Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD– cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a ∼150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.

Developing Dictyostelium cells aggregate to form fruiting bodies containing typically 2 ؋ 10 4 cells. To prevent the formation of an excessively large fruiting body, streams of aggregating cells break up into groups if there are too many cells. The breakup is regulated by a secreted complex of polypeptides called counting factor (CF). Countin and CF50 are two of the components of CF. Disrupting the expression of either of these proteins results in cells secreting very little detectable CF activity, and as a result, aggregation streams remain intact and form large fruiting bodies, which invariably collapse. We find that disrupting the gene encoding a third protein present in crude CF, CF45-1, also results in the formation of large groups when cells are grown with bacteria on agar plates and then starve. However, unlike countin ؊ and cf50 ؊ cells, cf45-1 ؊ cells sometimes form smaller groups than wild-type cells when the cells are starved on filter pads. The predicted amino acid sequence of CF45-1 has some similarity to that of lysozyme, but recombinant CF45-1 has no detectable lysozyme activity. In the exudates from starved cells, CF45-1 is present in a ϳ450-kDa fraction that also contains countin and CF50, suggesting that it is part of a complex. Recombinant CF45-1 decreases group size in colonies of cf45-1 ؊ cells with a 50% effective concentration (EC 50 ) of ϳ8 ng/ml and in colonies of wild-type and cf50؊ cells with an EC 50 of ϳ40 ng/ml. Like countin ؊ and cf50 ؊ cells, cf45-1 ؊ cells have high levels of cytosolic glucose, high cell-cell adhesion, and low cell motility. Together, the data suggest that CF45-1 participates in group size regulation in Dictyostelium.

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Initial differentiation in Dictyostelium involves both asymmetric cell division and a cell cycle-dependent mechanism. We previously identified a gene, rtoA, which when disrupted randomizes the cell cycle-dependent mechanism without affecting either the underlying cell cycle or asymmetric differentiation. We find that in wild-type cells, RtoA levels vary during the cell cycle. Cytosolic pH, which normally varies with the cell cycle, is randomized in rtoA cells. The middle 60% of the RtoA protein is 10 tandem repeats of an 11 peptide-long serine-rich motif, which we find has a random coil structure. This domain catalyzes the fusion of phospholipid vesicles in vitro. Conversely, rtoA cells have a defect in the fusion of endocytic vesicles. They also have a decreased exocytosis rate, a decreased pH of endocytic/ exocytic vesicles, and an increased average cytosolic pH. Our data indicate that the serine-rich domain of RtoA can mediate membrane fusion and that RtoA can increase the rate of vesicle fusion during processing of endoctyic vesicles. We hypothesize that RtoA modulates initial cell type choice by linking vegetative cell physiology to the cell cycle.Little is known about how a set of undifferentiated cells can break symmetry and differentiate into distinct cell types. One of the simplest systems that exhibits this behavior is the eukaryote Dictyostelium discoideum. Dictyostelium lives as a single cell on soil surfaces that eats bacteria and divides by fission. When the cells overgrow an area and starve, they form an aggregate of ϳ2 ϫ 10 4 cells, which develops into a fruiting body consisting of two cell types: an approximately 2-mm column of stalk cells supporting a mass of spore cells.The initial differentiation of Dictyostelium cells occurs by a combination of asymmetric cell division and a musical chairs mechanism based on the cell cycle (1-8). At the time of starvation, each pair of sister cells in S or early G 2 phase differentiates into a prestalk cell and a null cell (a cell type that initially expresses neither prespore nor prestalk markers). Each pair of sister cells in the late G 2 or M phase differentiates into a prespore cell and a null cell (Dictyostelium has an undetectable G 1 phase; Ref. 9). This mechanism regulates only initial differentiation. The eventual fate of the cell is still plastic, and a variety of factors such as adenosine, ammonia, a chlorinated hydrocarbon called differentiation-inducing factor, and oxygen can change the final fate (10 -17).Using shotgun antisense (18), we identified a gene involved in the initial differentiation of Dictyostelium cells that we called rtoA. rtoA codes for a 39.8-kDa protein, with 11 perfect repeats of a 10-amino acid-long serine-rich acidic sequence, a possible N-terminal transmembrane domain, and a possible ATP/GTP-binding domain. The gene disruption mutant of rtoA has a high prestalk:prespore ratio while maintaining a normal cell cycle. However, prestalk and prespore cells from rtoA disruptants originate from cells in any phase of the cell cycle...

Although we were unable to obtain cells lacking CF60, decreasing CF60 levels by antisense resulted in large groups, and overexpressing CF60 resulted in small groups. When added to wild-type cells, conditioned starvation medium from CF60 overexpressor cells as well as recombinant CF60 caused the formation of small groups. The ability of recombinant CF60 to decrease group size did not require the presence of the CF component CF45-1 or countin but did require the presence of CF50. Recombinant CF60 does not have acid phosphatase activity, indicating that the CF60 bioactivity is not due to a phosphatase activity. Together, the data suggest that CF60 is a component of CF, and thus this secreted signal has four different protein components.