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Fig. 6 Mechanism of chemokine-induced polarization. (A) Rac1 activity is required for ligand-induced polarity establishment. Localization of Ezrin (green, Upper), Esyt2a (green, second panel), MTs (green, third panel), and Lifeact (magenta, Lower) in DN-Rac1-expressing PGCs in the presence of a chemokine gradient. Graphs show the polarity index of Ezrin (Upper), Esyt2a (Middle), and the MTs (Lower). (B) Localization of Ezrin (green, Left), Esyt2a (green, Middle), and MTs (green, Right) in DN-ROCK-expressing PGCs in the presence of a chemokine gradient. Lifeact is marked in magenta. Inhibition of contractility by DN-ROCK affects the definition of the cell rear in these cells. (C) Enhancement of contractility by expression of a constitutively active RhoA (CA-RhoA) in the presence of the chemokine gradient with Lifeact (magenta) and Ezrin (Left), Esyt2a (Middle), and MTs (Right) in green. (D) The angle of protrusions relative to the position of the attractant source in wild-type cells (Left, n = 72), and in PGCs with increased contractility (Right, n = 50). In the rose plot, 0° indicates the source of the chemokine. (E and E′) Cxcr4b expression in control (E) and CA-RhoA-expressing cells (E′). The plasma membrane is labeled with a farnesylation signal and Cxcr4b is an in-frame fusion with GFP (z-projection). In A–D the distribution of the chemokine (Cxcl12a) is illustrated by the gradient in the red rectangle. (Scale bars, 10 µm.)