaxis of motile cells were visualized at the basal plasma membrane by the use of a confocal microscope. BM-DCs were tracked over a period of 5 minutes at 2 seconds per frame. Retrograde actin flow was analyzed by kymograph analysis and line scan analysis (B-D,F-H) of yellow lines (in panels A and E, respectively) using ImageJ. (I) Quantification of retrograde actin flow velocities of migrating CD81^−/− and wild-type BM-DCs, respectively. (J) Quantification of retrograde actin flow velocities of migrating Talin1 RNAi BM-DCs and control BM-DCs, respectively. Three kymographs per cell were analyzed; each dot represents the value of 1 single kymograph (I-J). Shown data are representative for 1 experiment of 3. Error bars indicate ± SD. ***P ≤ .001. Bars represent 10 μm.