A model for the prestalk/prespore patterning in the slug of the slime mold Dictyostelium discoideum

Abstract. We propose that the prestalk/prespore pattern in Dictyostelium is generated in two steps: In a first process, an intermingled, non-position dependent prestalk/prespore pattern is generated by a cell-restricted autocatalysis and the antagonistic action of a long-ranging substrate which becomes depleted during this autocatalysis. By computer simulations we show that the assumed interaction accounts for several experimentally observed features of the prestalk/ prespore pattern: The size-independent ratio of both cell types, the pattern regulation after removal of one cell type, the development towards one or the other pathway before the slug obtains its final shape or even before aggregation is completed. Our hypothetical substrate may be identical with an experimentally found differentiation-inducing factor (DIF). Alternative molecular realizations of the basic mechanism are discussed. A second process leads to the aggregation of the prestalk cells in a particular region of the aggregate, the future tip region. Interactions which en-able tip formation and the coupling between the prestalk/prespore and the tip-forming system are discussed. Our model shows that the formation of a single large patch of differentiated cells and its size regulation requires conflicting parameters. By a separation into a mechanism which determines the position and a second one which determines the size of a structure, each mechanism can be optimized individually without requiring compromises for the other. Such a separation also seems to occur in other developmental systems.     

Biochemical characterization of human SET and MYND domain-containing protein 2 methyltransferase

SET and MYND domain-containing protein 2 (SMYD2) is a protein lysine methyltransferase that catalyzes the transfer of methyl groups from S-adenosylmethionine (AdoMet) to acceptor lysine residues on histones and other proteins. To understand the kinetic mechanism and the function of individual domains, human SMYD2 was overexpressed, purified, and characterized. Substrate specificity and product analysis studies established SMYD2 as a monomethyltransferase that prefers nonmethylated p53 peptide substrate. Steady-state kinetic and product inhibition studies showed that SMYD2 operates via a rapid equilibrium random Bi Bi mechanism at a rate of 0.048 ± 0.001 s(-1), with K(M)s for AdoMet and the p53 peptide of 0.031 ± 0.01 μM and 0.68 ± 0.22 μM, respectively. Metal analyses revealed that SMYD2 contains three tightly bound zinc ions that are important for maintaining the structural integrity and catalytic activity of SMYD2. Catalytic activity was also shown to be dependent on the GxG motif in the S-sequence of the split SET domain, as a G18A/G20A double mutant and a sequence deletion within the conserved motif impaired AdoMet binding and significantly decreased enzymatic activity. The functional importance of other SMYD2 domains including the MYND domain, the cysteine-rich post-SET domain, and the C-terminal domain (CTD), were also investigated. Taken together, these results demonstrated the functional importance of distinct domains in the SMYD family of proteins and further advanced our understanding of the catalytic mechanism of this family.     

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.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.     

The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways

Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium.     

Cell behavior during formation of prestalk/prespore pattern in submerged agglomerates of Dictyostelium discoideum

When cells dissociated from Dictyostelium discoideum slugs were cultured in roller tubes, they formed agglomerates in which prestalk cells were initially dispersed but soon sorted out to the center and then moved to the edge to reconstitute the prestalk/prespore pattern. To examine the mechanism of sorting out, individual prestalk cells were traced by a videotape recorder. The radial component of the rate of movement toward the center of the presumptive prestalk region was calculated. Prestalk cells did not move randomly, but rather directionally toward the center. Their movement was pulsatile, with a period of ca. 15 min, and accompanied by occasional formation of cell streams, thus resembling the movement observable during cell aggregation. These results favor the idea that prestalk cells sort out to the prestalk region due to differential chemotaxis rather than differential adhesiveness. After formation of the prestalk/prespore pattern, the prestalk region rotated along the circumference of the agglomerates. This appears comparable to migration of slugs on the substratum, the rate of rotation being similar to that of slug migration. To examine the processes of pattern formation during development, washed vegetative cells were cultured in roller tubes. Prespore cells identified by antispore immunoglobulin initially appeared randomly within the agglomerates, but then nonprespore cells accumulated in the center and finally moved to the edge to establish the prestalk/prespore pattern, the processes being similar to those of pattern reconstruction with differentiated prestalk and prespore cells.     

Heat shock protein 90 regulates development in Dictyostelium discoideum

Cytosolic heat shock protein 90 (Hsp90) has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, development, and morphological evolution. Model systems available for studying Hsp90 function either allow ease of manipulation for biochemical studies or facilitate a phenomenological study of its role in influencing phenotype. In this work, we have explored the use of the cellular slime mold Dictyostelium discoideum to examine cellular functions of Hsp90 in relation to its multicellular development. In addition to cloning, purification, biochemical characterization, and examination of its crystal structure, our studies, using a pharmacological inhibitor of Hsp90, demonstrate a role for the cytoplasmic isoform (HspD) in D. discoideum development. Inhibition of HspD function using geldanamycin (GA) resulted in delayed aggregation and arrest of D. discoideum development at the ‘mound’ stage. Crystal structure of the amino-terminal domain of HspD showed a binding pocket similar to that described for yeast Hsp90. Fluorescence spectroscopy, as well as GA-coupled beads affinity pulldown, confirmed a specific interaction between HspD and GA. The results presented here provide an important insight into the function of HspD in D. discoideum development and emphasize the potential of the cellular slime mold to serve as an effective model for studying the many roles of Hsp90 at cellular and organismal levels.     

A cell number counting factor regulates Akt/protein kinase B to regulate Dictyostelium discoideum group size

Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of approximately 20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin- cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB.     

A lysine-rich protein functions as an H1 histone in Dictyostelium discoideum chromatin.

Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp). Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion. This was confirmed by stripping the protein from chromatin with Dowex resin. Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion. Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes. Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes. Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome. Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx. 10 kilodaltons. Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease. Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.     

SET7/9 interacts and methylates the ribosomal protein,eL42 and regulates protein synthesis

Methylation of proteins is emerging to be an important regulator of protein function. SET7/9, a protein lysine methyltransferase, catalyses methylation of several proteins involved in diverse biological processes. SET7/9-mediated methylation often regulates the stability, sub-cellular localization and protein-protein interactions of its substrate proteins. Here, we aimed to identify novel biological processes regulated by SET7/9 by identifying new interaction partners. For this we used yeast two-hybrid screening and identified the large subunit ribosomal protein, eL42 as a potential interactor of SET7/9. We confirmed the SET7/9-eL42 interaction by co-immunoprecipitation and GST pulldown studies. The N-terminal MORN domain of SET7/9 is essential for its interaction with eL42. Importantly, we identified that SET7/9 methylates eL42 at three different lysines - Lys53, Lys80 and Lys100 through site-directed mutagenesis. By puromycin incorporation assay, we find that SET7/9-mediated methylation of eL42 affects global translation. This study identifies a new role of the functionally versatile SET7/9 lysine methyltransferase in the regulation of global protein synthesis.     

Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain‐containing 3 and induces activator protein 1 activation

Hepatitis C virus (HCV) non‐structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A‐interacting protein, SET and MYND domain‐containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon‐harboring and HCV‐infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N‐SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A‐SMYD3 interaction. NS5A co‐localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP‐1) activity, this being potentiated by co‐expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP‐1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP‐1 activation in HCV‐infected cells.     

Prestalk/prespore differentiation tendency of Dictyostelium discoideum cells as detected by a stalk-specific monoclonal antibody

By the use of a prestalk- and stalk-specific monoclonal antibody, production of prestalk antigen was examined with non-glucose grown [G(-)] and glucose grown [G(+)] cells of Dictyostelium discoideum AX2. Unlike wild type (NC4), some growth phase cells of AX2 were reactive with the antibody. However, G(-) cells contained much more antigen than G(+) cells and the difference between the two remained during the preaggregation period. Besides glucose, the addition of metabolizable, but not nonmetabolizable sugars to both growth phase and preaggregation cells suppressed the production of the prestalk antigen on the one hand and stimulated the accumulation of glycogen on the other hand. When mixed, G(-) cells which produced more prestalk antigen during the preaggregation period remained prestalk cells after aggregation, while G(+) cells which produced less antigen were converted to prespore cells. G(+) cells collected at the stationary phase [G(+)st] were stronger in prestalk sorting tendency than G(+) cells but weaker than G(-) cells. The prestalk antigen content of G(+)st cells prior to aggregation was an intermediate between those of G(-) and G(+) cells. These lead to the conclusion that the prestalk antigen content of preaggregation cells reflect the tendency of the cells toward either prestalk or prespore differentiation after aggregation.     

A density-sensing factor regulates signal transduction in Dictyostelium

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.     

Prestalk/prespore Differentiation of Dictyostelium Cells under the Conditions Favoring Stalk or Spore Cell Formation*

The differentiation processes of Dictyostelium discoideum cells under the conditions which favored either stalk or spore cell formation were examined by the use of prestalk- and prespore-specific antibodies. In stalk cell-forming conditions, cells reactive with prestalk-specific monoclonal antibody (C1) increased rapidly early in development and later differentiated into stalk cells. No or only a few cells became reactive with prespore-specific monoclonal (B6) and polyclonal (antispore) antibodies. Despite the fact that most cells terminally became spores under spore cell-forming conditions, cells were first stained with the C1 antibody before becoming reactive with the B6 antibody. Unlike the case of normal development where cells coincidentally become reactive with the B6 and antispore antibodies, the appearance of the cells reactive with the latter was either delayed or suppressed. In conclusion, under either spore or stalk cell-forming conditions, the appearance of the prestalk antigen preceded that of the prespore one, which is consistent with normal development.     

The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity

The SET protein and the cell cycle inhibitor p21(Cip1) interact in vivo and in vitro. We identified here the domain (157)LIF(159) of p21(Cip1) as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21(Cip1), one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21(Cip1) co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G(2)/M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21(Cip1) cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G(2)/M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G(2)/M transition by modulating cyclin B-CDK1 activity.     

Correlations between tip dominance, prestalk/prespore pattern, and CAMP-relay efficiency in slugs of Dictyostelium discoideum

Abstract. It is very likely that oscillatory cAMP secretion and cAMP relay organize postaggregative cell movement in the cellular slime molds. We present evidence indicating that cAMP signaling may also be involved in the formation of the prestalk/prespore pattern in slugs of Dictyostelium discoideum. Reduction of cAMP relay in slugs caused by caffeine increased the proportion of prespore tissue. An even stronger increase was observed in a mutant with a very low CAMP-relay response. The effects on pattern resulting from a reduction of cAMP relay are not due to a reduction in the amount of cAMP in the slug, but to an as yet undefined property of oscillatory cAMP signaling.