Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as "slugs." Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Characterization and complementation analysis of sporogenous mutants of Dictyostelium discoideum

Sporogenous mutants of the cellular slime mold Dictyostelium discoideum are defined as mutants which are able to undergo terminal differentiation into spores in monolayer cultures in the presence of millimolar amounts of exogenous cyclic AMP. We describe the morphological development and cellular differentiation of a collection of 12 independently isolated sporogenous mutants of strain V12 M2. All mutants develop more rapidly than do wild-type at an air-water interface, display aberrant morphogenesis, and show overt spore and stalk differentiation as soon as 4 hr after starvation. All mutants differentiate in submerged monolayer culture in the presence of cAMP into variable proportions of spores and stalk cells. A number of the mutants also form both stalk cells and spores in submerged culture in the absence of exogenous cAMP. The spores formed by many of the mutants have a greatly reduced viability. Using parasexual genetics, we have found that two of the 12 mutants analyzed are dominant to wild-type and the remaining ten fall into a minimum of four complementation groups, the overall analysis thus yielding a minimum of four and a maximum of seven complementation groups. Intracellular cAMP levels in vegetative cells are significantly elevated in the two dominant mutants but are similar to wild type in all the other mutants.     

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.     

Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discoideum: evidence for two populations of prestalk cells

Cells from the pseudoplasmodial stage of Dictyostelium discoideum differentiation were dispersed and separated on Percoll gradients into prestalk and prespore cells. The requirements for stalk cell formation in low-density monolayers from the two cell types were determined. The isolated prespore cells required both the Differentiation Inducing Factor (DIF) and cyclic AMP for stalk cell formation. In contrast, only part of the isolated prestalk cell population required both cyclic AMP and DIF, the remainder requiring DIF alone, suggesting the possibility that there were two populations of prestalk cells, one independent of cyclic AMP and one dependent on cyclic AMP for stalk cell formation. The finding that part of the prestalk cell population required only a brief incubation in the presence of DIF to induce stalk cell formation, whilst the remainder required a considerably longer incubation in the presence of both DIF and cyclic AMP was consistent with this idea. In addition, stalk cell formation from cyclic-AMP-dependent prestalk cells was relatively more sensitive to caffeine inhibition than stalk cell formation from cyclic-AMP-independent prestalk cells. The latter cells were enriched in the most anterior portion of the migrating pseudoplasmodium, indicating that there is spatial segregation of the two prestalk cell populations. The conversion of prespore cells to stalk cells took longer and was more sensitive to caffeine when compared to stalk cell formation from cyclic-AMP-dependent prestalk cells.     

Stalk cell formation in monolayers of Dictyostelium discoideum V12-M2

Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation. During in vivo development cells first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells. There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modelling Dictyostelium discoideum morphogenesis: the culmination

The culmination of the morphogenesis of the cellular slime mould Dictyostelium discoideum involves complex cell movements which transform a mound of cells into a globule of spores on a slender stalk. We show that cyclic AMP signalling and differential adhesion, combined with cell differentiation and slime production, are sufficient to produce the morphogenetic cell movements which lead to culmination. We have simulated the process of culmination using a hybrid cellular automata/partial differential equation model. With our model we have been able to reproduce the main features that occur during culmination, namely the straight downward elongation of the stalk, its anchoring to the substratum and the formation of the long thin stalk topped by the spore head. We conclude that the cyclic AMP signalling system is responsible for the elongation and anchoring of the stalk, but in a roundabout way: pressure waves that are induced by the chemotaxis towards cyclic AMP squeeze the stalk through the cell mass. This mechanism forces the stalk to elongate precisely in the direction opposite to that of the chemotactically moving cells. The process turns out to be ‘guided’ by inactive ‘pathfinder’ cells, which form the tip of the stalk. We show that the entire development is enacted by means of the aforementioned building blocks. This means that no global gradients or different modes of chemotaxis are needed to complete the culmination. MPEG movies of the simulations are available on-line: http://www-binf.bio. uu.nl/stan/bmb.     

Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium

We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria. Cells in which mfeA(-) is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media. Bacterially grown mfeA(-) cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal. Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP-induced gene, csaA. As a consequence, they do not acquire developmentally regulated EDTA-resistant cell-cell adhesion. When mutant cells are mixed with wild-type cells and allowed to develop together, they co-aggregate and differentiate into both spores and stalk cells. Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay.     

Morphogenesis and differentiation of Dictyostelium cells interacting with immobilized glucosides: dependence on DIF production

Previous work has shown that multicellular morphogenesis of submerged Dictyostelium cells is inhibited when they bind to glucosides covalently linked to polyacrylamide gels. The amoebae aggregate normally, but then the aggregates repeatedly disperse and reaggregate, whereas control cells go on to form tight aggregates. We have investigated the role of the stalk cell differentiation inducing factors (DIFs) in this process. In the presence of cyclic AMP, amoebae submerged at high cell density accumulate DIF and differentiate into stalk cells. We find that stalk cell differentiation is inhibited by interaction of the cells with glucoside gels in these conditions, but can be restored by the addition of exogenous DIF-1. Since the responsiveness of cells to DIF-1 is not altered, it appears likely that the effect of the glucoside gel is to block DIF-1 production. Further, the addition of DIF-1 or DIF-2 stimulates the formation of tight aggregates by cells developing on glucoside gels in the absence of cyclic AMP, thus preventing the rounds of aggregation and disaggregation otherwise seen. This suggests a role for DIF in morphogenesis as well as in controlling cell differentiation. We propose a model in which immobilized glucosides activate a specific receptor ("food sensor") which drives the amoebae toward the vegetative state and inhibits DIF accumulation. DIF, on the other hand, induces tight aggregate formation and so locks the amoebae into the developmental program.     

Inhibitors of intracellular cyclic AMP accumulation affect differentiation of sporogenous mutants of Dictyostelium discoideum

Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.     

THE EFFECTS OF CYCLIC AMP ON DIFFERENTIATION OF A MUTANT DICTYOSTELIUM DISCOIDEUM CAPABLE OF DEVELOPING WITHOUT MORPHOGENESIS

The dev 1510 mutant of Dictyostelium discoideum differs from the wild type in that unaggregated cells are capable of differentiating into either spores or stalk cells depending on the culture conditions (12). Taking advantage of this fact, the effects of cyclic AMP (cAMP) on differentiation of the mutant cells were examined under conditions that prevent normal morphogenesis. In the presence of low concentrations of exogenous cAMP, the cells differentiated into only stalk cells, whereas in the presence of high concentrations they differentiated into only spores. Untreated cells formed stalk cells, but this was inhibited by addition of phosphodiesterase, indicating that it was induced by a low concentration of cAMP which they produced themselves. Cyclic GMP and dibutyryl cAMP also induced spore formation though less effectively, while 5'AMP, ADP and ATP had no effect. During development, the cells increased in sensitivity to cAMP in that spore formation was induced at lower concentration of cAMP after 4 hr of starvation. Treatment of cells that had been starved for 6hr with 10⁻⁴M cAMP for as short a time as 30 min was enough to induce 8% of the cells to form spores.
The effects on cAMP-induced differentiation of chemicals that are known to influence development of the wild type were also examined. Both NH₄Cl and KCl inhibited cAMP-induced stalk formation, but had no effect on spore formation. In the presence of arginine, spore formation was induced at a lower concentration of cAMP with higher efficiency. CaCl₂, LiCl and KF had no effect on cAMP-induced differentiation.     

A retinoblastoma ortholog controls stalk/spore preference in Dictyostelium

We describe rblA, the Dictyostelium ortholog of the retinoblastoma susceptibility gene Rb. In the growth phase, rblA expression is correlated with several factors that lead to 'preference' for the spore pathway. During multicellular development, expression increases 200-fold in differentiating spores. rblA-null strains differentiate stalk cells and spores normally, but in chimeras with wild type, the mutant shows a strong preference for the stalk pathway. rblA-null cells are hypersensitive to the stalk morphogen DIF, suggesting that rblA normally suppresses the DIF response in cells destined for the spore pathway. rblA overexpression during growth leads to G1 arrest, but as growing Dictyostelium are overwhelmingly in G2 phase, rblA does not seem to be important in the normal cell cycle. rblA-null cells show reduced cell size and a premature growth-development transition; the latter appears anomalous but may reflect selection pressures acting on social ameba.     

Developmental commitment in Dictyostelium discoideum

Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.     

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(-)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Developmental regulation of a stalk cell differentiation-inducing factor in Dictyostelium discoideum

Previous work has shown that cells developing at high density release a low-molecular-weight factor that can induce isolated Dictyostelium discoideum amoebae of strain V12M2 to differentiate into stalk cells in the presence of cyclic AMP. We now show that this differentiation-inducing factor, called DIF, can be extracted from cells during normal development and that its production is strongly developmentally regulated. DIF is not detectable in vegetative cells but rises dramatically after aggregation to reach a peak during slug migration. DIF levels are very low in two mutants defective in aggregation. The postaggregative synthesis of DIF is stimulated by the addition of extracellular cyclic AMP. We propose that DIF is a morphogen controlling prestalk cell differentiation.     

Purification and properties of a cyclic-AMP phosphodiesterase that is active in only one cell type during the multicellular development of Dictyostelium discoideum

Cyclic-AMP phosphodiesterase (PDE) accumulates during the aggregation stage of Dictyostelium where it functions in maintaining extracellular levels of cyclic AMP (cAMP). The activity decreases during the subsequent multicellular slug stage and then accumulates again during sorocarp construction, but the enzyme is active only in the developing stalk. Because of the possible significance of this localized activity in only one of the two cell types, we have purified the enzyme from the multicellular stage in order to understand its mode of regulation in vivo. We find that the enzyme which is localized in the prestalk cells is similar in many respects to the extracellular PDE which is active at the aggregation stage. The enzyme from both stages is inhibited by a low molecular weight protein. The mechanism of this inhibition is through a shift in the apparent Km for cAMP from micromolar to millimolar levels. The inhibited form of the enzyme can be activated by preincubation with MgSO4 and dithiothreitol (DTT). This activation treatment releases the inhibitor from the enzyme, thus restoring the low Km form, changes the molecular weight of the culmination stage enzyme from 95 000-100 000 to 68 000 by releasing the Mr 35 000-40 000 inhibitor protein, and causes irreversible loss of inhibitor activity. Although the inhibitor could be obtained in high yield from the aggregation stage by simply heating the extracellular fluid, it could not be detected from culmination stage extracts when prepared by this method. However, inclusion of calcium in the extraction buffer resulted in release of inhibitor from both heated and nonheated samples. The results indicate that the stalk cell specific PDE is regulated similarly to the aggregation stage PDE and opens the possibility of differential regulation of PDE in the two cell types.     

The requirement for DIF for prestalk and stalk cell formation in Dictyostelium discoideum: a comparison of in vivo and in vitro differentiation conditions

In Dictyostelium discoideum stalk cell formation is induced by cyclic AMP and differentiation-inducing factor (DIF) when cells are plated in in vitro monolayers (Kay et al., 1979, Differentiation 13: 7-14). The in vivo developmental stages at which cells became independent of these factors were determined. Independence was defined as the stage at which dispersed cells no longer required the factors for stalk cell formation in low density monolayers. Cyclic AMP independent cells were first detected at around 12 hr of development, a time that corresponds to the transition between the tipped aggregate and the first finger stages. In contrast cells did not become independent of DIF until late culmination. The prestalk cell-specific isozyme acid phosphatase II and a stalk cell-specific 41,000 Mr antigen (ST 41) were expressed during differentiation in low density monolayers in the presence of both cyclic AMP and DIF, but neither component was expressed in the presence of cyclic AMP alone. This result implies that DIF is essential for both prestalk and stalk cell formation. The two components were expressed within 2 hr of each other during differentiation in vitro, whereas during development in vivo acid phosphatase II was first detected at the first finger stage and ST 41 was first detected during late culmination, 8-12 hr later. These contrasting results suggest that the conversion of prestalk cells to stalk cells is unrestrained in monolayers, following directly after prestalk cell induction, but restrained in vivo until the culmination stage. This interpretation is consistent with the finding that cells become independent of DIF early during in vitro differentiation (A. Sobolewski, N. Neave, and G. Weeks, 1983, Differentiation 25, 93-100), but do not become independent of DIF until the culmination stage when differentiating in vivo.     

A MUTANT OF DICTYOSTELIUM DISCOIDEUM CAPABLE OF DIFFERENTIATING WITHOUT MORPHOGENESIS

A mutant which is capable of differentiating into spores and stalk cells without forming a cell aggregate was isolated from the cellular slime mould, Dictyostelium discoideum. The mutant stopped developing at various stages, before formation of mature fruits, and the cells differentiated into spores and stalk cells at whichever stage the development stopped. Unaggregated cells also differentiated into spores or stalk cells, depending on the culture conditions; differentiation into spores predominated in nutrient rich medium, while differentiation into stalk cells predominated in nutrient poor medium. The ratio of spores to stalk cells or of prespores to total cells in cell masses depended on the terminal structures formed; the ratio was unusually high or unusually low in a structure which stopped developing before papilla formation, while the ratio was normal in a structure formed after that stage. When isolated from a cell mass, prespore cells of the mutant did not dedifferentiate or resumed vegetative growth, indicating that they had lost plasticity of differentiation. The conditioned medium in which the mutant cells had grown was effective in inducing differentiation of wild type slug cells into spore-like or stalk-like cells.     

CulB,a putative ubiquitin ligase subunit,regulates prestalk cell differentiation and morphogenesis in Dictyostelium spp

Dictyostelium amoebae accomplish a starvation-induced developmental process by aggregating into a mound and forming a single fruiting body with terminally differentiated spores and stalk cells. culB was identified as the gene disrupted in a developmental mutant with an aberrant prestalk cell differentiation phenotype. The culB gene product appears to be a homolog of the cullin family of proteins that are known to be involved in ubiquitin-mediated protein degradation. The culB mutants form supernumerary prestalk tips atop each developing mound that result in the formation of multiple small fruiting bodies. The prestalk-specific gene ecmA is expressed precociously in culB mutants, suggesting that prestalk cell differentiation occurs earlier than normal. In addition, when culB mutant cells are mixed with wild-type cells, they display a cell-autonomous propensity to form stalk cells. Thus, CulB appears to ensure that the proper number of prestalk cells differentiate at the appropriate time in development. Activation of cyclic AMP-dependent protein kinase (PKA) by disruption of the regulatory subunit gene (pkaR) or by overexpression of the catalytic subunit gene (pkaC) enhances the prestalk/stalk cell differentiation phenotype of the culB mutant. For example, culB⁻ pkaR⁻ cells form stalk cells without obvious multicellular morphogenesis and are more sensitive to the prestalk O (pstO) cell inducer DIF-1. The sensitized condition of PKA activation reveals that CulB may govern prestalk cell differentiation in Dictyostelium, in part by controlling the sensitivity of cells to DIF-1, possibly by regulating the levels of one or more proteins that are rate limiting for prestalk differentiation.