Schematic diagram of an acidic vesicle with a bafilomycin-sensitive vesicular proton pump and an ATP-dependent Ca²⁺/H⁺ exchanger. Protons are transferred into the vesicle by the proton pump, and the Ca²⁺/H⁺-ATPase pumps Ca²⁺ into the vesicles in exchange for the protons. The thin arrows illustrate how weak bases such as ammonia oppose Ca²⁺ sequestration by diffusing into the vesicles in the uncharged form (e.g., NH₃), picking up protons, and exiting in the charged form (NH₄⁺), thus creating a steady proton leak. Ca²⁺ sequestration is also sensitive to vanadate, indicating that it is dependent on a Ca²⁺-ATPase rather than a simple Ca²⁺/H⁺ antiporter. The Ca²⁺ pump is a PMCA-type ATPase (50) that is inhibited by tBHQ but not thapsigargin (60), and the acidic Ca²⁺ stores seem to be of two kinds, so-called contractile vacuoles and acidocalcisomes (45). The term acidic vesicle is used to refer to vesicles that have an associated vacuolar H⁺-ATPase whether or not their lumens are markedly acidic. In fact, contractile vacuoles are not stained markedly by neutral red, indicating that they are not strongly acidic. (Figure prepared with the assistance of Anthony Morgan.)

Tipped aggregate and slug. Cells with acidified vesicles are indicated schematically by the red dots, and cells whose acidic vesicles are poorly acidified are indicated by the gray dots. According to the view presented here, scroll waves of cAMP propagate from the tip through the prestalk zone of aggregates, leading to forward movement of the aggregates, but the waves are unable to propagate through the prespore zone because the cells of this zone do not have acidified vesicles. Only prestalk cells, therefore, are thought to experience Ca²⁺ transients, and these transients drive prestalk cell-specific gene expression and inhibit prespore cell-specific gene expression. Prespore cells contain a constitutively active adenylyl cyclase, ACG, responsible for prespore-specific gene expression (1). The highly acidified autophagic vacuoles of prestalk cells break down proteins and RNA, generating substantial amounts of ammonia that diffuses rapidly, and is proposed to form an anterior-posterior gradient that contributes to the inhibition of vesicle acidification in the prespore cells. Prestalk cells also create a local acidic environment consisting of protons—or some kind of weak acid—that protects the anterior cells from the inhibitory effects of the ammonia and promotes vesicle acidification. Prespore cells may move together with the prestalk cells by a follow-my-leader type of mechanism, as well as by being constrained within the aggregate by the slime sheath. (Figure prepared with the assistance of Anthony Morgan.)

Neutral red-stained slugs. The prestalk zones are strongly stained. The rear regions of the prespore zones are also stained, weakly, perhaps because ammonia diffuses away from the backs of the slugs and permits some acidification of acidic compartments. (Reproduced from reference 32 with permission.)

Simplified diagram of the proposed network underlying signaling. Binding of cAMP to G-protein-coupled cell surface receptors (car1) activates a series of components including CRAC, Aimless, Erk2, and Pianissimo, leading to transitory stimulation of the aggregative adenylyl cyclase ACA (44), and much of the cAMP produced is released from the cell to propagate the cAMP signal. This signaling loop has traditionally attracted the most attention. However, according to the model presented here, equally essential for signaling is the generation of a Ca²⁺ transient resulting from the release of Ca²⁺ from acidic vesicles (AV). Ca²⁺ release presumably involves the production of some unidentified agent that binds to the membrane of the acidic vesicles. I propose that the Ca²⁺ transients activate Ca²⁺-dependent proteins, one or more of which are required directly or indirectly to activate ACA, as well as to induce prestalk cell-specific gene expression. Ca²⁺ is returned to the acidic vesicles by the action of the V-type H⁺-ATPase and Ca²⁺/H⁺-ATPase (NB, both transporters are actually in the acidic vesicle membrane), and this sequestration is opposed by the action of weak bases, which therefore reduce the responsiveness of cells to cAMP signals. (Figure prepared with the assistance of Anna Gross.)