A chemoattractant-mediated Gi-coupled pathway activates adenylyl cyclase in human neutrophils.

Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to Galpha(s)-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical Galpha(s)-meditated pathway.

S-adenosylhomocysteine hydrolase is localized at the front of chemotaxing cells, suggesting a role for transmethylation during migration.

Chemotaxis of bacteria requires regulated methylation of chemoreceptors. However, despite considerable effort in the 1980s, transmethylation has never been established as a component of eukaryotic cell chemotaxis. S-adenosylhomocysteine (SAH), the product formed when the methyl group of the universal donor S-adenosylmethionine (SAM) is transferred to an acceptor molecule, is a potent inhibitor of all transmethylation reactions. In eukaryotic cells, this inhibition is relieved by hydrolysis of SAH to adenosine and homocysteine catalyzed by SAH hydrolase (SAHH). We now report that SAHH, which is diffuse in the cytoplasm of nonmotile Dictyostelium amoebae and human neutrophils, concentrates with F-actin in pseudopods at the front of motile, chemotaxing cells, but is not present in filopodia or at the very leading edge. Tubercidin, an inhibitor of SAHH, inhibits both chemotaxis and chemotaxis-dependent cell streaming of Dictyostelium, and chemotaxis of neutrophils at concentrations that have little effect on cell viability. Tubercidin does not inhibit starvation-induced expression of the cAMP receptor, cAR1, or G protein-mediated stimulation of adenylyl cyclase activity and actin polymerization in Dictyostelium. Tubercidin has no effect on either capping of Con A receptors or phagocytosis in Dictyostelium. These results add SAHH to the list of proteins that redistribute in response to chemotactic signals in Dictyostelium and neutrophils and strongly suggest a role for transmethylation in chemotaxis of eukaryotic cells.

Signal relay during the life cycle of Dictyostelium.

A fundamental property of multicellular organisms is signal relay, the process by which information is transmitted from one cell to another. The integration of external information, such as nutritional status or developmental cues, is critical to the function of organisms. In addition, the spatial organizations of multicellular organisms require intricate signal relay mechanisms. Signal relay is remarkably exhibited during the life cycle of the social amoebae Dictyostelium discoideum, a eukaryote that retains a simple way of life, yet it has greatly contributed to our knowledge of the mechanisms cells use to communicate and integrate information. This chapter focuses on the molecules and mechanisms that Dictyostelium employs during its life cycle to relay temporal and spatial cues that are required for survival.

Selenocysteine tRNA identification in the model organisms Dictyostelium discoideum and Tetrahymena thermophila.

Characterizing Sec tRNAs that decode UGA provides one of the most direct and easiest means of determining whether an organism possesses the ability to insert selenocysteine (Sec) into protein. Herein, we used a combination of two techniques, computational to identify Sec tRNA genes and RT-PCR to sequence the gene products, to unequivocally demonstrate that two widely studied, model protozoans, Dictyostelium discoideum and Tetrahymena thermophila, encode Sec tRNA in their genomes. The advantage of using both procedures is that computationally we could easily detect potential Sec tRNA genes and then confirm by sequencing that the Sec tRNA was present in the tRNA population, and thus the identified gene was not a pseudogene. Sec tRNAs from both organisms decode UGA. T. thermophila Sec tRNA, like all other sequenced Sec tRNAs, is 90 nucleotides in length, while that from D. discoideum is 91 nucleotides long making it the longest eukaryotic sequenced to date. Evolutionary analyses of known Sec tRNAs reveal the two forms identified herein are the most divergent eukaryotic Sec tRNAs thus far sequenced.

Transcriptional transitions during Dictyostelium spore germination.

Many protozoa form spores in response to adversity; therefore, spore germination is a key process in their life cycle. Dictyostelium discoideum sporulates in response to starvation following a developmental program. Germination is characterized by two visible changes, spore swelling and the emergence of amoeba from the spore capsule. Several studies have indicated that an additional process termed spore activation is also required, but the physiological changes that characterize the three phases are largely uncharacterized. We used microarrays to monitor global transcriptional transitions as a surrogate measure of the physiological changes that occur during germination. Using two independent methods to induce germination, we identified changes in mRNA levels that characterized the germination process rather than changes that resulted from the induction method. We found that germination is characterized by three transitions. The first transition occurs during activation, while the spores appear dormant, the largest transition occurs when swelling begins, and the third transition occurs when emergence begins. These findings indicate that activation and swelling are not passive occurrences, such as dilution of inhibitors or spore rehydration, but are active processes that are accompanied by dramatic events in mRNA degradation and de novo transcription. These findings confirm and extend earlier reports that genes such as celA are regulated during spore germination. We also found by mutation analysis that the unconventional myosin gene myoI, which is induced during early germination, plays roles in the maintenance of dormancy and in spore swelling. This finding suggests that some of the observed transcriptional changes are required for spore germination.

The Dictyostelium discoideum prespore-specific catalase B functions to control late development and to protect spore viability.

Changes in the levels of reactive oxygen species (ROS) have been associated previously with cell differentiation and development in several systems. Thus, there is interest in studying the developmental regulation of antioxidant enzymes, whose activities may modulate ROS levels and subsequent oxidant-mediated signal transduction events in specific tissues. Our recent identification in Dictyostelium discoideum of the prespore-specific catalase B (CatB) enzyme suggested (a) that the CatB enzyme functions to provide protection to the mature spores, and (b) that the CatB enzyme may have a regulatory role in cell differentiation and morphogenesis. We have now confirmed both these hypotheses. We specifically disrupted the catB gene by homologous recombination. The resulting catB null strain displays a 4-h delay in development at the time of normal catB gene expression, followed by slow and asynchronous development of fruiting bodies, taking 10 h longer than the isogenic parent strain. The expression of both prestalk- and prespore-specific genes was altered in the mutant both temporally and quantitatively, and the resultant mutant spores had increased sensitivity to H(2)O(2). This study supports the idea that CatB functions in the development of D. discoideum by regulating the level of ROS, and adds to the growing body of evidence for regulatory roles for ROS.

Electron microscopy of actin rods and bundles in Dictyostelium discoideum by high-pressure freezing.

A new type of actin rods comprising actin tubules appears in dormant spores of Dictyostelium discoideum. Occasionally, the rods in the nucleus were observed in an amorphous state using a combination of high-pressure freezing and freeze-substitutions. Also in the case of actin bundles formed in the nudeus of vegetative cells exposed to dimethyl sulphoxide, actin filaments seemed to be embedded in matrices. The karyoplasm of spores fixed by the above method appeared to be denser than that obtained by other methods. As soluble materials may be efficiently retained in the nucleus, actin tubules or actin filaments embedded in those materials may result in hazy images of actin rods and bundles.

Galpha3 and protein kinase A represent cross-talking pathways for gene expression in Dictyostelium discoideum.

Heterotrimeric G proteins and protein kinase A (PKA) are regulators of development in Dictyostelium discoideum. It has been reported that disruption of the Dictyostelium Galpha3 gene (galpha3-) blocks development and expression of several early development genes, characteristics that are reminiscent of mutants lacking the catalytic subunit of PKA (pkac-). The hypothesis that Galpha3 and PKA signaling pathways may interact to control developmental gene expression was tested by comparing the regulation of seven genes expressed early in development in the wild-type and in galpha3- and pkac- mutants, and comparing PKA activity in the wild-type and in a galpha3- mutant. The expression patterns of six genes were affected similarly by the Galpha3 and PKA mutations, while the expression of only one gene, the cAMP receptor 1 (cAR1), differed between the mutants. PKA activity, measured by phosphorylation of the PKA-specific substrate Kemptide, was higher in galpha3- cells than in wild-type cells, suggesting that Galpha3 normally exerts an inhibitory effect on PKA activity. Although some early development genes appear to require both Galpha3 and PKA for expression, the differing response of cAR1 expression and the inhibitory effect of Galpha3 on PKA activity suggest that Galpha3 and PKA are members of interacting pathways controlling gene expression early in development.