Real-time measurements of cAMP production in live Dictyostelium cells

Abstract

Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP sensor that possesses high cAMP sensitivity and great temporal resolution. We show the transient profile of cAMP accumulation in live Dictyostelium cells and establish that chemoattractants control intracellular cAMP dynamics by regulating synthesis via the adenylyl cyclase ACA. aca– cells show no significant change in FRET response following chemoattractant addition. Furthermore, cells lacking ACB, the other adenylyl cyclase expressed in chemotaxing cells, behave similarly to wild-type cells. We also establish that the RegA is the major phosphodiesterase that degrades intracellular cAMP in chemotaxis-competent cells. Interestingly, we failed to measure intracellular cAMP compartmentalization in actively chemotaxing cells. We conclude that cytosolic cAMP, which is destined to activate PKA, is regulated by ACA and RegA and does not compartmentalize during chemotaxis.