Starved Dictyostelium cells lacking both guanylyl cyclases (GCase A and GCase), but expressing GCase with an inactivated catalytic domain (sGCΔCat) and in medium supplemented with 60 µM LY294002 (a PI3K inhibitor) and 1µM cAMP migrate continuously 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, 24045). 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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