Characterization GxcM overexpressing cells. (A) Top: Trajectories of randomly migrating cells (n = 64 for GFP/WT and 50 for GxcM-GFP/WT). Bottom: Summary of motility parameters. Data were from three independent experiments; mean ± SEM. (B) Quantification of TRITC dextran (TD) uptake. Data were from three independent experiments; the scatter plot shows data points with mean ± SEM; n, number of cells analyzed. (C) Left: GFP/WT and GxcM-GFP/WT cells grown on a cell culture plate were fixed and stained with DAPI to visualize the nuclei. Right: Quantification of nuclei. n, number of cells analyzed. (D) Time-lapse imaging of randomly migrating cells expressing GFP-ArpC4 and untagged GxcM. (E) Cells expressing GxcM-GFP from an expression cassette integrated into the genome as a stable single copy via restriction enzyme-mediated integration (REMI) was immunostained with an anti-GFP antibody. (F–I) Localization of GFP-Coronin in randomly migrating cells expressing RFP (F), GxcM-RFP (G), GxcM^N790-RFP (H), or GxcM^4A-RFP (I). (J and K) Localization of GFP-Coronin and RFP (J) or GxcM-RFP (K) in WT cells migrating under agarose along a folic acid gradient. Angle-series plots on the right show fluorescent intensity distribution of the indicated proteins along the perimeter of the cell, with 0° and +180°/−180° corresponding to the migrating front and rear, respectively. Solid lines represent the mean and shades represent mean ± SD. n, number of cells analyzed. (L) Top: Trajectories of cells migrating under 0.5% agarose along a folic acid gradient (n = 30 for GFP/WT and 29 for GxcM-GFP/WT). Bottom: Summary of chemotaxis parameters. Data were from at least three movies; means ± SEM. Scale bars, 5 μm.