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First published online 28 January 2009
doi: 10.1242/dev.032607

Development 136, 803-812 (2009)
Published by The Company of Biologists 2009
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Steroids initiate a signaling cascade that triggers rapid sporulation in Dictyostelium

Christophe Anjard, Yongxuan Su and William F. Loomis*

Center for Molecular Genetics, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.


Figure 1
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  		Fig. 1. Synergy between Dictyostelium discoideum strains unable to produce SDF-2. (Above) Mutant strains lacking one of six proteins essential for production of SDF-2 were mixed in equal proportions with cells lacking another of the indicated proteins. After developing together for 24 hours, fruiting bodies were collected in cAMP buffer and the supernatant tested for SDF-2 activity. Minus indicates that less than 0.2 units of SDF-2/103 cells were recovered, whereas plus indicates that more than 5000 units of SDF-2/103 cells were produced. Strains that failed to synergize to produce SDF-2 constituted a synergy group. (Below) A signaling cascade is proposed to connect the two groups. GrlA and GrlE are GPCRs and GadA is glutamate decarboxylase. G{alpha}4 and G{alpha}7 are trimeric G protein components. AcbA, acyl-CoA-binding protein. GABA, {gamma}-aminobutyric acid. SDF-2 is a 34 amino acid signaling peptide.

 

Figure 2
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  		Fig. 2. Time course of spore formation and SDF-2 production after induction by SDF-3. (A) Induction of spore formation by SDF-2 or SDF-3. KP cells were plated in 24-well plates at 1x103 cells/cm2 in cAMP buffer. After overnight incubation at 22°C, 10 pM SDF-2 or 10 units SDF-3 were added and the number of spores was scored every 10 minutes for 1 hour and at 90 minutes. Antibodies against GABA or AcbA were added prior to induction. Open circle, induction by SDF-2; black diamond, induction by SDF-3; open diamond, induction by SDF-3 in the presence of anti-AcbA antibodies (1/500); black circle, induction by SDF-3 in presence of anti-GABA antibodies (1/5000). Each experiment was repeated at least three times. (B) Induction of SDF-2 production by SDF-3, hydrocortisone or GABA. 10 nM GABA (triangle), 10 nM hydrocortisone (diamond) or 10 units SDF-3 (square) were added to KP cells and aliquots were taken at the indicated times. SDF-2 production was measured after purification over anion-exchange resin. In another set of experiments, anti-GABA antibodies were added just prior to SDF-3 (circle) or at the indicated time after SDF-3 addition (arrows). In these experiments, SDF-2 production was measured 11 minutes after SDF-3 addition. The arrow with a minus sign indicates the time when addition of anti-GABA antibodies prevented SDF-2 production, and the arrow with a plus sign indicates the time when addition of anti-GABA antibodies did not prevent SDF-2 production. The data for GABA are derived from Anjard and Loomis (Anjard and Loomis, 2005Go). (C) The delay between SDF-3 addition and SDF-2 production increases with the volume of the bioassay. KP cells were starved overnight in 500 µl cAMP buffer (standard conditions). Just before being used in the assay the volume was adjusted to that indicated, by adding or removing cAMP buffer. SDF-2 production was stimulated by addition of 2 units SDF-3/100 µl (black square) or 10 nM GABA (open square). Aliquots were taken every 30 seconds and tested for SDF-2 production. The earliest time SDF-2 could be detected for a given volume was plotted. Error bars corresponding to ±15 seconds are given. Each experiment was repeated two to three times.

 

Figure 3
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  		Fig. 3. SDF-2 production and spore formation in wild-type and mutant Dictyostelium strains. Cells of the indicated wild-type (A) and mutant (B-E) strains were developed on filters (non-nutrient agar for gpa4-) and harvested at the mid-culmination stage. The fruiting bodies were dissociated and the cells washed before plating at 1x104 cells/cm2 in cAMP buffer. The cells were then treated with no addition (none), 10 pM synthetic SDF-2, 10 nM GABA, 10 units of SDF-3 or 100 nM hydrocortisone. Spores were counted after 1 hour. An aliquot of the supernatant was harvested for each sample to measure the amount of SDF-2 produced after purification using anion-exchange resin. Minus indicates production of less than 0.2 units of SDF-2/103 cells, whereas plus indicates more than 5000 units of SDF-2/103 cells.

 

Figure 4
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  		Fig. 4. Sporulation induced by steroids. Developed KP cells were incubated with various concentrations of hydrocortisone or dexamethasone. The number of spores was determined after 1 hour of incubation. Each experiment was repeated three to five times. Antibodies against GABA (circle) or AcbA (open square) were added prior to the addition of (A) dexamethasone (black square) or (B) hydrocortisone (black square).

 

Figure 5
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  		Fig. 5. Competition between mifepristone and either hydrocortisone or SDF-3. KP cells were plated at low-density in cAMP buffer and incubated overnight. The indicated amounts of mifepristone and either SDF-3 (A) or hydrocortisone (B) were added. The number of spores was scored 1 hour later. One unit (black square), 10 units (open square), or 100 units (circle) of SDF-3 were added together with the indicated amounts of mifepristone. Hydrocortisone was added to 10 nM (black square), 100 nM (open square) or 1 µM (circle) together with the indicated amounts of mifepristone. Each experiment was repeated at least three times.

 

Figure 6
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  		Fig. 6. SDF-3 and hydrocortisone decrease the level of GABA required to overcome glutamate inhibition. Low-density KP cells were incubated overnight in cAMP buffer before addition of 1 mM glutamate. The indicated amount of GABA was then added with 2 units of SDF-3 (black circle) or 10 nM hydrocortisone (open circle) or left untreated (black square). Spores were counted after 1 hour. Each experiment was repeated at least three times. Almost identical results were obtained in the presence of 10 mM glutamate.

 

Figure 7
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  		Fig. 7. Involvement of GABA transaminase in SDF-2 production. (A) Low-density KP cells were incubated overnight (~18 hours) before the addition of the indicated compounds. The following concentrations were used: 1 µM vigabatrin, 10 units of SDF-3, 10 nM CGP55845, 1/5000 anti-GABA antibodies (final dilution), 1/500 anti-AcbA antibodies (final dilution). The number of spores was scored 1 hour after the addition of SDF-3 or 2 hours after the addition of vigabatrin. An aliquot of the cell supernatants was harvested and SDF-2 purified using anion-exchange resin before quantification on fresh KP cells. (B) Low-density gabT-null KP cells lacking functional GABA transaminase were incubated for 17 hours in cAMP buffer. The number of spores was scored before and again 3 hours after addition of 1/5000 anti-GABA antibodies (final dilution), 1/500 anti-AcbA antibodies (final dilution), or 10 nM CGP55845. Aliquots of the cell supernatants were harvested and SDF-2 purified using anion-exchange resin before quantification on fresh KP cells. Minus indicates production of less than 0.2 units of SDF-2/103 cells, whereas plus indicates more than 5000 units of SDF-2/103 cells.

 

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