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First published online 21 September 2005
doi: 10.1242/dev.02032

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A secreted factor represses cell proliferation in Dictyostelium

Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005-1892, USA

View larger version (37K): [in a new window]   Fig. 1. (A) The predicted amino acid sequence of AprA. The amino acid sequences from tryptic peptides of AprA are shown by grey boxes. The SignalP program predicted that amino acids 1-18 are a signal sequence, with a maximum cleavage site probability (0.933) between amino acids 18 and 19. Asterisks indicate potential N-and O-linked glycosylation sites. A double underline indicates a potential ATP-GTP binding site. The broken underline indicates the region with some similarity to human proteins. (B) The partial sequence of AprA (top line) compared with a short region of similarity in three different human ESTs.

View larger version (32K): [in a new window]   Fig. 2. AprA is secreted by starving Dictyostelium cells, but is not part of the CF complex. Conditioned starvation medium from wild-type cells was fractionated by sieving gel filtration chromatography. Western blots of the fractions were stained with affinity-purified rabbit antibodies against CF50, countin and AprA. On the SDS-polyacrylamide gels, the upper band stained with anti-countin antibodies is at 40 kDa, the CF50 band is at 50 kDa and the heavy AprA band is at 60 kDa. The affinity-purified anti-AprA antibodies stained a band at 56 kDa in fractions 28-30. Besides this band and the heavy band at 60 kDa, the anti-AprA antibodies did not stain any other band. See Fig. 3 for validation that the anti-AprA antibodies are staining the product of the aprA gene.

View larger version (35K): [in a new window]   Fig. 3. AprA is present in and is secreted by vegetative cells. (A) Cells growing in shaking culture (vegetative cells), conditioned growth medium, and conditioned starvation buffer were electrophoresed on an SDS-polyacrylamide gel. A western blot of the gel was stained with affinity-purified anti-AprA antibodies. `aprA-/a15::aprA' indicates aprA-/actin15::aprA. (B) Growing cells were stained for AprA by immunofluorescence. Scale bar: 20 µm.

View larger version (47K): [in a new window]   Fig. 4. Disruption of aprA by homologous recombination causes cells to form large abnormal structures during development. The phenotype can be rescued by expression of the aprA cDNA under control of an actin15 promoter (right panel). WT indicates the wild-type parental cells. Scale bar: 0.5 mm.

View larger version (25K): [in a new window]   Fig. 5. AprA is expressed in vegetative cells and during early development. Ax2 wild-type cells were starved on filters and samples were harvested at the indicated hours after starvation according to Brock et al. (Brock et al., 2002). (A) A Northern blot of RNA extracted from cells harvested at the indicated times in hours after starvation (V indicates vegetative cells) was probed for aprA mRNA. (B) A western blot of whole cell lysates from cells harvested at the indicated times was stained with anti-AprA antibodies.

View larger version (31K): [in a new window]   Fig. 6. The subcellular distribution of AprA. Cells from preparations similar to those shown in Fig. 3B were imaged with a Zeiss Axioplan II deconvolution microscope. Scale bar: 5 µm.

View larger version (22K): [in a new window]   Fig. 7. AprA slows the proliferation of cells. (A) Cells were diluted to 2x105 cells/ml in HL5 and the cell density was measured daily. The graph shows means±s.e.m. from three independent experiments. WT indicates wild type. The insert shows the data from the first two days plotted using a log scale for the density. The saturation densities (in units of 107 cells/ml) were 3.24±0.06 for aprA-, 1.94±0.07 for wild type and 0.69±0.10 for aprA-/actin15::aprA. The differences between the wild-type saturation density and either the aprA- or the aprA-/actin15::aprA saturation density were significant (P<0.01; one-way ANOVA; Dunnett's test). (B) For each cell type, three 100 mm petri dishes with SM/5 agar were spread with a lawn of bacteria mixed with 1000 Dictyostelium cells. At the indicated times, the bacteria and cells were washed off one of the plates, and the Dictyostelium cells were counted. The graph shows means±s.e.m. from three independent experiments. At 72 hours, the difference between wild type and aprA-/actin15::aprA was significant (P<0.05) and the difference between wild type and aprA- was significant (P<0.01; one-way ANOVA, Dunnett's test). The absence of error bars indicates the error was smaller than the plot symbol.

View larger version (21K): [in a new window]   Fig. 8. Growing wild-type cells secrete a 150 kDa factor that represses cell proliferation, and aprA- cells do not accumulate this factor. Conditioned growth media from the indicated cell types were fractionated on the Superose gel filtration column used for Fig. 1. Indicated fractions (5 µl), as well as 5 µl of the column buffer (Buffer) were added to wild-type cells, and the cells were counted 24 hours later. Values are means±s.e.m. from three independent assays. The difference between the counts for fraction 29 of the exudates from wild type and aprA- is significant (P<0.025; t-test).

View larger version (53K): [in a new window]   Fig. 9. Purification of AprA by immunoprecipitation. AprA was immunoprecipitated from growth medium conditioned by the indicated cell types and electrophoresed on an SDS-polyacrylamide gel and stained with Coomassie blue (left panel). A western blot of a similar gel was stained with anti-AprA antibodies (right panel).

View larger version (66K): [in a new window]   Fig. 10. AprA helps to prevent the formation of multinucleate cells. (Top row) Cells growing in shaking culture were fixed with ethanol and stained with DAPI to show nuclear DNA (the background staining in the cells is mitochondrial DNA). The bottom row shows phase contrast images of the same cells. Multinucleate cells (white arrows) are present in the aprA- population. Scale bar: 20 µm.