A secreted counting factor (CF), regulates the size of Dictyostelium discoideum fruiting bodies in part by regulating cell-cell adhesion. Aggregation and the expression of adhesion molecules are mediated by relayed pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. A 1-min exposure of cells to purified CF increases the cAMP-induced cAMP pulse. CF does not affect the cAMP receptor or its interaction with its associated G proteins or the translocation of the cytosolic regulator of adenylyl cyclase to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, whereas reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The results suggest that regulating cell-cell adhesion, the cGMP pulse size, or the cAMP pulse size can control group size and that CF regulates all three of these independently.

Little is known about how a morphogenetic rearrangement of a tissue is affected by individual cells. Starving Dictyostelium discoideum cells aggregate to form dendritic streams, which then break up into groups of Ϸ2 ؋ 10 4 cells. Cell number is sensed at this developmental stage by using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF indicates that there is a large number of cells, which then causes the aggregation stream to break up. Computer simulations indicated that stream breakup could be caused by CF decreasing cell-cell adhesion and͞or increasing cell motility, and we observed that CF does indeed decrease cell-cell adhesion. We find here that CF increases cell motility. In Dictyostelium, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and the ABP-120 actin-crosslinking protein. Partially inhibiting motility by using drugs that interfere with actin polymerization reduces stream dissipation, resulting in fewer stream breaks and thus larger groups. CF also potentiates the phosphorylation and redistribution of myosin while repressing its basal level of assembly. The computer simulations indicated that a narrower distribution of group sizes results when a secreted factor modulates both adhesion and motility. CF thus seems to induce the morphogenesis of streams into evenly sized groups by increasing actin polymerization, ABP-120 levels, and myosin phosphorylation and decreasing adhesion and myosin polymerization.

In Dictyostelium discoideum counting factor (CF), a secreted ϳ450-kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups, with an EC 50 of ϳ3 ng/ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1-min exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway.125 I-Labeled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have ϳ53 cell-surface sites that bind countin with a K D of ϳ1.5 ng/ml or 60 pM. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities.