CAX regulates cell substrate attachment. (a and b) Effect of CAX knockdown on chemotaxis. (a) Tracks of individual explants migrating toward beads soaked in Sdf-1 over the entire period of recording (left) or the initial 100 min (right). (b) Summary data (from three knockdowns) quantifying forward motion index, persistence, and velocity of migration. (c–g) Effect of CAX knockdown on cell dispersion. (c) Summary data from three knockdowns quantifying the size of control and morphant explants 30 min after plating. (d) Representative color-coded triangulation diagrams at time 0 and 6 h from control or morphant explants. (e) Time courses (from three knockdowns) quantifying triangle area. Data are normalized to control explants at time 0. (f) Transmitted light micrographs showing spreading of control or morphant explants over a 20-min period. Panels on the right show an overlay of the area occupied by the explants where red represents the extent of spreading. Bar, 500 µm. (g) Summary data from three knockdowns quantifying spread area. (h and i) Effect of Ca²⁺ buffering on cell dispersion. Triangulation (h) and spreading (i) analysis of explants treated with cell-permeable Ca²⁺ chelators (50 µM BAPTA/EGTA-AM) or DMSO 30 min before imaging. (j and k) Live cell imaging of CAX. Stills from time-lapse confocal imaging of explants coexpressing mRFP-CAX and either membrane GFP (j; bar, 10 µm) or focal adhesion kinase–GFP (k). Arrowheads mark small CAX-positive vesicles. (k) Bars: (top) 4 µm; (bottom) 1 µm. Heat map summarizes the mean projection for CAX-positive vesicles relative to the centroid of focal adhesions (intersection of white lines) within 5 × 5–µm regions of interest. Data are from 28 focal adhesions from two expression experiments. (l–n) Effect of CAX knockdown on focal adhesions. (l) Expression of focal adhesion kinase–GFP (left) and immunocytochemistry analysis using an antibody to phosphopaxillin (middle) in control and morphant explants. Overlays (right) show costaining of phalloidin. Bar, 20 µm. (m) Summary data (94–422 cells from two to three knockdowns) quantifying the number and size of labeled structures. (n) Lifetime analysis of focal adhesion kinase–GFP in control and morphant explants (30–58 focal adhesions from two knockdowns). 10 ng AMO1 was used for all knockdowns. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.