A Cell Number-counting Factor Regulates Group Size inDictyostelium by Differentially Modulating cAMP-induced cAMP and cGMP Pulse Sizes

Lei Tang,Robin Ammann,Tong Gao,Richard H Gomer

doi:10.1074/jbc.m102205200

Lei Tang, Robin Ammann + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m102205200

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Abstract

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.

Highlights

Controlling the size of a group of cells is critical for the development of multicellular organisms and for the maintenance of their normal functions [1]
To determine if counting factor (CF) regulates group size by regulating the cAMPinduced cGMP pulse, we examined the cAMP-induced cGMP pulse in Ax4 parental, smlAϪ, and countinϪ cells
We find that the breakup into groups of a specific size may be caused in part by the secreted CF altering cAMP signal transduction

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Cell culture, development of cells, and preparation of CM from starving cells were performed as described by Jain et al [37]. To study the effect of cAMP pulses on cAMP-induced cGMP pulses, Ax4 cells were treated with beef heart cAMP PDE (0.01 unit/ml, Sigma), PBM, or cAMP pulses (final concentration, 30 nM) every 6 min for 5 h starting 1 h after starvation. The effect of cAMP on the binding of [3H]GTP to membranes was measured following Snaar-Jagalska and Van Haastert [40] with the exception that instead of spinning membranes through silicone oil, membranes were collected by centrifugation for 2 min at 14,000 ϫ g. Guanylyl Cyclase and cGMP-specific PDE Assays—GTP␥S-stimulated guanylyl cyclase activity was measured following Snaar-Jagalska and Van Haastert [42] with the exception that cells were starved in PBM at 1 ϫ 106 cells/ml for 6 h, collected by centrifugation, washed twice, and resuspended in 40 mM HEPES/NaOH, pH 7.0, at 1.5 ϫ 108 cells/ml. Adhesion was measured by counting both the total number of cells and the number of single cells with a hemacytometer

RESULTS

Normalized cAMP stimulation of GTPase

DISCUSSION