Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

The dual-specificity protein phosphatase MkpB, homologous to mammalian MKP phosphatases, is required for D. discoideum post-aggregative development and cisplatin response

Dual-specificity protein phosphatases participate in signal transduction pathways inactivating mitogen-activated protein kinases (MAP kinases). These signaling pathways are of critical importance in the regulation of numerous biological processes, including cell proliferation, differentiation and development. The social ameba Dictyostelium discoideum harbors 14 genes coding for proteins containing regions very similar to the dual-specificity protein phosphatase domain. One of these genes, mkpB, additionally codes for a region similar to the Rhodanase domain, characteristic of animal MAP kinase-phosphatases, in its N-terminal region. Cells that over-express this gene show increased protein phosphatase activity. mkpB is expressed in D. discoideum ameba at growth but it is greatly induced at 12h of multicellular development. Although it is expressed in all the cells of developmental structures, mkpB mRNA is enriched in cells with a distribution typical of anterior-like cells. Cells that express a catalytically inactive mutant of MkpB grow and aggregate like wild-type cells but show a greatly impaired post-aggregative development. In addition, the expression of cell-type specific genes is very delayed, indicating that this protein plays an important role in cell differentiation and development. Cells expressing the MkpB catalytically inactive mutant show increased sensitivity to cisplatin, while cells over-expressing wild type MkpB, or MkpA, proteins or mutated in the MAP kinase erkB gene are more resistant to this chemotherapeutic drug, as also shown in human tumor cells.     

The role of Src family kinases in egg activation

Solitary amoebae of Dictyostelium discoideum are frequently exposed to stressful conditions in nature, and their multicellular development is one response to environmental stress. Here we analyzed an aggregation stage abundant gene, krsA, homologous to human krs1 (kinase responsive to stress 1) to understand the mechanisms for the initiation of development and cell fate determination. The krsA- cells exhibited reduced viability under hyperosmotic conditions. They produced smaller aggregates on membrane filters and did not form aggregation streams on a plastic surface under submerged starvation conditions, but were normal in sexual development. During early asexual development, the expression of cAMP-related genes peaked earlier in the knockout mutants. Neither cAMP oscillation in starved cells nor an increase in the cAMP level following osmotic stress was observed in krsA-. The nuclear export signal, as well as the kinase domain, in KrsA was necessary for stream formation. These results strongly suggest that krsA is involved in cAMP relay, and that signaling pathways for multicellular development have evolved in unison with the stress response.     

Pleiotropic roles of a ribosomal protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over- or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over- or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect--specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.     

The Dictyostelium repertoire of seven transmembrane domain receptors

The availability of fully sequenced genomes allows the in silico analysis of whole gene families in a given genome. A particularly large and interesting gene family is the G-protein-coupled receptor family. These receptors detect a variety of extracellular signals and transduce them, generally via heterotrimeric G-proteins, to effector proteins inside the cell and thus elicit a physiological response. G-protein-coupled receptors are found in all eukaryotes and constitute in vertebrates 3-5% of all genes. They are also very important drug targets and approximately 25 of the top 100 selling drugs are directed against these receptors. The Dictyostelium discoideum genome contains a surprisingly high number of 55 such receptors, approximately 0.5% of the encoded genes. Besides the four well-studied cAMP receptors the genome encodes eight additional cAMP receptor-like proteins and one of these is distinguished by a novel domain structure, one secretin-like receptor, 17 GABA(B)-like and 25 Frizzled-like receptors. The existence of the latter three types of receptors in D. discoideum was surprising because they had not been observed outside the animal kingdom before. Their presence suggests unprecedentedly complex and so far unknown signaling activities in this lower eukaryote.     

Efficient transformation of Dictyostelium discoideum amoebae.

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.     

Lipopolysaccharide induction of autophagy is associated with enhanced bactericidal activity in Dictyostelium discoideum

Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum.     

Gamete fusion and cytokinesis preceding zygote establishment in the sexual process of Dictyostelium discoideum

Cells of Dictyostelium discoideum become sexually mature under submerged and dark conditions, and fuse with opposite mating-type cells to form zygote giant cells, which gather surrounding cells and finally develop into dormant structures called macrocysts. In the present study, we found that the multinuclear fused cells formed during this process frequently underwent cytokinesis driven by random local movements. The split cells were capable of re-fusion, and repeated cytokinesis. These radical behaviors continued until the extensive cell aggregation started around the giant cells. Thus, gamete fusion and initiation of zygote development do not coincide in the mating of D. discoideum. Analyses by confocal microscopy and flow cytometry indicated that the cessation of the random movement followed pronuclear fusion, and that microtubule organizing centers (MTOC), abundant in the fused cells at the beginning, gradually decreased and only one of them remained within the developed macrocyst. Some of the genes known to control cell movement, such as rasGEFB and rasS, increased shortly before the cessation of repeated fusion-cytokinesis and initiation of phagocytosis. These results suggest that the sequential molecular events are necessary in D. discoideum after gamete fusion to establish a new individuality of zygotes.     

A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, different PI kinases regulate distinct cellular processes, including cell growth, development, and protein trafficking.     

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.     

A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.

We are studying cell differentiation in Dictyostelium discoideum by examining the regulation of genes that are preferentially expressed in different cell types. A system has been established in which prestalk- and prespore-cell-specific genes are expressed in single cells in response to culture conditions. We confirm our previous results showing that cyclic AMP induces prestalk genes and now show that it is also required for prespore gene induction. The expression of both classes of genes is additionally dependent on the presence of a factor(s) secreted by developing cells which we call conditioned medium factor(s). An assay for conditioned medium factor(s) shows that it is detectable within 2.5 h after the onset of development. Conditioned medium factor(s) also promotes the expression of genes induced early in development, but has no detectable effect on the expression of actin genes and a gene expressed maximally in vegetative cells. In the presence of conditioned medium factor(s), exogenous cyclic AMP at the onset of starvation fails to induce the prespore and prestalk genes. The addition of cyclic AMP between 2 and 12 h of starvation results in rapid prestalk gene expression, whereas prespore genes are induced at an invarient time (approximately 18 h after the onset of starvation). These data suggest that cyclic AMP and conditioned medium factor(s) are sufficient for prestalk gene induction, whereas an additional parameter(s) is involved in the control of prespore gene induction. In contrast to several previous studies, we show that multicellularity is not essential for the expression of either prespore or prestalk genes. These data indicate that prespore and prestalk genes have cell-type-specific as well as shared regulatory factors.     

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.     

Density-dependent induction of discoidin-I synthesis in exponentially growing cells of Dictyostelium discoideum

The synthesis of the lectin, discoidin I, by vegetative cells of Dictyostelium discoideum (strain NC4) was monitored using immunoblot analysis and indirect immunofluorescence. Suspension cultures were used, so that the D. discoideum cell density and the concentration of bacteria could be controlled. Discoidin-I production was found to be a function of the relative densities of D. discoideum cells and food bacteria. Synthesis was initiated in exponentially growing D. discoideum cells approximately three generations before depletion of the food supply. In the growth medium of cells producing discoidin I, a soluble activity was detected that caused low-density cells to begin discoidin-I synthesis. This activity was not dialyzable and was destroyed by heat. A similar activity was produced by AX3 cells during axenic growth. Density-dependent induction of other 'early developmental' proteins was also detected in wild-type cells. These findings suggest that the expression of several 'early developmental' genes is regulated by a mechanism that measures cell density relative to food supply, not by starvation per se.     

Nonsense suppression in Dictyostelium discoideum

We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes. These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D. discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D. discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active beta-galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth.     

A vesicle surface tyrosine kinase regulates phagosome maturation

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.     

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.