Regulation of cell migration by fiber width and dimensionality. (A) Triplets (bracketed) of 1, 2.5, 5, 15, and 20 µm-wide lines used for cell migration tracking. (B) Biphasic effect of fiber width on fibrillar migration rate. (C) TIRF images of GFP-vinculin containing adhesions on a 2.5-µm line show two parallel sets of adhesions. The arrow indicates the direction of migration. (D) Fibroblasts migrating with a 3D/1D phenotype along multiple 1-µm lines. (E) 1D to 2D ECM transition induces spreading of the leading edge (arrow) and formation of many cell processes (arrowheads). The red box indicates ROI in time lapse below. (F) Rac siRNA knockdown in 2D induces a uniaxial phenotype lacking the single linear adhesion seen in 1D and does not increase migration rate. *, P < 0.05 versus all conditions except multilines; +, P < 0.05 versus 15–40 µm. Con, control. Error bars indicate SEM. Bars, 10 µm.