Cellular and subcellular distribution of a cAMP-regulated prestalk protein and prespore protein in Dictyostelium discoideum: a study on the ontogeny of prestalk and prespore cells.

We have analyzed a developmentally and spatially regulated prestalk-specific gene and a prespore-specific gene from Dictyostelium. The prestalk gene, pst-cathepsin, encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. The prespore gene encodes a protein with some homology to the anti-bacterial toxin crambin and has been designated beejin. Using the lambda gtll system, we have made polyclonal antibodies directed against a portion of the protein encoded by pst-cathepsin and other antibodies directed against the beejin protein. Both antibodies stain single bands on Western blots. By immunofluorescence and Western blots, pst-cathepsin is not present in vegetative cells or developing cells during the first approximately 10 h of development. It then appears with a punctate distribution in a subset of developing cells. Beejin is detected only after approximately 15 h of development, also in a subset of cells. Pst-cathepsin is distributed in the anterior approximately 1/10 of migrating slugs and on the peripheral posterior surfaces of slugs. Beejin is distributed in the posterior region of slugs. Expression of both pst-cathepsin and beejin can be induced in subsets of isolated cultured cells by a combination of conditioned medium and extracellular cAMP in agreement with the regulation of the mRNAs encoding these proteins. We have used the antibodies as markers for cell type to examine the ontogeny and the spatial distribution of prestalk and prespore cells throughout multicellular development. Our findings suggest that prestalk cell differentiation is independent of position within the aggregate and that the spatial localization of prestalk cells within the multicellular aggregate arises from sorting of the prestalk cells after their induction. We have also found a class of cell in developing aggregates that contains neither the prestalk nor the prespore markers.

PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.

Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium.

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3' kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain-containing proteins. We demonstrate that in response to chemoattractants three PH domain-containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium.

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.

Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.

Phosphatidylinositide 3-kinases (PI3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum. DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (delta ddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, delta ddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme alpha-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that delta ddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, delta ddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.

Cell-autonomous determination of cell-type choice in Dictyostelium development by cell-cycle phase.

The developmental fate of individual cells has been examined in a system that allows Dictyostelium discoideum cells to differentiate in the absence of aggregation. The results show that the propensity of single amoebae to differentiate into either prespore or prestalk cells occurs by a cell-autonomous mechanism dependent on the cell's position in the cell cycle at the initiation of development. Cells that divide between approximately 1 1/2 hours before and approximately 40 minutes after the differentiation-inducing starvation become prestalk, whereas cells dividing at other times become prespore cells. These results suggest mechanisms by which an initial proportioning of the two cell types within the aggregate is achieved.

Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes.

When Dictyostelium cells that have initiated their developmental program upon starvation are returned to growth medium, there is a rapid and transient de novo tyrosine phosphorylation of a 43-kilodalton protein. This protein was found to be actin. Most of the phosphorylation occurred in a single, minor acidic isoform of actin. Developing cells that had been returned to growth medium lost their pseudopod extensions, became round, and had reduced adhesion to the substratum. These effects occurred with kinetics that matched the increase in tyrosine phosphorylation of actin. In mutant cell lines in which the gene for the phosphotyrosine phosphatase PTP1 had been disrupted, tyrosine phosphorylation of actin was rapid and more prolonged. These cells responded with proportionally accelerated kinetics of cell rounding. Cell lines overexpressing PTP1 had diminished amplitude and duration of actin tyrosine phosphorylation and exhibited diminished cell-shape change and an accelerated return to the extended cell-shape morphology seen in starved cells.

Signaling pathways controlling cell polarity and chemotaxis.

Many important biological processes, including chemotaxis (directional cell movement up a chemoattractant gradient), require a clearly established cell polarity and the ability of the cell to respond to a directional signal. Recent advances using Dictyostelium cells and mammalian leukocytes have provided insights into the biochemical and molecular pathways that control chemotaxis. Phosphoinositide 3-kinase plays a central and possibly pivotal role in establishing and maintaining cell polarity by regulating the subcellular localization and activation of downstream effectors that are essential for regulating cell polarity and proper chemotaxis. This review outlines our present understanding of these pathways.

Interacting signaling pathways controlling multicellular development in Dictyostelium.

cAMP functions as the key extracellular signaling molecule controlling Dictyostelium development acting through classic G-protein-coupled/serpentine receptors. Whereas aggregation is controlled by nanomolar pulses of cAMP, a more continuous micromolar signal controls multicellular differentiation by activating a transcriptional cascade via a receptor-mediated but non G-protein-coupled pathway. Potential mechanisms by which extracellular cAMP functions to differentially control aggregation followed by morphogenesis and cell-type differentiation are discussed. This review also summarizes new findings elucidating pathways controlling cell-type regulation in this organism, including signaling cascades mediated by glycogen synthase kinase 3 and cAMP-dependent protein kinase, key regulators of cell-type differentiation in metazoans, and newly identified transcription factors.

cAMP signal transduction pathways regulating development of Dictyostelium discoideum.

Dictyostelium discoideum development is regulated through receptor/G protein signal transduction using cAMP as a primary extracellular signal. Signaling pathways will be discussed as well as the regulation and function of individual cAMP receptors and G alpha subunits. Finally potential downstream targets including protein kinases and nuclear events will be explored.

Dictyostelium: a model for regulated cell movement during morphogenesis.

Dictyostelium has played an important role in unraveling the pathways that control cell movement and chemotaxis. Recent studies have started to elucidate the pathways that control cell sorting, morphogenesis, and the establishment of spatial patterning in this system. In doing so, they provide new insights into how cell movements within a multicellular organism are regulated and the importance of pathways that are similar to those that regulate chemotaxis of cells on two-dimensional surfaces during aggregation.

Phosphorelay signalling: new tricks for an ancient pathway.

'Two-component' phosphorelay systems, once thought to be used exclusively by prokaryotes, have in the past few years been shown to exist in eukaryotes. A two-component system in Dictyostelium has now been shown to modulate protein kinase A, a key regulator of multicellular development in this organism.

Intercellular signaling. A kinase for cell-fate determination?

GSK-3, a ubiquitous kinase regulated by tyrosine phosphorylation, controls cell-fate decisions in both Drosophila and Dictyostelium; genetic analysis of its interactions with other signaling pathways is now possible.

A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium.

BACKGROUND: Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. RESULTS: PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. CONCLUSIONS: Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development.

Signal transduction pathways controlling multicellular development in Dictyostelium.

The importance of signal transduction pathways in regulating developmental processes in a number of organisms has become evident in recent years. This is exceptionally clear for Dictyostelium, which uses soluble factors to regulate morphogenesis and cellular differentiation. It is now known that many of these processes are controlled by signal transduction pathways mediated by cyclic AMP through cell surface receptors coupled to G proteins, and that others are mediated by the morphogen DIF.

Just the right size: cell counting in Dictyostelium.

The regulation of tissue and organism size plays an essential, but poorly understood, role in multicellular development. Genes have been identified that affect body and organ size in a number of animals. Two recently identified genes, smlA and countin, are required for the proper function of a cell-counting mechanism that regulates organism size in the eukaryotic microorganism Dictyostelium discoideum. The discovery of this process now allows the study of size regulation in a simple multicellular system.

Genomic instability and mobile genetic elements in regions surrounding two discoidin I genes of Dictyostelium discoideum.

We have found that the genomic regions surrounding the linked discoidin I genes of various Dictyostelium discoideum strains have undergone rapid changes. Wild-type strain NC-4 has three complete discoidin I genes; its axenic derivative strain Ax-3L has duplicated a region starting approximately 1 kilobase upstream from the two linked genes and extending for at least 8 kilobases past the genes. A separately maintained stock, strain Ax-3K, does not have this duplication but has undergone a different rearrangement approximately 3 kilobases farther upstream. We show that there are repeat elements in these rapidly changing regions. At least two of these elements, Tdd-2 and Tdd-3, have characteristics associated with mobile genetic elements. The Tdd-3 element is found in different locations in related strains and causes a 9- to 10-base-pair duplication of the target site DNA. The Tdd-2 and Tdd-3 elements do not cross-hybridize, but they share a 22-base-pair homology near one end. At two separate sites, the Tdd-3 element has transposed into the Tdd-2 element, directly adjacent to the 22-base-pair homology. The Tdd-3 element may use this 22-base-pair region as a preferential site of insertion.

Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum.

We have cloned and analyzed a developmentally and spatially regulated prestalk cell-specific gene from Dictyostelium discoideum. The gene encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. Amino acid comparisons between these enzymes showed that the active-site amino acids were conserved, as were amino acids known to be important for catalysis and residues which form the intramolecular cysteine bridges. We have constructed a series of internal deletions, duplications, and linker scanner mutations within the region 300 base pairs 5' to the cap site. Analysis of expression of the mutations in transformants identified a approximately 35-base pair GC-rich region containing a dAdC/dGdT palindromic repeat and a G-rich box which is homologous to the 3' GT half of the palindromic repeat. Deletion or disruption of the G box resulted in a approximately 50-fold drop in the level of expression of the gene fusion in transformants in response to cyclic AMP in single-cell culture but did not affect the temporal pattern of regulation or control by cyclic AMP. The expression of such constructs during normal multicellular differentiation paralleled that of the endogenous gene; however, the level of RNA from the constructs was only approximately 10-fold lower than that of constructs containing the G box. Deletion of the 3' half of the palindromic sequence and the G box region resulted in a dramatic decrease in the level of transcription, although the constructs still showed proper temporal expression. These results suggest that this 35-base-pair region acts as an important part of the regulatory region for cell type and cyclic AMP regulation.

Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.

We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that expression of many early genes in D. discoideum is regulated via the cell surface cAMP receptor. We expect that many of these genes may play essential roles in early Dictyostelium development and could code for elements of the cAMP signal transduction pathway involved in aggregation.

Sequence organization and developmental expression of an interspersed, repetitive element and associated single-copy DNA sequences in Dictyostelium discoideum.

We have examined the genomic organization and developmental expression pattern of a short, transcribed, interspersed repeat element and its associated single-copy sequences. We have previously shown that 1% of the polyadenylated [poly(A)+] RNA from vegetative cells contains sequences that hybridize to this repeat. The complementary RNA is heterogeneous in size, and 90% of its mass hybridizes to single-copy DNA. In this study, we examined a series of genomic DNAs and cDNAs derived from poly(A)+ RNAs which are complementary to the repeat. Comparisons of sequence data from various genomic and cDNA clones indicated that (AAC)n X (GTT)n is the common sequence element. The tandem repeat occurred in approximately 100 short segments (approximately 35 to 150 base pairs) per haploid genome interspersed with single-copy DNA. Probes from regions adjacent to this element hybridized to unique restriction fragments on DNA blots and unique poly(A)+ RNA species on RNA blots. The (AAC)n X (GTT)n sequence was asymmetrically transcribed with only (AAC)n sequences represented in RNA. The repeat was localized within the transcribed regions of several genes and 70 base pairs 5' to the transcription initiation site of another gene. Individual (AAC)n-containing RNAs exhibited a developmental pattern of expression suggestive of the coordinate expression of many AAC gene family members.