Starved Dictyostelium cells lacking cGMP-binding protein C (gbpC-; no guanylyl cyclase activity) and in medium supplemented with 60 µM LY294002 (a PI3K inhibitor) and 1µM cAMP migrate toward the anode (right). A direct-current electric field of 10 Vpcm was applied and the cells were observed for 20 min at 5-sec intervals. (Scale bar, 50 µm.) In contrast, cathode-directed migration is strongly attenuated in wild-type cells in the same medium. Compare with wild-type control video (CIL# 24043) and other mutants lacking guanylyl cyclase activity (CIL#24044 and 24046). Videos are supporting information in Proc Natl Acad Sci (2009). 106: 6667-6672.
Starved cells were suspended in buffer containing 10 mM Na/K PO4, 2 mM MgSO4, and 0.2 mM CaCl2, (pH 6.5) and 4 mM caffeine to inhibit adenylyl cyclase activity (which also reduced cell–cell interactions). A chamber was constructed to allow application of a direct-current electric field (Sato et al. 2007. BioSystems). The cells in the chamber were observed with an Olympus IX-71 inverted microscope with phase contrast optics. Cell behavior was recorded with a cooled CCD camera (MicroMax; Princeton Instruments) and software (MetaMorph; Molecular Devices).
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