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First published online 31 January 2007
doi: 10.1242/dev.02775

Development 134, 959-966 (2007)
Published by The Company of Biologists 2007
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cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs

Elisa Alvarez-Curto, Shweta Saran, Marcel Meima, Jenny Zobel, Claire Scott and Pauline Schaap*

Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, Angus DD1 5EH, UK.


Figure 1
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  		Fig. 1. ACG promoter activity in developing structures. (A-C) D. discoideum wild-type cells were transformed with vector ACG::gal, which contains a gene fusion of the ACG promoter and the ß-galactosidase reporter gene. Cells were starved on nitrocellulose filters supported by PB agar and developing structures were fixed and stained with X-Gal. (A) aggregate; (B) standing slug; (C) mature fruiting body. (D,E) D. discoideum wild-type cells were transformed with vector ACG::ile-gal, where ile-gal encodes a labile form of ß-galactosidase with a 30-minute half-life. Slugs (D) and mid-culminants (E) were stained with X-Gal. Scale bars: 100 µm.

 

Figure 2
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  		Fig. 2. Developmental regulation of ACG transcription and ACG protein accumulation. (A,B). Dictyostelium wild-type (WT) cells (A), the acrA mutant (B) and both cell lines transformed with the ACG::ile-gal construct were incubated for 24 hours on PB agar. Every 2 hours, WT and acrA- cells were harvested, lysed in SDS-PAGE sample buffer and immunoblotted with {alpha}ACG antibody. The {alpha}ACG antibody reacts with a single band of around 98 kD, which is the predicted size of ACG. (C) The ACG-ile-gal transformed cells were lysed and assayed for ß-galactosidase activity using a spectrophotometric assay (black and white squares). The data are expressed as percentage of ß-galactosidase activity measured at 24 hours in wild-type cells. The means of two experiments assayed in triplicate are presented. This panel also shows data obtained from a densitometric scan of the ACG bands in the immunoblots in A,B (black and white circles). The optical density values of the scan are expressed as percentage of the value obtained for WT at 24 hours. (D) Total RNA was extracted from developing WT cells at 2-hour intervals and subjected to RT-PCR for 25, 30 and 35 cycles using primers that yield a product that spans the two introns of the ACG gene. Product was first detectable after 30 cycles (shown here) from both cDNA (lower band) and contaminating gDNA (upper band) amplification. The control reaction lacked RNA.

 

Figure 3
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  		Fig. 3. ACG protein in intact structures and cells. Intact wild-type slugs (A), fruiting bodies (B) and spores (C) were fixed in methanol and stained with {alpha}ACG antibody and FITC-conjugated goat-anti-rabbit IgG (GARFITC). (D-F). Slugs were dissociated into single cells, which were first stained with {alpha}ACG antibody and GARFITC, and subsequently with mouse monoclonal antibody mAb83.5 that was raised against the spore coat protein SP85 (Zhang et al., 1999Go) and Texas Red-conjugated goat-anti-mouse IgG. Intact structures and cells were photographed using a Leica TCS SP2 confocal laser scanning microscope, using 596 nm excitation and 620 nm emission for Texas Red, and 495 nm excitation and 520 nm emission for FITC. D and E are superimposed in F to show colocalization of ACG and SP85. Scale bars: A,B, 100 µm; C, 1 µm; D, 10 µm.

 

Figure 4
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  		Fig. 4. Prespore and spore differentiation in adenylyl cyclase mutants. (A) Slugs of wild-type, acrA-, acg- and acrA-/A15::ACG{Delta}cat cells that had migrated for 2-3 hours were dissociated and stained with a spore-specific antiserum and GARFITC. Cell nuclei were counterstained with DAPI. The percentage of prespore cells (cells with at least 3-4 fluorescent vacuoles) relative to DAPI-stained cells was determined. Means and SE of four experiments are presented. Significant differences (P>0.95) between datasets connected by brackets, as determined by Kruskal-Wallis ANOVA on ranks using SigmaStat software (Systat, San Jose, US), are indicated by asterisks. (B) Wild-type, acrA-, acg- and acrA-/A15::ACG{Delta}cat cells were developed on PB agar until fruiting bodies had formed. Total RNA was extracted at 2-hour intervals, size-fractionated on 1.5% agarose gels containing 2.2 M formaldehyde and transferred to nylon membranes (Nellen et al., 1987Go). The four northern blots were hybridized in the same batch to a [32P]dATP-labelled CotB probe at 65oC, then stripped and reprobed with the constitutively expressed gene 1G7 (Williams et al., 1987Go). (C) Three-day-old fruiting bodies of acrA-, acg- and acrA-/A15::ACG{Delta}cat cells were transferred to a glass slide and stained with the cellulose dye Calcofluor at 0.03% (w/v) final concentration. The preparations were photographed under UV by fluorescence microscopy to visualize the Calcofluor-stained spores in the presence of a low level of transillumination to obtain a phase contrast image of the remaining amoebae. Scale bar: 10 µm.

 

Figure 5
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  		Fig. 5. Induction of prespore gene expression in adenylyl cyclase mutants. Aggregation-competent wild-type, acrA-, acg- and acrA-/A15::ACG{Delta}cat cells were shaken for 8 hours in the presence or absence of 300 µM cAMP, added every hour. Total RNA was isolated at 2-hour intervals and all RNA samples were size-fractionated on a single gel and transferred to a single membrane, which was successively probed with [32P]dATP-labelled CotB, PsA and 1G7 DNA probes.

 

Figure 6
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  		Fig. 6. AcrA expression in wild-type and acg- cells. (A,B) AcrA expression detected by in-situ hybridization. Wild-type cells were starved on dialysis membrane supported by PB agar. AcrA mRNA was visualized in migrating slugs (A) and mid-culminants (B) by in-situ hybridization to a DIG-labelled AcrA RNA-probe. (C,D) Wild-type (C) and acg- (D) cells were transformed with vector AcrA::ala-gal, which contains a gene fusion of the AcrA promoter and ala-gal, which encodes a stable form of ß-galactosidase. Migrating slugs were fixed and stained with X-Gal to visualize ß-galactosidase activity. AcrA-ile-gal-transformed cells yielded the same pattern, but the staining intensity was very low (data not shown). Scale bars: 100 µm.

 

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© The Company of Biologists Ltd 2007