Many eukaryotic cells can elicit intracellular signaling relays to produce pseudopodia and move up to the chemoattractant gradient (chemotaxis) or move randomly in the absence of extracellular stimuli and nutrients (random movement). A precise spatiotemporal regulation of Ras-GTPases, such as Ras and Rap, is crucial to induce pseudopodia formation and cellular adhesion during the chemotaxis and random movement. Here, we describe biochemical and real-time imaging methods for usingDictyosteliumto understand the signaling events important for chemotaxis and random cell movement. The chapter includes (1) a biochemical method to assess Ras and Rap1 activation in response to chemoattractant, (2) an imaging method to detect endogenous Ras and Rap1 activation in moving cells, and (3) a simultaneous imaging method to decipher the precise order and localization of these signaling events. With a combination of powerfulDictyosteliumgenetics, these methods will facilitate to elucidate a dynamic activation of Ras proteins and their inter relay with other signaling molecules during chemotaxis and random movement.
