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An F-Box/WD40 Repeat-Containing Protein Important for Dictyostelium Cell-Type Proportioning, Slug Behaviour, and Culmination
Abstract
FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA− cells), the spore mass fails to fully ascend the stalk. In addition, fbxA− slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA− cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA− slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.
... CulA was cloned as described in Materials and Methods. The cloning and preliminary analysis of fbxA, also called chtA, has been described previously (Ennis et al. 2000;Nelson et al. 2000). The sequence of the CulA ORF indicates that it is a member of the cullin family of proteins ( Fig. 1A; GenBank accession no. ...
... Previous results show that fbxA is preferentially expressed in prestalk cells and is required in these cells for proper cell-type proportioning. fbxA null cells have a propensity to differentiate into prespore cells and exhibit a reduction in prestalk gene expression and a reduced number of prestalk cells ( Fig. 4A ; Ennis et al. 2000;Nelson et al. 2000). Unlike culA null cells, the fbxA null cell phenotypes suggest that FbxA is required predominantly during the multicellular stages, as fbxA null cells do not exhibit aggregation-stage chemotaxis or gene expression defects. ...
... Unlike culA null cells, the fbxA null cell phenotypes suggest that FbxA is required predominantly during the multicellular stages, as fbxA null cells do not exhibit aggregation-stage chemotaxis or gene expression defects. [Note: the fbxA null phenotypes vary in severity depending on the genetic background and are most severe in strain KAx-3, the parental strain used in this study (data not shown; Nelson et al. 2000)]. In this strain, most fbxA null aggregates arrest at the mound stage (data not shown; Nelson et al. 2000), ecmA prestalk and SP60/ cotC gene expression is delayed, and the level of ecmA expression is reduced compared with that in wild-type cells (Fig. 4), consistent with previous findings (Nelson et al. 2000). ...
Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity.
... We were able to select a mutant, which we called cheater (chtA), with these characteristics (10,12). Nelson et al. (25) recovered a mutant affected in the same locus, which they called the fbxA mutant, because the gene codes for an F-box protein. To simplify nomenclature and to aid annotation projects, we will convert to the fbxA designation. ...
... The promoter of fbxA was obtained from genomic DNA of REMI (restriction enzyme-mediated integration) (18) mutant 82. This mutant arose from the insertion of plasmid pJB1 near the 3Ј end of the gene to create the fbxA mutation (25). The original chtA/fbxA mutant arose from an insertion of a different plasmid at the same site in strain AX3-1 (12). ...
... The outline of the locus is shown in Fig. 1. Plasmids pBSR3 and pJB1 integrated at a common DpnII site during REMI mutagenesis to yield fbxA derivatives of AX3 (12) and DH1 (25). A second insertion, into the F-box coding domain of the gene (Fig. 1), conferred the same phenotype as the more 3Ј insertion (data not shown). ...
Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality.
In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells.
To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it
is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation
of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor,
there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined.
The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected
on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of
RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.
... The culmination of the migrating slugs is controlled by external factors such as light, temperature and humidity and internal factors such as ammonia and cAMP (Newell et al., 1969;Schindler and Sussman, 1977). These factors have been identified by the studies of slugger mutants which are unable to culminate under normal condition (Davies et al., 1993;Fukuzawa et al., 1997;Fukuzawa and Williams, 2002;Gee et al., 1994;Nelson et al., 2000;Newell and Ross, 1982;Singleton et al., 1998). ...
... The aberration of specific cell-type differentiation has been found in some slugger mutants (Fukuzawa et al., 1997;Nelson et al., 2000;Singleton et al., 1998). To verify cell-type differentiation in Grx1 OE cells, the expression pattern of various cell-type-specific genes was examined by Northern blot analysis, as shown in Fig. 3. ...
... However, when the slugs were exposed to a lateral light source, the migration distance of Grx1 OE slugs was indistinguishable from that of KAx3 slugs (data not shown), indicating that the increased migration of Grx1 OE slugs may be due to the delayed culmination rather than increased mobility. Previous studies have demonstrated that many slugger mutants display hypersensitivity toward ammonia and have a problem in prestalk cell differentiation (Gee et al., 1994;Hopper et al., 1993;Nelson et al., 2000;Singleton et al., 1998). Although Grx1 OE cells were also sensitive to ammonia inhibition (data not shown), they formed fruiting bodies normally under overhead light, which can trigger culmination, and showed the defective expression of some prespore genes. ...
Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate.
... A known substrate for Dictyostelium P4H1 is Skp1 (van der Wel et al., 2005), a subunit of the SCF-class of E3 Ubligases. E3 SCF Ub-ligases regulate the cell cycle, nutrient sensing, physiology and development in many organisms (Willems et al., 2004), including the latter in Dictyostelium (Ennis et al., 2000;Nelson et al., 2000;Mohanty et al., 2001;Tekinay et al., 2003). Dd-Skp1 is modified at Pro143, which is replaced by Glu in chordate Skp1s. ...
... Forward genetic screens previously implicated cullins and F-box proteins, partners with Skp1 in SCF complexes, in the regulation of multiple developmental transitions in Dictyostelium (Ennis et al., 2000;Nelson et al., 2000;Mohanty et al., 2001;Wang and Kuspa, 2002). Studies on regulation of E3 SCF Ub-ligases, conducted primarily in yeast and mammals, have focused on the E2, RING, cullin, and the F-box protein subunits (Deshaies and Joazeiro, 2009). ...
O(2) regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O(2)-threshold for culmination implicating it as a direct O(2)-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3(SCF)ubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O(2)-signal. Here we show that genetically induced changes in Skp1 levels also affect the O(2)-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3(SCF)ubiquitin-ligase activity modulate O(2)-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O(2)-sensing.
... Dictyostelium has proven an interesting system for the study of regulated protein destruction and development (Ennis et al., 2000Mohanty et al., 2001;Nelson et al., 2000;Tekinay et al., 2003;Wang and Kuspa, 2002). Differentiation and morphogenesis of Dictyostelium are regulated by chemotactic and morphogenic signaling mediated by extracellular cAMP (Kimmel and Firtel, 2004;Kimmel and Parent, 2003). ...
... Yet, we do not know the specific actions of CSN5 or the COP9 holocomplex during the growth of Dictyostelium. Dictyostelium have at least four cullins; the two characterized, culA and culB, are dispensable for growth (Mohanty et al., 2001;Nelson et al., 2000;Wang and Kuspa, 2002), but are both required for development. It should therefore be emphasized that we first identified CSN5 in a yeast twohybrid screen as an interacting partner of cAMP receptor CAR3 that is only active during development and that is not required for growth (Kim et al., 1999;Kimmel and Firtel, 2004;Plyte et al., 1999). ...
Regulated protein destruction involving SCF (Skp1/Cullin/F-box, E3 ubiquitin ligase) complexes is required for multicellular development of Dictyostelium discoideum. Dynamic modification of cullin by nedd8 is required for the proper action of SCF. The COP9 signalosome (CSN), first identified in a signaling pathway for light response in plants, functions as a large multi-protein complex that regulates cullin neddylation in eukaryotes. Still, there is extreme sequence divergence of CSN subunits of the yeasts in comparison to the multicellular plants and animals. Using the yeast two-hybrid system, we have identified the CSN5 subunit as a potential interacting partner of a cell surface receptor of Dictyostelium. We further identified and characterized all 8 CSN subunits in Dictyostelium discoideum. Remarkably, despite the ancient origin of Dictyostelium, its CSN proteins cluster very closely with their plant and animal counterparts. We additionally show that the Dictyostelium subunits, like those of other systems are capable of multi-protein interactions within the CSN complex. Our data also indicate that CSN5 (and CSN2) are essential for cell proliferation in Dictyostelium, a phenotype similar to that of multicellular organisms, but distinct from that of the yeasts. Finally, we speculate on a potential role of CSN in cullin function and regulated protein destruction during multicellular development of Dictyostelium.
... When these markers were examined post-aggregation in culA − cells, ecmA expression was absent and cotC expression was decreased (Mohanty et al., 2001). In addition, an altered ratio of pre-stalk to pre-spore cells was reported in fbxA − cells and overexpression of FLAG-tagged FbxD has been shown to delay fruiting body formation (Nelson et al., 2000;Sheikh et al., 2015). Finally, inhibiting neddylation with MLN4924 was shown to impair fruiting body formation . ...
“Cullins (CULs) are a core components” of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba Dictyostelium discoideum , has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in D. discoideum , which supports previous work that revealed the cullin interactome in D. discoideum and the roles of cullins and the CSN in regulating cellular and developmental processes during the D. discoideum life cycle. Here, we review the roles of cullins, neddylation, and the CSN in D. discoideum to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.
... The culmination of migrating slugs is regulated by internal (e.g., ammonia and cAMP) and external (e.g., light, temperature, and humidity) factors (Newell et al., http://dx.doi.org/10.1016/j.biocel.2017.07.019 Received 2 April 2017; Received in revised form 13 July 2017; Accepted 27 July 2017 1969; Schindler and Sussman, 1977), which have been studied by mutating slugs to be defective in the culmination phase (Davies et al., 1993;Fukuzawa et al., 1997;Gee et al., 1994;Nelson et al., 2000;Singleton et al., 1998). However, despite the high resistance of D. discoideum to H 2 O 2 , little is known about oxidative stress-induced responses associated with methylglyoxal (MG), its inducing partner, and slug migration (Garcia et al., 2000). ...
... It could reflect the fact that different parental strains were used: AX4 in the original study and Ax2 here. There are other examples where disruption of the same gene produces a more severe phenotype in AX4 (Nelson et al., 2000; Schilde et al., 2004). Alternatively, it may result from differences in the genetic lesion; the original REMI mutant and the re-disrupted mutant were single site insertions while the new mutant is a deletion mutant that removes a significant part of the gene including most of the MIF4G domain (Fig. 4). ...
DIF-1 (differentiation-inducing factor1) is a polyketide produced by Dictyostelium prespore cells which induces initially uncommitted cells to differentiate as prestalk cells. Exposure of cells to DIF-1 causes transitory hypo-phosphorylation of seven serine residues in YelA, a protein with a region of strong homology to the MIF4G domain of the eukaryotic initiation factor eIF4G. Based upon its domain architecture, which in one important aspect closely resembles that of Death-Associated Protein 5 (DAP5), we predict a role in stimulating internal ribosome entry-driven mRNA translation. The two paradigmatic DIF-1 inducible genes are ecmA (extracellular matrixA) and ecmB. In support of a YelA function in DIF-1 signaling, a YelA null strain showed greatly increased expression of ecmA and ecmB in response to DIF-1. Also, during normal development in the null strain, expression of the two genes is accelerated. This is particularly evident for ecmB, a marker of stalk tube and supporting structure differentiation. Mutants in DIF-1 bio-synthesis or signaling display a rudimentary or no basal disc and, conversely, YelA null mutants produce fruiting bodies with a highly enlarged basal disc that ectopically expresses a stalk tube-specific marker. Thus YelA acts as an antagonist of DIF-1 signaling, with a consequent effect on cell type proportioning and it is predicted to act as a translational regulator.
... Ubiquitylation represents an evolutionarily conserved regulatory mechanism. RING finger ubiquitin ligases and ancillary proteins have been shown to be important for normal Dictyostelium development (Clark et al., 1997;Ennis et al., 2000;Nelson et al., 2000;Wang and Kuspa, 2002;Whitney et al., 2006) and even for slug phototactic migration (Whitney et al., 2006). Searches indicate that the Dictyostelium genome is likely to encode six HECT ligases, each containing a C-terminal HECT domain and most an identifiable N-terminal domain that might be responsible for binding to its specific target. ...
Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and ‘salt and pepper’ patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA− pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA− pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA− background rescues pstO cell localisation. hfnA− cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on ‘salt and pepper’ differentiation and sorting out.
... The relative proportions of these prestalk subtypes were found to be affected by mutations in several different genes. Mutations in the gene encoding adenosine monophosphate deaminaase, amdA, resulted in twice the number of PstA cells (Chae, Fuller and Loomis, 2001) while mutations in the F-box genes mkkA and fbxA affected the proportions of PstO cells (Chung et al., 1998;Nelson et al., 2000). These mutations act in a cell autonomous manner ruling out any role in production of an intercellular signal but consistent with affecting the response to such signals. ...
Cells grow, move, expand, shrink and die in the process of generating the characteristic shapes of organisms. Although the structures generated during development of the social amoeba Dictyostelium discoideum look nothing like the structures seen in metazoan embryogenesis, some of the morphogenetic processes used in their making are surprisingly similar. Recent advances in understanding the molecular basis for directed cell migration, cell type specific sorting, differential adhesion, secretion of matrix components, pattern formation, regulation and terminal differentiation are reviewed. Genes involved in Dictyostelium aggregation, slug formation, and culmination of fruiting bodies are discussed.
Copyright © 2015. Published by Elsevier Inc.
... Specifically, early tipped mound formation has been reported in strains overexpressing cyclin C, cyclin-dependent kinase 8, or the G-protein alpha 5 subunit, and in knockout mutants of histidine kinase C, a metabotropic glutamate receptor-like protein, protein inhibitor of STAT, and SCAR/WAVE [70][71][72][73][74][75]. Several knockout mutants that display increased slug migration have been described, including mutants for genes important for oxysterol binding, the assembly of mitochondrial complex I, and the targeting of proteins for degradation via proteasomes [76][77][78]. This phenotype has also been observed in cells overexpressing histidine kinase C or in cells where calcium-binding protein 3 expression has been knocked down by RNAi [72,79]. ...
... Specifically, early tipped mound formation has been reported in strains overexpressing cyclin C, cyclin-dependent kinase 8, or the G-protein alpha 5 subunit, and in knockout mutants of histidine kinase C, a metabotropic glutamate receptor-like protein, protein inhibitor of STAT, and SCAR/WAVE [70][71][72][73][74][75]. Several knockout mutants that display increased slug migration have been described, including mutants for genes important for oxysterol binding, the assembly of mitochondrial complex I, and the targeting of proteins for degradation via proteasomes [76][77][78]. This phenotype has also been observed in cells overexpressing histidine kinase C or in cells where calcium-binding protein 3 expression has been knocked down by RNAi [72,79]. ...
The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3- cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3- cells was precocious and cln3- slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3- cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3- cells, strongly supports the use of this new model for JNCL research.
... Distinct developmental phenotypes appear in other pathway mutants (16), reinforcing the concept that global Skp1 functions, perhaps hydroxylation-dependent but not necessarily O 2 -regu-lated, are affected by Skp1 modification. Skp1 is an adaptor in the multisubunit SCF complex family, with potentially 50 members based on the number of predicted FBP genes, 3 some of which are active in Dictyostelium development (33)(34)(35)(36). Studies from other organisms indicate that in addition to interacting with F-box proteins and cullins and the modification enzymes discussed here, Skp1 can homodimerize (37) and interact with other proteins such as Sgt1 and Siah-interacting protein (SIP). ...
Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.
... Ubiquitylation represents an evolutionarily conserved regulatory mechanism. RING finger ubiquitin ligases and ancillary proteins have been shown to be important for normal Dictyostelium development (Clark et al., 1997;Ennis et al., 2000;Nelson et al., 2000;Wang and Kuspa, 2002;Whitney et al., 2006) and even for slug phototactic migration (Whitney et al., 2006). Searches indicate that the Dictyostelium genome is likely to encode six HECT ligases, each containing a C-terminal HECT domain and most an identifiable N-terminal domain that might be responsible for binding to its specific target. ...
Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.
... In cells, Skp1 forms a stable complex with F-box proteins (e.g., Fig. 3C), which are generally insoluble when expressed separately from Skp1 (36). To test whether heterodimerization with an F-box protein affected activity, Skp1 was coexpressed in E. coli with His 6 ⊗N-FbxA, a previously described N-terminally truncated form of a Dictyostelium F-box protein important for slug morphogenesis (23,37). As expected, a fraction of His 6 ⊗N-FbxA was soluble when expressed with Skp1, but not in its absence (data not shown), suggesting formation of a possible native complex as occurs in cells. ...
The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.
... Serial selections of REMI mutagenized cells have allowed the identification of genes that, when inactivated , lead to a cheater phenotype. The most frequently occurring and best characterized of these, fbxA, encodes an F box protein (Ennis et al. 2000; Nelson et al. 2000). The F box family of ubiquitin ligases targets proteins for ubiquitination and degradation. ...
Any established or aspiring model organism must justify itself using two criteria: does the model organism offer experimental advantages not offered by competing systems? And will any discoveries made using the model be of wider relevance? This review addresses these issues for the social amoeba Dictyostelium and highlights some of the organisms more recent applications. These cover a remarkably wide gamut, ranging from sociobiological to medical research with much else in between.
... Though the majority of steady-state Skp1 appears to be modified throughout the life cycle, nascent Skp1-myc, induced under developmental control of a prespore cell promoter, is very inefficiently hydroxylated [14], suggesting that modification of newly synthesized Skp1 may be rate limiting at the time of O 2 -sensing. In addition, proteins that potentially interact with Skp1 in SCF complexes, including CulA, CulB, and FbxA, are required for normal development [12,[60][61][62], though effects on the O 2 -threshold remain to be investigated. Future work is needed to determine whether these proteins interfere with or promote Skp1 modifications, and if O 2 signals via the SCF complex(es) to regulate culmination. ...
The soil amoeba Dictyostelium is an obligate aerobe that monitors O(2) for informational purposes in addition to utilizing it for oxidative metabolism. Whereas low O(2) suffices for proliferation, a higher level is required for slugs to culminate into fruiting bodies, and O(2) influences slug polarity, slug migration, and cell-type proportioning. Dictyostelium expresses a cytoplasmic prolyl 4-hydroxylase (P4H1) known to mediate O(2)-sensing in animals, but lacks HIFalpha, a major hydroxylation target whose accumulation directly induces animal hypoxia-dependent transcriptional changes. The O(2)-requirement for culmination is increased by P4H1-gene disruption and reduced by P4H1 overexpression. A target of Dictyostelium P4H1 is Skp1, a subunit of the SCF-class of E3-ubiquitin ligases related to the VBC-class that mediates hydroxylation-dependent degradation of animal HIFalpha. Skp1 is a target of a novel cytoplasmic O-glycosylation pathway that modifies HyPro143 with a pentasaccharide, and glycosyltransferase mutants reveal that glycosylation intermediates have antagonistic effects toward P4H1 in O(2)-signaling. Current evidence indicates that Skp1 is the only glycosylation target in cells, based on metabolic labeling, biochemical complementation, and enzyme specificity studies. Bioinformatics studies suggest that the HyPro-modification pathway existed in the ancestral eukaryotic lineage and was retained in selected modern day unicellular organisms whose life cycles experience varying degrees of hypoxia. It is proposed that, in Dictyostelium and other protists including the agent for human toxoplasmosis Toxoplasma gondii, prolyl hydroxylation and glycosylation mediate O(2)-signaling in hierarchical fashion via Skp1 to control the proteome, directly via degradation rather than indirectly via transcription as found in animals.
... Different SCF complexes, which vary in their F-box proteins, are specific for the ubiquitination of individual cell cycle regulatory proteins, transcriptional factors, and other signaling proteins. F-box proteins and a cullin have recently been identified in Dictyostelium (Chung et al., 1998; Ennis et al., 2000; Nelson et al., 2000). The precise function of Skp1 has yet to be elucidated. ...
Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on M(r)-shift analysis using SDS-polyacrylamide gel electrophoresis/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on reverse-transcription polymerase chain reactions. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based M(r)-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration.
... Disruption of a gene encoding a protein with seven ankyrin repeats and a homeobox, wriA, also results in expansion of the PST-O domain at the expense of the prespore domain (Han and Firtel, 1998). On the other hand, disruption of fbxA, a gene encoding five WD40 repeats and an F-box, results in fewer PST-O cells, while overexpression of this gene increases the number of PST-O cells (Nelson et al., 2000). It is not clear whether these genes participate in a common network controlling cell-type proportions. ...
The proportions of prespore and prestalk cells in Dictyostelium discoideum are regulated so that they are size invariant and can adjust when the ratio is perturbed. We have found that disruption of the gene amdA that encodes AMP deaminase results in a significantly increased proportion of prestalk cells. Strains lacking AMP deaminase form short, thick stalks and glassy sori with less than 5% the normal number of spores. The levels of prestalk-specific mRNAs in amdA(-) cells are more than twice as high as those in wild-type strains and prespore-specific mRNAs are reduced. Using an ecmA::lacZ construct to mark prestalk cells, we found that amdA(-) null slugs have twice the normal number of prestalk cells. The number of cells expressing an ecmO::lacZ construct was not affected by loss of AmdA, indicating that the mutation results in an increase in PST-A prestalk cells rather than PST-O cells. This alteration in cell-type proportioning is a cell-autonomous consequence of the loss of AMP deaminase since mutant cells developed together with wild-type cells still produced excess prestalk cells and wild-type cells carrying the ecmA::lacZ construct formed normal numbers of prestalk cells when developed together with an equal number of amdA(-) mutant cells.
... However, only in plants, animals and multicellular protists (D. discoideum) are START domains found in association with domains having established functions in signal transduction or transcriptional control, consistent with the idea that START evolved as a regulatory domain in multicellular eukaryotes. The cellular slime mold D. discoideum, which progresses from unicellular to multicellular developmental stages, contains an unusual START-domain protein [8] which has so far not been found in any other organism: FbxA/CheaterA (ChtA), an F-Box/WD40 repeat-containing protein [12,13]. FbxA/ChtA is thought to encode a component of an SCF E3 ubiquitin ligase implicated in cyclic AMP metabolism and histidine kinase signaling during development [14]. ...
In animals, steroid hormones regulate gene expression by binding to nuclear receptors. Plants lack genes for nuclear receptors, yet genetic evidence from Arabidopsis suggests developmental roles for lipids/sterols analogous to those in animals. In contrast to nuclear receptors, the lipid/sterol-binding StAR-related lipid transfer (START) protein domains are conserved, making them candidates for involvement in both animal and plant lipid/sterol signal transduction.
We surveyed putative START domains from the genomes of Arabidopsis, rice, animals, protists and bacteria. START domains are more common in plants than in animals and in plants are primarily found within homeodomain (HD) transcription factors. The largest subfamily of HD-START proteins is characterized by an HD amino-terminal to a plant-specific leucine zipper with an internal loop, whereas in a smaller subfamily the HD precedes a classic leucine zipper. The START domains in plant HD-START proteins are not closely related to those of animals, implying collateral evolution to accommodate organism-specific lipids/sterols. Using crystal structures of mammalian START proteins, we show structural conservation of the mammalian phosphatidylcholine transfer protein (PCTP) START domain in plants, consistent with a common role in lipid transport and metabolism. We also describe putative START-domain proteins from bacteria and unicellular protists.
The majority of START domains in plants belong to a novel class of putative lipid/sterol-binding transcription factors, the HD-START family, which is conserved across the plant kingdom. HD-START proteins are confined to plants, suggesting a mechanism by which lipid/sterol ligands can directly modulate transcription in plants.
... Other proportioning mutants also display a change in the extent of the pstO region while retaining a normally sized pstA zone (Han and Firtel, 1998;Chung et al., 1998;Nelson et al., 2000;Ennis et al., 2000). However, the mybE-strain has a more severe defect than any of these strains. ...
PstA and pstO cells are the two major populations in the prestalk region of the Dictyostelium slug and DIF-1 is a low molecular weight signalling molecule that selectively induces pstO cell-specific gene expression. The two cell types are defined by their differential use of spatially separated regions of the ecmA promoter. Additionally, there are anterior-like cells (ALCs) scattered throughout the rear, prespore region of the slug. They, like the pstO cells, use a cap-site distal ecmA promoter segment termed the ecmO region. When multimerised, a 22-nucleotide subsegment of the ecmO region directs expression in pstA cells, pstO cells and ALCs. It also directs DIF-inducible gene expression. The 22-nucleotide region was used to purify MybE, a protein with a single MYB DNA-binding domain of a type previously found only in a large family of plant transcription factors. Slugs of a mybE-null (mybE-) strain express an ecmAO:lacZ fusion gene (i.e. a reporter construct containing the ecmA and ecmO promoter regions) in pstA cells but there is little or no expression in pstO cells and ALCs. The ecmA gene is not induced by DIF-1 in a mybE-strain. Thus, MybE is necessary for DIF-1 responsiveness and for the correct differentiation of pstO cells and ALCs.
... As slugger mutants have been reported to be hypersensitive to the inhibitory effect of ammonia (Gee et al., 1994;Singleton et al., 1998;Nelson et al., 2000), we examined whether Trr OE cells also possess this property. The cells were deposited on black filters resting on the support pads soaked with KK2 buffer containing NH4Cl. ...
The thioredoxin system, consisting of thioredoxin, thioredoxin reductase and NADPH, has been well established to be critical for the redox regulation of protein function and signalling. To investigate the role of thioredoxin reductase (Trr) in Dictyostelium discoideum, we generated mutant cells that underexpress or overexpress Trr. Trr-underexpressing cells exhibited severe defects in axenic growth and development. Trr-overexpressing (TrrOE) cells formed very tiny plaques on a bacterial lawn and had a lower rate of bacterial uptake. When developed in the dark, TrrOE cells exhibited a slugger phenotype, defined by a prolonged migrating slug stage. Like other slugger mutants, they were hypersensitive to ammonia, which has been known to inhibit culmination by raising the pH of intracellular acidic compartments. Interestingly, TrrOE cells showed defective acidification of intracellular compartments and decreased activity of vacuolar H+-ATPase which functions in the acidification of intracellular compartments. Moreover, biochemical studies revealed that the thioredoxin system can directly reduce the catalytic subunit of vacuolar H+-ATPase whose activity is regulated by reversible disulphide bond formation. Taken together, these results suggest that Dictyostelium Trr may be essential for growth and play a role in regulation of phagocytosis and culmination, possibly through the modulation of vacuolar H+-ATPase activity.
... Skp1 is the only substrate that accumulates in P4H1cells based on an indirect assay with rP4H1 and rGnT1 (our unpublished data). A role for Skp1 is further supported by evidence that CulA and FbxA, proteins that physically associate with Skp1 in SCF-type E3 Ub-ligase complexes (Willems et al., 2004), are also required for multiple developmental steps, including culmination (Mohanty et al., 2001;Nelson et al., 2000;Ennis et al., 2000). However, P4H1slugs do not accumulate the E3(SCF FbxA )Ub-ligase substrate RegA (not shown), as occurs in culA-and fbxA-null cells (Mohanty et al., 2001). ...
Development in multicellular organisms is subject to both environmental and internal signals. In Dictyostelium, starvation induces amoebae to form migratory slugs that translocate from subterranean areas to exposed sites, where they culminate to form sessile fruiting bodies. Culmination, thought to be regulated by anterior tip cells, is selectively suppressed by mild hypoxia by a mechanism that can be partially overridden by another environmental signal, overhead light, or genetic activation of protein kinase A. Dictyostelium expresses, in all cells, an O2-dependent prolyl 4-hydroxylase (P4H1) required for O-glycosylation of Skp1, a subunit of E3SCF-Ub-ligases. P4H1-null cells differentiate the basic pre-stalk and pre-spore cell types but exhibit a selectively increased O2 requirement for culmination, from approximately 12% to near or above ambient (21%) levels. Overexpression of P4H1 reduces the O2 requirement to <5%. The requirement for P4H1 can be met by forced expression of the active enzyme in either pre-stalk (anterior) or pre-spore (posterior) cells, or replaced by protein kinase A activation or addition of small numbers of wild-type cells. P4H1-expressing cells accumulate at the anterior end, suggesting that P4H1 enables transcellular signaling by the tip. The evidence provides novel genetic support for the animal-derived O2-sensor model of prolyl 4-hydroxylase function, in an organism that lacks the canonical HIFalpha transcriptional factor subunit substrate target that is a feature of animal hypoxic signaling.
In social situations, opportunities arise for some individuals to
take advantage of others. This happens in wild populations of the social amoeba
Dictyostelium discoideum.
The oxysterol binding proteins (OSBPs) are believed to control cholesterol homeostasis but their precise mechanism of action is not well understood. The Dictyostelium osbA gene encodes a predicted OSBP, OSBPa, which lacks the PH domain that in most other OSBPs directs targetting to the Golgi. OSBPa instead localises selectively to the cell periphery and also, in some cells, to the perinuclear region. OSBPa null strains form normal fruiting bodies but are defective in the regulation of the transition from slug migration to culmination. Thus a plasma membrane-enriched OSBP family member is essential for correct regulation of the slug-fruiting body switch.
We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.
Cell-fate decisions and spatial patterning in Dictyostelium are regulated by a number of genes. Our studies have implicated a gene called fbxA, which codes for an F-box protein, in these pathways. The FbxA protein is one of the controls on a cAMP phosphodiesterase called RegA, mediating its degradation via ubiquitin-linked proteolysis. Using marked strains, we showed that the fbxA- mutant has defective cell-type proportioning, with a dearth of prestalk cells compared to prespore cells. In this work, we show that this effect occurs earlier during the 24 hour developmental cycle than previously thought. The normal sorting of the prestalk and prespore cells in aggregates and mounds is not affected by the mutation. The mutant cells sort abnormally at the tipped mound stage, when prespore and prestalk cells normally distribute into their proper compartments. The fbxA- mutant forms pre-stalk cells in low numbers when not in chimeras, but in the presence of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form non-viable stalk cells. In an attempt to identify the signal transduction pathway that mediates proportionality in prestalk and prespore cells, we asked whether certain signal transduction mutants were immune to the effects of the fbxA- cells and formed spores in chimeras.
The control of cheating is important for understanding major transitions in evolution, from the simplest genes to the most complex societies. Cooperative systems can be ruined if cheaters that lower group productivity are able to spread. Kin-selection theory predicts that high genetic relatedness can limit cheating, because separation of cheaters and cooperators limits opportunities to cheat and promotes selection against low-fitness groups of cheaters. Here, we confirm this prediction for the social amoeba Dictyostelium discoideum; relatedness in natural wild groups is so high that socially destructive cheaters should not spread. We illustrate in the laboratory how high relatedness can control a mutant that would destroy cooperation at low relatedness. Finally, we demonstrate that, as predicted, mutant cheaters do not normally harm cooperation in a natural population. Our findings show how altruism is preserved from the disruptive effects of such mutant cheaters and how exceptionally high relatedness among cells is important in promoting the cooperation that underlies multicellular development.
• altruism
• cellular slime molds
• conflict
• Dictyostelium discoideum
• kin selection
Introduction of restriction enzyme along with linearized plasmid results in integration of plasmid DNA at genomic restriction sites in a high proportion of the resulting transformants. We have found that electroporating BamHI or EcoRI together with pyr5-6 plasmids cut with the same enzyme stimulates the efficiency of transformation in Dictyostelium discoideum more than 20-fold over the rate seen when plasmid DNA alone is introduced. Restriction enzyme-mediated integration generates insertions into genomic restriction sites in an apparently random manner, some of which cause mutations. About 1 in 400 of the Dictyostelium transformants displayed arrested or aberrant development. The integrated plasmid, along with flanking genomic DNA, was excised from some of these mutants, cloned in Escherichia coli, and used to transform other Dictyostelium cells. Homologous recombination within the flanking sequences resulted in the same phenotypes displayed by the original mutants, directly demonstrating that the affected genes were responsible for the specific morphological phenotypes. This method of insertional mutagenesis should be useful for tagging, and subsequent cloning, of many developmentally important genes that can be identified by their mutant phenotypes.
We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.
The contact site A (csA) glycoprotein is a developmentally regulated cell adhesion molecule which mediates EDTA-stable cell contacts during the aggregation stage of Dictyostelium discoideum. A transformation vector was constructed which allows overexpression of the csA protein during the growth phase. In that stage the csA protein is normally not expressed; in the transformants it was transported to the cell surface and carried all modifications investigated, including a phospholipid anchor and two types of oligosaccharide chain. csA expression enabled the normal non-aggregative growth-phase cells to form EDTA-stable contacts in suspension and to assemble into three-dimensional aggregates when moving on a substratum. After prolonged cultivation of csA overexpressing transformants in nutrient medium the developmental program was found to be turned on, as it normally occurs only in starving cells. During later development of transformed cells, the csA glycoprotein remained present on the cell surface, while it is down-regulated in the wild type. It was detected in both the prestalk and prespore regions of the multicellular slugs made from transformed cells.
The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon
after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly
enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence
from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene.
There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account
for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich
and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus,
which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol
link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct
containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We
found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.
The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member
of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed
transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon
Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15
vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200
copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed
from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences
required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5' noncoding
DNA for actin 15 and 0.7 kilobases of 5' noncoding DNA for actin 6.
We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter
gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418.
This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7)
cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable
for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin
phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover,
analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated
that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream
flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene
during D. discoideum development. These results indicate that this system can be used to examine control of gene expression
during D. discoideum development.
Reciprocal transplants were performed between slugs of two strains of Dictyostelium discoideum. Slugs of one strain (NP84 'slugger') showed prolonged migration, while slugs of the other strain (AX3 'fruiter') migrated for a short period only. The transplant experiments showed that the 'slug/fruit' characteristic is tip dependent, since an NP84 tip induced an AX3 rear to migrate for a prolonged period, while an AX3 tip induced the rear of a NP84 slug to fruit without migration. These findings are not consistent with the hypothesis that tips of all stages release only one signal which is interpreted differently by the rest of the cells in the aggregate at different stages. Rather, we propose that the tip of a D. discoideum aggregate may release a number of signals, one of which is stage dependent, and triggers the 'slug/fruit' switch.
The weak base ammonia inhibits aggregation and culmination of wild-type amoebae of Dictyostelium discoideum. Here we have examined its effect on a series of 'slugger' mutants previously assigned to 10 complementation groups, and so-called because they remain as slugs for extended periods. We show that the mutants accumulate normal levels of ammonia and hence may be abnormally susceptible to the ammonia they produce. In agreement with this we find that representatives of the slugger complementation groups are hypersensitive to ammonia inhibition at three clearly recognisable morphological stages of development: aggregation, tip formation and culmination. This finding suggests that a common ammonia-sensitive process underlies each of these developmental events.
The unusual floral organs (ufo) mutant of Arabidopsis has flowers with variable homeotic organ transformations and inflorescence-like characteristics. To determine the relationship between UFO and previously characterized meristem and organ identity genes, we cloned UFO and determined its expression pattern. The UFO gene shows extensive homology with FIMBRIATA (FIM), a gene mediating between meristem and organ identity genes in Antirrhinum. All three UFO mutant alleles that we sequenced are predicted to produce truncated proteins. UFO transcripts were first detected in early floral meristems, before organ identity genes had been activated. At later developmental stages, UFO expression is restricted to the junction between sepal and petal primordia. Phenotypic, genetic, and expression pattern comparisons between UFO and FIM suggest that they are cognate homologs and play a similar role in mediating between meristem and organ identity genes. However, some differences in the functions and genetic interactions of UFO and FIM were apparent, indicating that changes in partially redundant pathways have occurred during the evolutionary divergence of Arabidopsis and Antirrhinum.
High resolution gene maps of the six chromosomes of Dictyostelium discoideum have been generated by a combination of physical mapping techniques. A set of yeast artificial chromosome clones has been ordered into overlapping arrays that cover >98% of the 34-magabase pair genome. Clones were grouped and ordered according to the genes they carried, as determined by hybridization analyses with DNA fragments from several hundred genes. Congruence of the gene order within each arrangement of clones with the gene order determined from whole genome restriction site mapping indicates that a high degree of confidence can be placed on the clone map. This clone-based description of the Dictyostelium chromosomes should be useful for the physical mapping and subcloning of new genes and should facilitate more detailed analyses of this genome. cost of silicon-based construction and in the efficient sample handling afforded by component integration.
The Dictyostelium cudA gene encodes a nucleoplasmic protein that is essential for normal culmination. There are no functionally characterised homologues in other organisms but there is a related gene of unknown function in Entamoeba histolytica. The cudA gene is expressed by the prestalk cells that constitute the slug tip (the pstA cells), it is not detectably expressed in the band of prestalk cells that lies behind the tip (the pstO cells) but it is expressed in the prespore cells. This unusual pattern of expression suggests a role on both the stalk and spore pathways of differentiation and cudA- mutant cells are indeed defective in both stalk and spore formation. Furthermore, the slugs formed by cudA- cells continue to migrate under environmental conditions where normal slugs culminate immediately. This aspect of their behaviour can be reversed when the cudA gene is selectively expressed in the pstA cells. This shows that processes occurring in the pstA cells regulate entry into culmination.
We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. The encoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conjugating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiquitination and possibly degradation during Dictyostelium development. Consistent with the homology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitination is altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute the developmental transition that occurs between the induction of postaggregative gene expression during mound formation and the induction of cell-type differentiation and subsequent morphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however, they remain at this stage for approximately 10 h before forming multiple tips and fingers that then arrest. Under other conditions, some of the fingers form migrating slugs, but no culmination is observed. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levels and do not decrease significantly with time as they do in wild-type cells. Expression of cell-type-specific genes is very delayed, with the level of prespore-specific gene expression being significantly reduced compared with that in wild-type cells. lacZ reporter studies using developmentally regulated and cell-type-specific promoters suggest that ubcB-null cells show an unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, a regulatory cell population found scattered throughout the aggregate, and reduced staining of a prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. In contrast, in chimeras containing only a small fraction of wild-type cells, the mature fruiting body is very small and composed almost exclusively of wild-type cells, with the ubcB-null cells being present as a mass of cells located in extreme posterior of the developing organism. The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis of the developmental phenotypes suggest that tip formation and subsequent development requires specific protein ubiquitination, and possibly degradation.
The fimbriata (fim) gene of Antirrhinum affects both the identity and arrangement of organs within the flower, and encodes a protein with an F-box motif. We show that FIM associates with a family of proteins, termed FAPs (FIM-associated proteins), that are closely related to human and yeast Skp1 proteins. These proteins form complexes with F-box-containing partners to promote protein degradation and cell cycle progression. The fap genes are expressed in inflorescence and floral meristems in a pattern that incorporates the domain of fim expression, supporting an in vivo role for a FIM-FAP complex. Analysis of a series of novel fim alleles shows that fim plays a key role in the activation of organ identity genes. In addition, fim acts in the regions between floral organs to specify the correct positioning and maintenance of morphological boundaries. Taking these results together, we propose that FIM-FAP complexes affect both gene expression and cell division, perhaps by promoting selective degradation of regulatory proteins. This may provide a mechanism by which morphological boundaries can be aligned with domains of gene expression during floral development.
Mutations that influence lin-12 activity in Caenorhabditis elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins. We describe genetic evidence indicating that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. We propose that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins, and discuss potential roles for the regulation of lin-12/Notch activity by sel-10 in cell fate decisions and tumorigenesis.
A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum. Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated. nosA contains seven exons and codes for a developmentally regulated 3.5-kb mRNA. The 125-kDa NOSA protein is present in the cytosol at constant levels during growth and development. The C-terminal region of NOSA has homology with ubiquitin fusion degradation protein-2 (UFD2) of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans. UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype. In accord with the homology to UFD2, we found differences in the ubiquitination patterns between nosA mutants and their parental cell line. While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants. These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle.
We have identified a developmentally regulated, putative MEK kinase (MEKKalpha) that contains an F-box and WD40 repeats and plays a complex role in regulating cell-type differentiation and spatial patterning. Cells deficient in MEKKalpha develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKalpha or MEKKalpha lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. Prespore and prestalk cells are interspersed throughout the slug. Analysis of chimeric organisms suggests that MEKKalpha function is required for the proper induction and maintenance of prespore cell differentiation. We show that the WD40 repeats target MEKKalpha to the cortical region of the cell, whereas the F-box/WD40 repeats direct ubiquitin-mediated MEKKalpha degradation. We identify a UBC and a UBP (ubiquitin hydrolase) that interact with the F-box/WD40 repeats. Our findings indicate that cells lacking the ubiquitin hydrolase have phenotypes similar to those of MEKKalpha null (mekkalpha-) cells, further supporting a direct genetic and biochemical interaction between MEKKalpha, the UBC, and the UBP. We demonstrate that UBC and UBP differentially control MEKKalpha ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally regulated manner. Our results represent a novel mechanism that includes targeted protein degradation by which MAP kinase cascade components can be controlled. More importantly, our findings suggest a new paradigm of spatial and temporal control of the kinase activity controlling spatial patterning during multicellular development, which parallels the temporally regulated degradation of proteins required for cell-cycle progression.
The ecmA gene of Dictyostelium encodes an extracellular matrix protein and is selectively expressed in prestalk cells. We show that its promoter contains discrete elements that direct expression in different subpopulations of prestalk cells. Prestalk(pst)A cells occupy the front half of the prestalk region. Expression in pstA cells requires DNA sequences close to the cap site of the gene and a separate, upstream region that acts in combination with the gene proximal sequences. PstO cells are situated in the rear half of the prestalk region and at least two separate and redundant promoter regions direct expression within them. All constructs that are expressed in pstO cells are also expressed in anteriorlike cells (ALCs); cells that resemble prestalk cells but which are scattered throughout the prespore region.
This observation suggests that pstO cells and ALCs may be very similar in their properties. If development occurs under conditions in which a migratory slug is not formed, there is an ordered movement of cells into the stalk tube. PstA cells enter the stalk tube first, followed by a proportion of the pstO cells. The remainder of the pstO cells contribute to the upper cup, an ALC-derived subpopulation of prestalk cells which is located at the apex of the spore head. After prolonged slug migration, a discrete pstO zone appears not to be maintained and, at culmination, pstO cells are found scattered throughout the stalk.
Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IκBα. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-κB inhibitor protein IκBα. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3- ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCF complex, SCF(β-TRCP), that specifically recognizes a 19-amino-acid destruction motif in IκBα (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in β-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IκBα-ubiquitin ligase activity cofractionates with SCF(β-TRCP). Furthermore, recombinant SCF(β-TRCP) assembled in mammalian cells contains phospho-IκBα-specific ubiquitin ligase activity. Our results suggest that an SCF(β-TRCP) complex functions in multiple transcriptional programs by activating the NF-κB pathway and inhibiting the β-catenin pathway.
HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human βTrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4–Vpu–βTrCP ternary complexes have been detected by coimmunoprecipitation. βTrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In βTrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of βTrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that βTrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.
During the course of development, cells of Dictyostelium discoideum are able to produce a multicellular body (a ‘slug’) which is capable of movement over the substratum. This phase, which is induced by production of ammonia by the starving cells, may last for hours or days depending on environmental stimuli. In order to probe the regulatory system controlling formation and duration of the slug phase, mutants were isolated that remained in the slug phase for an extended period. Thirty-two such 'slugger’ mutants were analysed by parasexual genetic techniques and placed into 10 complementation groups (slgA-slgJ). The linkage groups bearing representatives of these complementation groups were determined by segregation of diploids formed between mutants and tester strains. Phenotypic studies of mutants indicated that members of slgD, slgE and slgG were over-sensitive to the ammonia slug-inducing stimulus.
The prestalk region of the Dictyostelium slug has recently been shown by Williams and his collaborators to consist of two distinct cell types, pstA and pstB cells. Here the movement of these cells in both the slug and culmination stages has been examined with the use of vital dyes. In the slug some of the pstB cells are continually lost from the prestalk region as small clusters of cells. These cells move through the prespore region and temporarily lie in the rearguard region at the posterior end of the slug. They are finally left in the slug's slime track as single cells or groups of a few cells. When culmination is initiated the pstB cells move as a whole from the prestalk region to the base where they join the rearguard cells to form the basal disc of the fruiting body. Transplantation experiments reveal that the rearguard cells form an outer ring portion of the basal disc and the pstB cells form an inner portion to which the stalk attaches. The continuous loss of one cell type during the slug stage without any change in cell type proportions suggests that cell types are redifferentiating. Grafting and transplantation experiments reveal that there is a unidirectional flow of cells through successive steps of cell type conversion. Prespore cells redifferentiate as anterior-like cells which migrate to the prestalk region and become pstA cells. The pstA cells then replace the pstB cells that are lost from the slug.
A Dictyostelium discoideum repetitive element composed of long repeats of the codon (AAC) is found in developmentally regulated transcripts. The concentration of (AAC) sequences is low in mRNA from dormant spores and growing cells and increases markedly during spore germination and multicellular development. The sequence hybridizes to many different sized Dictyostelium DNA restriction fragments indicating that it is scattered throughout the genome. Four cDNA clones isolated contain (AAC) sequences in the deduced coding region. Interestingly, the (AAC)-rich sequences are present in all three reading frames in the deduced proteins, i.e., AAC (asparagine), ACA (threonine) and CAA (glutamine). Three of the clones contain only one of these in-frame so that the individual proteins carry either asparagine, threonine, or glutamine clusters, not mixtures. However, one clone is both glutamine- and asparagine-rich. The (AAC) portion of the transcripts are reiterated 300 times in the haploid genome while the other portions of the cDNAs represent single copy genes, whose sequences show no similarity other than the (AAC) repeats. The repeated sequence is similar to the opa or M sequence found in Drosophila melanogaster notch and homeo box genes and in fly developmentally regulated transcripts. The transcripts are present on polysomes suggesting that they are translated. Although the function of these repeats is unknown, long amino acid repeats are a characteristic feature of extracellular proteins of lower eukaryotes.
There are cells acattered in the rear, prespore region of the Dictyostelium slug that share many of the properties of the prestalk cells and that are therefore called anterior-like cells (ALCs). By placing the gene encoding a cell surface protein under the control of an ALC-specific promoter and immunologically labeling the living cells, we analyze the movement of ALCs within the slug. There is a posterior to anterior cellular flow, and the ALCs change their movement pattern as they enter the prestalk zone. Prestalk cells are periodically shed from the migrating slug. They must be replaced if the correct ratio of prestalk to prespore cells is to be maintained, and we present evidence for the trans-differentiation of prespore into prestalk cells, with ALCs functioning as intermediates in the transition. The slug has, therefore, a surprisingly dynamic structure, both with respect to cellular differentiation and cell movement.
Previous work (Newell et al., 1969) had indicated that the transformation of a newly formed multicellular aggregate into a migrating slug rather than a fruiting body depended on the local accumulation of a diffusible metabolite excreted before and during aggregation. The subsequent shift back to the fruiting mode could be accounted for by the disappearance or removal of the metabolite. All these morphogenetic transformations were associated with profound and consistent changes in the patterns of enzyme accumulation and disappearance (Sussman et al., 1975). In the present study, evidence is presented to support the conclusion that the metabolite in question is NH3. Thus: 1.(a) Cell populations accumulated and excreted significant amounts of NH3 + NH4+ before and during cell aggregation under the conditions employed.2.(b) Excretory products collected from such cells induced newly formed aggregates to develop into migrating slugs under conditions which otherwise would permit them to construct fruiting bodies directly. The inducing activity of such preparations was precisely correlated with the NH3 + NH4+ concentration and was destroyed by incorporation of the NH3 into glutamic acid in the presence of glutamate dehydrogenase.3.(c) The slug-inducing activities of the extracellular metabolite preparations and purified fractions thereof were mimicked by equimolar solutions of ammonium carbonate at identical pH values.4.(d) Isolated migrating slugs treated with 0.5-μ1 samples of a reaction mixture containing glutamate dehydrogenase, α-ketoglutarate and NADH immediately stopped migrating and constructed fruiting bodies. Samples lacking a single component or containing boiled enzyme were ineffective.5.(e) By several criteria, the biological and biochemical performances of autoinduced slugs and of those induced by the metabolite preparations or by ammonium carbonate solutions were indistinguishable.
Taking advantage of the fact that differentiation of the prespore cell of Dictyostelium discoideum is characterized by synthesis of a prespore specific antigen, the process of its differentiation during the course of morphogenesis was quantitatively studied by determining the proportion of prespore cells and their cellular contents of the antigen, using the method of microfluorometry in combination with immunocytochemistry with antispore serum. The cells synthesizing the antigen became first detectable in the early aggregation center which was about to form a papilla. As the papilla elongated, the number of prespore cells rapidly increased up to the stationary level (70–80% of total cells) before completion of slug formation. During the process antigenic contents of prespore cells were gradually increased and leveled off in the early migration stage. When culmination was induced, antigenic contents were markedly increased to the maximum, which was followed by a sudden decrease immediately before spore formation. On the other hand, the proportions of prespore to total cells were kept constant at the stationary level all through the migration and culmination stages, in spite of a persistent decrease during culmination in the total number of cells due to continuous differentiation of the prestalk into the mature stalk cells. These results were discussed in relation to possible mechanisms of differentiation in this organism.
We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.
The ecmA (pDd63) and ecmB (pDd56) genes encode extracellular matrix proteins of the slime sheath and stalk tube of Dictyostelium discoideum. Using fusion genes containing the promoter of one or other gene coupled to an immunologically detectable reporter, we previously identified two classes of prestalk cells in the tip of the migrating slug; a central core of pstB cells, which express the ecmB gene, surrounded by pstA cells, which express the ecmA gene. PstB cells lie at the position where stalk tube formation is initiated at culmination and we show that they act as its founders. As culmination proceeds, pstA cells transform into pstB cells by activating the ecmB gene as they enter the stalk tube. The prespore region of the slug contains a population of cells, termed anterior-like cells (ALC), which have the characteristics of prestalk cells. We show that the ecmA and ecmB genes are expressed at a low level in ALC during slug migration and that their expression in these cells is greatly elevated during culmination. Previous observations have shown that ALC sort to surround the prespore cells during culmination (Sternfeld and David, 1982 Devl Biol. 93, 111-118) and we find just such a distribution for pstB cells. We believe that the ecmB protein plays a structural role in the stalk tube and its presence, as a cradle around the spore head, suggests that it may play a further function, perhaps in ensuring integrity of the spore mass during elevation. If this interpretation is correct, then a primary role of anterior-like cells may be to form these structures at culmination. We previously identified a third class of prestalk cells, pstO cells, which lie behind pstA cells in the slug anterior and which appeared to express neither the ecmA nor the ecmB gene. Using B-galactosidase fusion constructs, which give more sensitive detection of gene expression, we now find that these cells express the ecmA gene but at a much lower level than pstA cells. We also show that expression of the ecmA gene becomes uniformly high throughout the prestalk zone when slugs are allowed to migrate in the light. Overhead light favours culmination and it may be that increased expression of the ecmA gene in the pst 'O' region is a preparatory step in the process.
The Dictyostelium ecmB gene encodes an extracellular matrix protein and is inducible by the stalk cell morphogen DIF. It is expressed in a subset of prestalk (pstB) cells in the slug and surrounding pstA cells first express it at culmination. A region of the ecmB promoter can direct transcription in all anterior prestalk cells, but a separate, downstream region acts to prevent its expression in pstA cells prior to culmination. This may be the site of interaction of a repressor, regulated by an extracellular antagonist to DIF. At culmination, expression of the ecmB gene also becomes greatly elevated in anterior-like cells as they move to surround the spore mass. A distal region of the ecmB promoter directs increased expression in those anterior-like cells that surmount the spore head. This divergence in gene expression suggests that anterior-like cells and anterior prestalk cells experience different inductive conditions at culmination.
We show that a fusion gene, containing the promoter and 5'-noncoding region of a Dictyostelium discoideum actin 6 gene linked to the Escherichia coli beta-galactosidase (beta Gal) gene (lacZ), directs the production of functionally active beta Gal in D. discoideum and that the enzyme can be detected by staining in situ; a procedure which will be of great value in analyzing cell-type-specific gene expression. We illustrate this by fusing lacZ to the promoter of the prespore-specific gene, D19, and localizing expressing cells in migrating slugs. Optimal expression requires the inclusion of termination and polyadenylylation signals and we describe pDDlac, a vector containing a multiple cloning site upstream from a lacZ-Dictyostelium terminator fusion, which can be used to analyze regulated promoters.
1. A simple axenic medium suitable for the growth of the myxamoebae of a strain of the cellular slime mould Dictyostelium discoideum is described. 2. Procedures suitable for the growth of this strain in liquid and on solid media are described. 3. Conditions suitable for initiating the cell differentiation of myxamoebae grown axenically are described.
A quantitative assay for estimating the proportion of prespore cells in D. discoideum slugs was established by labelling disaggregated slug cells with a prespore specific monoclonal antibody and analysing the cell population with a FACS-IV. The method is validated using a wild-type strain and its stalky mutant. "Wild-type" strains have different proportions of prespore cells and it is demonstrated that slugs of some strains have an increased percentage of prespore cells when migrated in the dark compared to the light and in the presence of EGTA. The technique is rapid and will make possible genetic analysis of proportion regulation in D. discoideum.
We present evidence that Dictyostelium slug tip cells, the pstA cells, may arise by positional differentiation, but at a site remote from that which they will eventually occupy. When first detectable, the pstA cells form a peripheral ring surrounding the other prestalk cell subtype, the pstO cells, but subsequently move above the pstO cells to form the tip. Because pstA cell differentiation requires a 10-fold higher concentration of differentiation-inducing factor, the stalk cell inducer, the initial patterning seems likely to reflect the existence of a morphogenetic gradient. The subsequent redistribution of the two cell types is explicable by their different rates of chemotaxis to cyclic AMP. These results help reconcile the two apparently opposing views of pattern formation in Dictyostelium, that there is positional differentiation and that pattern formation occurs by cell sorting.
We have been able to hybridize nonradioactive probes from cell-type-specific genes to fixed whole-mounts prepared at the mound, slug, and culminant stages of Dictyostelium development. The cellular patterns of labeling with probes from the prespore gene, cotB, and the prestalk genes, ecmA and ecmB, confirmed the patterns seen in strains carrying reporter constructs in which the regulatory regions of these genes drive beta-galactosidase. This technique permits the direct observation of protein synthetic capacity from characterized genes without the need of generating transformed lines carrying specific reporter constructs. Moreover, the pattern is not complicated by a previous developmental history of gene expression.
We describe a double-label in situ hybridization protocol based on the optimized synthesis of biotin-labeled RNA probes. Biotin-labeled probes are used in conjunction with digoxigenin-labeled probes to simultaneously visualize two different transcripts. One transcript is hybridized with a biotin-labeled RNA probe and visualized as a brown peroxidase reaction product, and the other transcript is hybridized with a digoxigenin-labeled RNA probe and visualized as a blue alkaline phosphatase reaction product. We present several examples in which this double-labeling method has proven useful in determining the spatial and temporal relationships between various transcripts expressed during Drosophila embryogenesis, indicating that this method should be of general use in establishing the relationship of two independent transcription patterns.
WD proteins are made up of highly conserved repeating units usually ending with Trp-Asp (WD). They are found in all eukaryotes but not in prokaryotes. They regulate cellular functions, such as cell division, cell-fate determination, gene transcription, transmembrane signalling, mRNA modification and vesicle fusion. Here we define the common features of the repeating units, and criteria for grouping such proteins into functional subfamilies.
The ecmA gene of Dictyostelium encodes an extracellular matrix protein and is selectively expressed in prestalk cells. We show that its promoter contains discrete elements that direct expression in different subpopulations of prestalk cells. Prestalk(pst)A cells occupy the front half of the prestalk region. Expression in pstA cells requires DNA sequences close to the cap site of the gene and a separate, upstream region that acts in combination with the gene proximal sequences. PstO cells are situated in the rear half of the prestalk region and at least two separate and redundant promoter regions direct expression within them. All constructs that are expressed in pstO cells are also expressed in anterior-like cells (ALCs); cells that resemble prestalk cells but which are scattered throughout the prespore region. This observation suggests that pstO cells and ALCs may be very similar in their properties. If development occurs under conditions in which a migratory slug is not formed, there is an ordered movement of cells into the stalk tube. PstA cells enter the stalk tube first, followed by a proportion of the pstO cells. The remainder of the pstO cells contribute to the upper cup, an ALC-derived subpopulation of prestalk cells which is located at the apex of the spore head. After prolonged slug migration, a discrete pstO zone appears not to be maintained and, at culmination, pstO cells are found scattered throughout the stalk.
A new selectable marker for transformation of Dictyostelium discoideum cells was constructed by using the bsr gene from Bacillus cereus, which confers resistance to Blasticidin S. The bsr gene was driven by Dictyostelium actin 15 promoter and Dictyostelium actin 8 terminator for expression in Dictyostelium cells. To demonstrate the feasibility of using the bsr marker, we constructed an extrachromosomal replication vector by replacing the Neor gene of pnDeI (B. Leiting and A. Noegel (1988) Plasmid 20, 241-248) with the bsr gene cassette. A mutant Dictyostelium actin 15 gene was constructed and inserted into the vector. Dictyostelium cells were transformed with the resulting vector and then transformants were selected with Blasticidin S. The selected cells showed high level expression of the mutant actin, indicating an efficient selection of transformed cells with the bsr marker.
We have identified the yeast and human homologs of the SKP1 gene as a suppressor of cdc4 mutants and as a cyclin F-binding protein. Skp1p indirectly binds cyclin A/Cdk2 through Skp2p, and directly binds Skp2p, cyclin F, and Cdc4p through a novel structural motif called the F-box. SKP1 is required for ubiquitin-mediated proteolysis of Cin2p, Clb5p, and the Cdk inhibitor Sic1p, and provides a link between these molecules and the proteolysis machinery. A large number of proteins contain the F-box motif and are thereby implicated in the ubiquitin pathway. Different skp1 mutants arrest cells in either G1 or G2, suggesting a connection between regulation of proteolysis in different stages of the cycle.
The dorsal-ventral pattern of the Drosophila embryo is established by three sequential signaling pathways. Each pathway transmits spatial information by localizing the activity of an extracellular signal, which acts as a ligand for a broadly distributed transmembrane receptor. The components of the first two pathways are encoded by maternal effect genes, while the third pathway is specified by genes expressed in the zygote. During oogenesis, the oocyte transmits a signal to the surrounding follicle cells by the gurken-torpedo pathway. After fertilization, the initial asymmetry of the egg chamber is used by the spätzle-Toll pathway to generate within the embryo a nuclear gradient of the transcription factor Dorsal, which regulates the regional expression of a set of zygotic genes. On the dorsal side of the embryo, the decapentaplegic-punt/thick veins pathway then establishes patterning of the amnioserosa and dorsal ectoderm. Each pathway uses a distinct strategy to achieve spatial localization of signaling activity.
Members of the Hedgehog (Hh) and Wnt/Wingless (Wg) families of secreted proteins control many aspects of growth and patterning during animal development. Hh signal transduction leads to increased stability of a transcription factor, Cubitus interruptus (Ci), whereas Wg signal transduction causes increased stability of Armadillo (Arm/beta-catenin), a possible co-factor for the transcriptional regulator Lef1/TCF. Here we describe a new gene, slimb (for supernumerary limbs), which negatively regulates both of these signal transduction pathways. Loss of function of slimb results in a cell-autonomous accumulation of high levels of both Ci and Arm, and the ectopic expression of both Hh- and Wg- responsive genes. The slimb gene encodes a conserved F-box/WD40-repeat protein related to Cdc4p, a protein in budding yeast that targets cell-cycle regulators for degradation by the ubiquitin/proteasome pathway. We propose that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression at least in part by inducing changes in Ci and Arm, which protect them from Slimb-mediated proteolysis.
The ubiquitin-dependent proteolytic pathway targets many key regulatory proteins for rapid intracellular degradation. Specificity in protein ubiquitination derives from E3 ubiquitin protein ligases, which recognize substrate proteins. Recently, analysis of the E3s that regulate cell division has revealed common themes in structure and function. One particularly versatile class of E3s, referred to as Skp1p-Cdc53p-F-box protein (SCF) complexes, utilizes substrate-specific adaptor subunits called F-box proteins to recruit various substrates to a core ubiquitination complex. A vast array of F-box proteins have been revealed by genome sequencing projects, and the early returns from genetic analysis in several organisms promise that F-box proteins will participate in the regulation of many processes, including cell division, transcription, signal transduction and development.
Shortly after initiation of Dictyostelium fruiting body formation, prespore cells begin to differentiate into non-motile spores. Although these cells lose their ability to move, they are, nevertheless, elevated to the tip of the stalk. Removal of the amoeboid anterior-like cells, located above the differentiating spores in the developing fruiting body, prevents further spore elevation although the stalk continues to elongate. Furthermore, replacement of the anterior-like cells with anterior-like cells from another fruiting body largely restores the ability to lift the spores to the top of the stalk. However, if amoeboid prestalk cells are used to replace the anterior-like cells, there is no restoration of spore elevation. Finally, when a droplet of mineral oil replaces differentiating spores, it is treated as are the spores: the mineral oil is elevated in the presence of anterior-like cells and becomes arrested on the stalk in the absence of anterior-like cells. Because a similar droplet of mineral oil is totally ignored by slug tissue, it appears that there is a dramatic transformation in the treatment of non-motile matter at this point in Dictyostelium development.
An early decision that a newly formed aggregate of Dictyostelium cells must make is whether to form a migrating slug or to proceed through culmination, the process of forming the mature fruiting body. The choice between these alternative morphological pathways is influenced by external and internal cues. dhkC was identified as a potential hybrid sensor kinase possessing domains homologous to the histidine kinase and receiver motifs of two-component signaling systems. Null strains of dhkC show a rapidly developing phenotype for aggregation through finger formation, and culmination commences immediately thereafter and proceeds at a normal rate to generate typical fruiting bodies. Ammonia, an endogenous regulator of the slug versus culmination choice, results in a prolonged slug stage for wild-type strains while the dhkC- strain bypasses the slug stage in the presence or absence of ammonia. Conversely, expression in wild-type cells of a modified DHKC protein composed of only the histidine kinase domain results in normal timing through early aggregation, but subsequent development is significantly delayed. The resulting fingers, once formed, readily convert to slugs that do not undergo culmination but instead migrate until their energy sources are depleted. The slugger phenotype is dependent on the presence of a functional response regulator REGA, and it is rescued by exogenously supplied cAMP. Together, the results indicate that DHKC contributes to the integration of environmental and cellular signals so that the appropriate choice is made between slug formation and culmination. We suggest that DHKC may function as a sensor for ammonia, and that it is the initial component of a phosphorelay signaling system that may modulate the activity of cAMP-dependent protein kinase to either inhibit or promote culmination. Additionally, dhkC- spores were found to be defective in germination, indicating a role for the DHKC signaling pathway in activating spore germination.
NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.
Signal-induced phosphorylation of IkappaBalpha targets this inhibitor of NF-kappaB for ubiquitination and subsequent degradation, thus allowing NF-kappaB to enter the nucleus to turn on its target genes. We report here the identification of an IkappaB-ubiquitin (Ub) ligase complex containing the F-box/WD40-repeat protein, beta-TrCP, a vertebrate homolog of Drosophila Slimb. beta-TrCP binds to IkappaBalpha only when the latter is specifically phosphorylated by an IkappaB kinase complex. Moreover, immunopurified beta-TrCP ubiquitinates phosphorylated IkappaBalpha at specific lysines in the presence of Ub-activating (E1) and -conjugating (Ubch5) enzymes. A beta-TrCP mutant lacking the F-box inhibits the signal-induced degradation of IkappaBalpha and subsequent activation of NF-kappaB-dependent transcription. Furthermore, Drosophila embryos deficient in slimb fail to activate twist and snail, two genes known to be regulated by the NF-kappaB homolog, Dorsal. These biochemical and genetic data strongly suggest that Slimb/beta-TrCP is the specificity determinant for the signal-induced ubiquitination of IkappaBalpha.
Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. Our results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway.