Cellulose is a major component of the extracellular matrices formed during development of the social amoeba, Dictyostelium discoideum. We isolated insertional mutants that failed to accumulate cellulose and had no cellulose synthase activity at any stage of development. Development proceeded normally in the null mutants up to the beginning of stalk formation, at which point the culminating structures collapsed onto themselves, then proceeded to attempt culmination again. No spores or stalk cells were ever made in the mutants, with all cells eventually lysing. The predicted product of the disrupted gene (dcsA) showed significant similarity to the catalytic subunit of cellulose synthases found in bacteria. Enzyme activity and normal development were recovered in strains transformed with a construct expressing the intact dcsA gene. Growing amoebae carrying the construct accumulated the protein product of dcsA, but did not make cellulose until they had developed for at least 10 hr. These studies show directly that the product of dcsA is necessary, but not sufficient, for synthesis of cellulose.

Using genome-wide microarrays, we recognized 172 genes that are highly expressed at one stage or another during multicellular development of Dictyostelium discoideum. When developed in shaken suspension, 125 of these genes were expressed if the cells were treated with cyclic AMP (cAMP) pulses at 6-min intervals between 2 and 6 h of development followed by high levels of exogenous cAMP. In the absence of cAMP treatment, only three genes, carA, gbaB, and pdsA, were consistently expressed. Surprisingly, 14 other genes were induced by cAMP treatment of mutant cells lacking the activatable adenylyl cyclase, ACA. However, these genes were not cAMP induced if both of the developmental adenylyl cyclases, ACA and ACR, were disrupted, showing that they depend on an internal source of cAMP. Constitutive activity of the cAMP-dependent protein kinase PKA was found to bypass the requirement of these genes for adenylyl cyclase and cAMP pulses, demonstrating the critical role of PKA in transducing the cAMP signal to early gene expression. In the absence of constitutive PKA activity, expression of later genes was strictly dependent on ACA in pulsed cells.

We used microarrays carrying most of the genes that are developmentally regulated in Dictyostelium to discover those that are preferentially expressed in prestalk cells. Prestalk cells are localized at the front of slugs and play crucial roles in morphogenesis and slug migration. Using whole-mount in situ hybridization, we were able to verify 104 prestalk genes. Three of these were found to be expressed only in cells at the very front of slugs, the PstA cell type. Another 10 genes were found to be expressed in the small number of cells that form a central core at the anterior, the PstAB cell type. The rest of the prestalk-specific genes are expressed in PstO cells, which are found immediately posterior to PstA cells but anterior to 80% of the slug that consists of prespore cells. Half of these are also expressed in PstA cells. At later stages of development, the patterns of expression of a considerable number of these prestalk genes changes significantly, allowing us to further subdivide them. Some are expressed at much higher levels during culmination, while others are repressed. These results demonstrate the extremely dynamic nature of cell-type-specific expression in Dictyostelium and further define the changing physiology of the cell types. One of the signals that affect gene expression in PstO cells is the hexaphenone DIF-1. We found that expression of about half of the PstO-specific genes were affected in a mutant that is unable to synthesize DIF-1, while the rest appeared to be DIF independent. These results indicate that differentiation of some aspects of PstO cells can occur in the absence of DIF-1.

Expression profiles of developmental genes in Dictyostelium were determined on microarrays during development of wild type cells and mutant cells lacking either the DNA binding protein GBF or the signaling protein LagC. We found that the mutant strains developed in suspension with added cAMP expressed the pulse-induced and early adenylyl cyclase (ACA)-dependent genes, but not the later ACA-dependent, post-aggregation genes. Since expression of lagC itself is dependent on GBF, expression of the post-aggregation genes might be controlled only by signaling from LagC. However, expression of lagC in a GBF-independent manner in a gbfA- null strain did not result in expression of the post-aggregation genes. Since GBF is necessary for accumulation of LagC and both the DNA binding protein and the LagC signal transduction pathway are necessary for expression of post-aggregation genes, GBF and LagC form a feed-forward loop. Such network architecture is a common motif in diverse organisms and can act as a filter for noisy inputs. Breaking the feed-forward loop by expressing lagC in a GBF-independent manner in a gbfA+ strain does not significantly affect the patterns of gene expression for cells developed in suspension with added cAMP, but results in a significant delay at the mound stage and asynchronous development on solid supports. This feed-forward loop can integrate temporal information with morphological signals to ensure that post-aggregation genes are only expressed after cell contacts have been made.

MEK/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase signaling is imperative for proper chemotaxis. Dictyostelium mek1؊ (MEK1 null) and erk1 ؊ cells exhibit severe defects in cell polarization and directional movement, but the molecules responsible for the mek1 ؊ and erk1 ؊ chemotaxis defects are unknown. Here, we describe a novel, evolutionarily conserved gene and protein (smkA and SMEK, respectively), whose loss partially suppresses the mek1 ؊ chemotaxis phenotypes. SMEK also has MEK1-independent functions: SMEK, but not MEK1, is required for proper cytokinesis during vegetative growth, timely exit from the mound stage during development, and myosin II assembly. SMEK localizes to the cell cortex through an EVH1 domain at its N terminus during vegetative growth. At the onset of development, SMEK translocates to the nucleus via a nuclear localization signal (NLS) at its C terminus. The importance of SMEK's nuclear localization is demonstrated by our findings that a mutant lacking the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the NLS does not. Microarray analysis reveals that some genes are precociously expressed in mek1 ؊ and erk1 ؊ cells. The misexpression of some of these genes is suppressed in the smkA deletion. These data suggest that loss of MEK1/ERK1 signaling compromises gene expression and chemotaxis in a SMEK-dependent manner.Chemotaxis is a conserved cellular process in which cells detect a chemical gradient, polarize, and proceed up the gradient by extending a pseudopod at their leading edge and retracting their posteriors (17,27,40). In both mammalian and Dictyostelium cells, mitogen-activated protein (MAP) kinase pathways are required for proper chemotaxis. Mouse knockout studies have shown that MEKK1, a MAP kinase kinase kinase that scaffolds and activates both the extracellular signal-regulated kinase (ERK) ERK1/2 and stress-activated Jun N-terminal protein kinase (JNK) MAP kinase pathways, is required for epithelial and fibroblast migration (65). Fibroblasts from mek1 Ϫ/Ϫ (MEK1 is a MAP kinase kinase that specifically activates ERK1/2) and jnk1 Ϫ/Ϫ jnk2 Ϫ/Ϫ mice also exhibit reduced directional motility in fibronectin and wound-induced migration assays (20,25). In addition, ERK1/2 activation is required for MDCK epithelial cells moving as a sheet or undergoing a partial epithelial-to-mesenchymal transition during tubulogenesis (36, 39).One MEK and two ERK family MAP kinases have been identified in Dictyostelium. Cells lacking MEK1 or ERK1 (mek1 Ϫ and erk1 Ϫ cells) exhibit a marked absence of polarity and severely reduced chemotaxis speed and directionality (34,50). Dictyostelium MEK1 is required for ERK1 activation and its localization to the cell cortex in response to chemoattractant stimulation (50). ERK2 is not regulated by MEK1 but has an established role in phosphorylating the RegA phosphodiesterase during starvation-induced cyclic AMP (cAMP) relay signaling (35).Although the ERK and JNK MAP kinase pathways are required for cell motility in...

Cell-type specific genes were recognized by interrogating microarrays carrying Dictyostelium gene fragments with probes prepared from fractions enriched in prestalk and prespore cells. Cell-type specific accumulation of mRNA from 17 newly identified genes was confirmed by Northern analyses. DNA microarrays carrying 690 targets were used to determine expression profiles during development. The profiles were fit to a biologically based kinetic equation to extract the times of transcription onset and cessation. Although the majority of the genes that were cell-type enriched at the slug stage were first expressed as the prespore and prestalk cells sorted out in aggregates, some were found to be expressed earlier before the cells had even aggregated. These early genes may have been initially expressed in all cells and then preferentially turned over in one or the other cell type. Alternatively, cell type divergence may start soon after the initiation of development.

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC ؊ , in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC ؊ cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC ؊ cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC ؊ cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC ؊ cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA ؊ /myoF ؊ myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC ؊ mutant are the immediate result of the absence of RasC function.The Ras GTPases function as molecular switches in the regulation of a variety of responses to extracellular signals (3,21,35,36). Dictyostelium discoideum, like higher eukaryotes, contains a number of Ras GTPases (6,21,22). Because of its attributes, Dictyostelium provides a unique experimental system for exploring the roles played by the Ras GTPases in cell motility and chemotaxis (6,21,49). First, because it is haploid, null mutations are readily generated and rescued (16,18). Second, because the behavior of Dictyostelium amoebae in buffer and in response to the temporal, spatial, and concentration components of the natural chemotactic wave has been characterized in detail by computer-assisted methods (33, 34), a unique contextual framework exists for identifying specific behavioral defects manifested in mutants (29) and for deducing from them the specific role played by mutated genes in motility and/or chemotaxis (4,8,10,42,45,50,51).In a previous study, it was demonstrated that rasC Ϫ cells could not progress through the early stages of development or form aggregates unless they were pulsed with the chemoattractant cyclic AMP (cAMP), indicating that RasC was necessary for signaling (20). rasC Ϫ cells artificially pulsed with cAMP were then capable of forming aggregates on filter pads. When mixed with a majority of normal cells, they could also enter aggr...

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.