GxcM localizes in the rear of Dictyostelium cells. (A) Schematic representation of GxcM, GxcM^4A, and GxcM^N790. The star indicates alanine mutations (aa 593–596) in the DH domain. (B) Time-lapse imaging of GxcM-GFP in cells migrating under agarose along a folic acid gradient. The white triangle indicates gradient direction. (C) Left: Schematic of the angle-series plot used to quantify protein distribution. The fluorescent intensity in the cortical region of a cell was plotted along the perimeter, with 0° and +180°/−180° corresponding to the migrating front and rear, respectively. Right: Angle-series plot of GxcM-GFP in under agarose chemotaxing cells as shown in B. Solid line represents the mean and shade represents mean ± SD. n, number of cells analyzed. (D, F, and H) Distribution of PHcrac-GFP and GxcM-RFP (D), GxcM^N790-RFP (F), or GxcM^4A-RFP (H) in randomly migrating cells. (E, G, and I) Translocation of GxcM-GFP (E), GxcM^N790-GFP (G), and GxcM^4A-GFP (I) in response to cAMP stimulation added at time 0 (pre: before stimulation). (J, K, N, and O) Distribution of LimEΔcoil-RFP and the GFP control (J), GxcM-GFP (K), GxcM^N790-GFP (N), or GxcM^4A-GFP (O) in randomly migrating cells. (L, M, P, and Q) Distribution of GFP-ArpC4 and the RFP control (L), GxcM-RFP (M), GxcM^N790-RFP (P), or GxcM^4A-RFP (Q) in randomly migrating cells. Scale bars, 5 μm.