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First published online 8 July 2008
doi: 10.1242/jcs.026682

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The secreted Dictyostelium protein CfaD is a chalone

Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892, USA

View larger version (42K): [in this window] [in a new window]   Fig. 1. cfaD– cells lack cfaD mRNA and CfaD protein. (A) Northern blot of RNA from wild type (WT) and cfaD– vegetative cells probed for cfaD. A loading-control gel stained with ethidium bromide showed apparently equal quantities and lack of degradation of the ribosomal RNA bands. (B) Western blot of total cell lysates from mid-log vegetative cells stained with affinity-purified anti-CfaD antibodies. Molecular weight markers (in kDa) are given at the right. A loading-control gel stained with Coomassie Blue showed apparently equal quantities of proteins in all samples. (C) Disruption of cfaD affects the appearance of fruiting bodies. Cells of the indicated strain were grown on bacterial lawns and fruiting bodies were photographed. Bar, 0.5 mm.

View larger version (25K): [in this window] [in a new window]   Fig. 2. CfaD slows the proliferation of cells growing in liquid shaking culture. (A) Cells were diluted to 2x105 cells/ml in HL5 and the cell density was measured daily. Values are the mean ± s.e.m. from six independent experiments. The absence of error bars indicates that the error was smaller than the plot symbol. WT, wild-type. At day 9, all of the cfaD– cells appeared to be dead. The saturation densities (in units of 107 cells/ml) were 2.4±0.1 for wild type, 3.6±0.3 for cfaD–, 1.5±0.1 for cfaDOE, and 2.2±0.1 for cfaD–/cfaDOE. The differences between all values are significant (P<0.05) except WT versus cfaD–/cfaDOE, which was not significant (1-way ANOVA, Tukey's test). (B) The data from the first 3 days were plotted using a log scale for the density. (C) Proliferation of cells growing on a lawn of bacteria was measured by plating 103 cells on bacteria and counting the number of cells at the indicated times. Values are the mean ± s.e.m. from three independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 3. The concentration of extracellular CfaD increases with cell density. (A) SDS-polyacrylamide gel of the rCfaD used for the calibration curve was stained with Coomassie Blue. Molecular mass markers (in kDa) are shown at left. (B) The amount of CfaD as a function of cell density was determined by staining western blots of conditioned growth medium from wild-type cells at the indicated densities together with standard amounts of rCfaD with affinity-purified anti-CfaD antibodies. Values are the mean ± s.e.m. from three independent experiments. The absence of error bars indicates that the error was smaller than the plot symbol. The insert shows a western blot of conditioned growth media harvested at the indicated densities (in units of 106 cells/ml) stained with affinity-purified anti-CfaD antibodies. Molecular mass markers (in kDa) are at left.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Size fractionation of complexes containing CfaD and AprA. Conditioned starvation medium (CSM) and conditioned growth medium (CGM) from the indicated cells were concentrated, and were then fractionated using molecular-sieve chromatography. WT, wild type. Western blots of the different fractions were stained with affinity-purified anti-CfaD and anti-AprA antibodies. At the top, numbers indicate fraction number, and the position of molecular-sieve molecular-mass markers is indicated. A 670 kDa marker eluted at fraction 30. For each of the western blots, the position of molecular mass markers is indicated at left.

View larger version (42K): [in this window] [in a new window]   Fig. 5. CfaD appears to interact with AprA. rHMCfaD and nickel-agarose beads were mixed overnight with conditioned growth medium (CGM) prepared from wild-type and aprA– cells. After washing with PBS, proteins bound to the beads were eluted with SDS sample buffer. Western blots of the bound proteins were stained with anti-Myc antibodies (top left panel) or anti-AprA antibodies (lower left panel). Similarly, rAprA was mixed with nickel-agarose beads and CGM from wild-type and cfaD– cells; western blots of the bound material were stained with anti-Myc antibodies (top right panel) or anti-CfaD antibodies (lower right panel). Molecular mass markers in kDa are shown in the middle.

View larger version (32K): [in this window] [in a new window]   Fig. 6. NC4 wild-type cells secrete AprA and CfaD. (A) NC4 cells were grown on a bacterial lawn on an agar plate. The cells were washed off and a piece of the agar was mixed with SDS sample buffer, heated and, while still hot and molten, the material corresponding to 10 µl of the agar was loaded on gels. Western blots of the gels were stained with affinity-purified anti-AprA or anti-CfaD antibodies. A 0.5 ng recombinant CfaD standard (His-tagged rCfaD) was also loaded on the CfaD gel. Molecular mass markers in kDa are shown at left. (B) Size fractionation of medium conditioned by NC4 cells growing in shaking culture with bacteria, and western blotting of the fractions, was done as described for Fig. 4.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Extracellular CfaD slows cell proliferation. His-tagged rCfaD at the indicated concentrations was added to cells growing in shaking culture, and cell densities were measured after 12 hours. For each experiment with each cell line, the proliferation at 12 hours was calculated as the density of cells treated with rCfaD as a percent of the density without rCfaD. Values are the mean ± s.e.m. from four separate experiments. The graphs show sigmoidal dose-response curves fit to the data; the calculated maximal inhibition and EC50 values for each cell line are given in Table 4.

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