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Fig. 7. The defects in the behavior of cells of the myosin heavy chain phosphorylation mutant 3XASP in the absence of attractant and in a spatial gradient of cAMP are remarkably similar to those of pten– cells. (A,B) Centroid and perimeter tracks of representative parental JH10 and representative 3XASP cells, respectively, migrating in buffer in a perfusion chamber in the absence of attractant. (Compare with data for corresponding pten– cells in Fig. 1B,D.) (C,D) Centroid tracks and perimeter tracks of representative JH10 and 3XASP cells, respectively, undergoing chemotaxis in a spatial gradient of cAMP. Arrows in A-D indicate net direction and + signs in C and D indicate a positive chemotactic index. (Compare with corresponding pten– data in Fig. 3B,D.) (E,F) 3D-DIAS reconstructions of a JH10 and 3XASP cell, respectively, translocating in buffer reveal that whereas the former extends a dominant anterior pseudopod with occasional lateral pseudopods, the latter extends multiple pseudopods from both anterior and posterior regions of the cell body. Color coding of reconstructions are the same as in Fig. 1. (Compare with corresponding data for AX2 and pten– in Fig. 1E and F, respectively.) (G) The defects in motility and chemotaxis reflected in measured parameters are remarkably similar for 3XASP and pten– cells. The ratio of the average mutant to parental parameter is presented for cells translocating in the absence of cAMP and for cells responding to a spatial gradient of cAMP. Instant. vel., instantaneous velocity; Dir. change, direction change; Pers., directional persistence; % Pos. chemotaxis, percentage positive chemotaxis. The 2D and 3D analyses of JH10 and 3XASP for A-F were performed anew for this study. The data for computing the parameter proportions for 3XASP and JH10 represent a combination from Heid et al. (Heid et al., 2004) and new experiments. The chemotactic index for 3XASP was recomputed from new data for which interval time was the same as that used for the pten– study.
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