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...