A putative Ariadne-like ubiquitin ligase is required for Dictyostelium discoideum development

The Dictyostelium rbrA gene encodes a putative Ariadne ubiquitin ligase. rbrA⁻ cells form defective slugs that cannot phototax. Prestalk cell numbers are reduced in rbrA⁻ slugs, and these prestalk cells do not localize to the tip of slugs. Chimeric slugs containing wild-type cells could phototax and form fruiting bodies.     

Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.     

A ubiquitin ligase complex regulates caspase activation during sperm differentiation in Drosophila

In both insects and mammals, spermatids eliminate their bulk cytoplasm as they undergo terminal differentiation. In Drosophila, this process of dramatic cellular remodeling requires apoptotic proteins, including caspases. To gain further insight into the regulation of caspases, we screened a large collection of sterile male flies for mutants that block effector caspase activation at the onset of spermatid individualization. Here, we describe the identification and characterization of a testis-specific, Cullin-3–dependent ubiquitin ligase complex that is required for caspase activation in spermatids. Mutations in either a testis-specific isoform of Cullin-3 (Cul3_Testis), the small RING protein Roc1b, or a Drosophila orthologue of the mammalian BTB-Kelch protein Klhl10 all reduce or eliminate effector caspase activation in spermatids. Importantly, all three genes encode proteins that can physically interact to form a ubiquitin ligase complex. Roc1b binds to the catalytic core of Cullin-3, and Klhl10 binds specifically to a unique testis-specific N-terminal Cullin-3 (TeNC) domain of Cul3_Testis that is required for activation of effector caspase in spermatids. Finally, the BIR domain region of the giant inhibitor of apoptosis–like protein dBruce is sufficient to bind to Klhl10, which is consistent with the idea that dBruce is a substrate for the Cullin-3-based E3-ligase complex. These findings reveal a novel role of Cullin-based ubiquitin ligases in caspase regulation.     

Occurrence of a putative SCF ubiquitin ligase complex in Drosophila

Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. We have identified in Drosophila melanogaster the closest homologues to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5. We show that putative Drosophila SCF core subunits dSkpA and dRbx1 both interact directly with dCu11 and the F-box protein Slmb. We also describe the direct interaction of the UbcH5 related protein UbcD1 with dCul1 and Slmb. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant showed that Drosophila Rbx1 is able to restore the yeast cells viability. Our results suggest that dRbx1, dSkpA, dCullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1.     

Evidence that a cell-type-specific efflux pump regulates cell differentiation in Dictyostelium

We have identified a cellular efflux pump, RhT, with the properties of an MDR transporter-a type of ATP-binding cassette transporter whose substrates include small hydrophobic molecules. RhT transports rhodamine 123 (Rh123) and is inhibited by low temperature, energy poisons, and several MDR transport inhibitors, such as verapamil. All vegetative cells have RhT activity, but during development prestalk cells lose RhT activity while prespore cells retain it. We also identified several RhT inhibitors. The most effective inhibitor is the stalk cell-inducing chlorinated alkyl phenone, DIF-1. The RhT inhibitors disrupted development, to varying degrees, and induced stalk cell formation in submerged culture. The inhibitors displayed the same rank order of pharmacological efficacy for stalk cell induction as they did for Rh123 transport inhibition. We also found that cerulenin, a specific inhibitor of DIF-1 biosynthesis (R. R. Kay, 1998, J. Biol. Chem. 273, 2669-2675), abolished the induction of stalk cells by each of the RhT inhibitors, and this effect could be reversed by DIF-1. Thus, DIF-1 synthesis appears to be required for the induction of stalk cells by the RhT inhibitors. Since DIF-1 is the most potent inhibitor of RhT activity, and thus a likely transport substrate itself, we propose that RhT inhibitors induce stalk cell differentiation by blocking DIF-1 export, causing DIF-1 to build up within cells. Our results provide evidence for a prespore-specific efflux pump that regulates cell fate determination, perhaps by regulating the cellular concentration of DIF-1.     

The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF(SLY1)-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.     

Immuno-localization and separation of multiple prestalk cell types in Dictyostelium

Abstract. The ecm A and ecm B genes of Dictyostelium encode closely related extracellular matrix proteins. The major prestalk cell population, pstA cells, expresses the ecm A gene but not the ecm B gene. PstAB cells, a minor prestalk cell population that we show to express both the ecm A and ecm B genes, form a core in the centre of the slug tip. The rear, prespore region of the slug contains amoebae, termed anterior-like cells (ALC), that display many of the properties of prestalk cells. The ecm A and B genes are weakly expressed in about 30% of the ALC and these comprise a mixture of pstA cells, pstAB cells and a third class, pstB cells. The latter cell type express the ecm B gene but show no detectable expression of the ecm A gene. The demonstration of the existence of pstB cells suggests a separate pathway of ecm B gene induction, wherein expression of the ecm A gene is absent or at a very low level. Pst A, AB and B cells most probably differ in their surface properties because they are partially separable by Counter Current Distribution (CCD), a chromatographic technique which, in the conditions used, is dependent upon differences in cell surface hydrophobicity.     

Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

Ozz-E3, a muscle-specific ubiquitin ligase, regulates beta-catenin degradation during myogenesis

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. Here we describe a RING-type E3 ligase complex specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. We identified beta-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated beta-catenin by mediating its degradation via the proteasome. Expression of beta-catenin mutants that reduce the binding of Ozz to endogenous beta-catenin leads to Mb-beta-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of Mb-beta-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation.     

RacB regulates cytoskeletal function in Dictyostelium spp

Thus far, 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. To investigate the specific function of RacB, we have conditionally expressed wild-type (WT-RacB), dominant negative (N17-RacB), and constitutively activated (V12-RacB) versions of the protein. On induction, cells expressing V12-RacB stopped growing, detached from the surface, and formed numerous spherical surface protrusions while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin-driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin II activity, which distinguishes them from blebs formed by wild-type cells under stress. Finally, we examined the functional consequences of expression of wild-type and mutant RacB. Phagocytosis, endocytosis, and fluid phase efflux rates were reduced in all cell lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin but the consequences of activation appear to be different from other Dictyostelium Rac proteins so far investigated, resulting in different morphological and functional changes in cells.     

Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation.

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.     

A nucleolar isoform of the Fbw7 ubiquitin ligase regulates c-Myc and cell size

The human tumor suppressor Fbw7/hCdc4 functions as a phosphoepitope-specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination of cyclin E , Notch , c-Jun and c-Myc . Fbw7 loss in cancer may thus have profound effects on the pathways that govern cell division, differentiation, apoptosis, and cell growth. Fbw7-inactivating mutations occur in human tumor cell lines and primary cancers , and Fbw7 loss in cultured cells causes genetic instability . In mice, deletion of Fbw7 leads to embryonic lethality associated with defective Notch and cyclin E regulation . The human Fbw7 locus encodes three protein isoforms (Fbw7alpha, Fbw7beta, and Fbw7gamma) . We find that these isoforms occupy discrete subcellular compartments and have identified cis-acting localization signals within each isoform. Surprisingly, the Fbw7gamma isoform is nucleolar, colocalizes with c-Myc when the proteasome is inhibited, and regulates nucleolar c-Myc accumulation. Moreover, we find that knockdown of Fbw7 increases cell size consistent with its ability to control c-Myc levels in the nucleolus. We suggest that interactions between c-Myc and Fbw7gamma within the nucleolus regulate c-Myc's growth-promoting function and that c-Myc activation is likely to be an important oncogenic consequence of Fbw7 loss in cancers.     

A novel E3 ubiquitin ligase TRAC-1 positively regulates T cell activation

TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn(2+)-chelating ability of its amino-terminal RING finger domain abolished TRAC-1's ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation.     

Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium

Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.     

Ca2+ signaling regulates ecmB expression, cell differentiation and slug regeneration in Dictyostelium

Ca(2+) regulates cell differentiation and morphogenesis in a diversity of organisms and dysregulation of Ca(2+) signal transduction pathways leads to many cellular pathologies. In Dictyostelium Ca(2+) induces ecmB expression and stalk cell differentiation in vitro. Here we have analyzed the pattern of ecmB expression in intact and bisected slugs and the effect of agents that affect Ca(2+) levels or antagonize calmodulin (CaM) on this expression pattern. We have shown that Ca(2+) and CaM regulate ecmB expression and pstAB/pstB cell differentiation in vivo. Agents that increase intracellular Ca(2+) levels increased ecmB expression and/or pstAB and pstB cell differentiation, while agents that decrease intracellular Ca(2+) or antagonize CaM decreased it. In isolated slug tips agents that affect Ca(2+) levels and antagonize CaM had differential effect on ecmB expression and cell differentiation in the anterior versus posterior zones. Agents that increase intracellular Ca(2+) levels increased the number of ecmB expressing cells in the anterior region of slugs, while agents that decrease intracellular Ca(2+) levels or antagonize CaM activity increased the number of ecmB expressing cells in the posterior. We have also demonstrated that agents that affect Ca(2+) levels or antagonize CaM affect cells motility and regeneration of shape in isolated slug tips and backs and regeneration of tips in isolated slug backs. To our knowledge, this is the first study detailing the pattern of ecmB expression in regenerating slugs as well as the role of Ca(2+) and CaM in the regeneration process and ecmB expression.     

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.     

EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis

Similarly to development, the process of regeneration requires that cells accurately sense and respond to their external environment. Thus, intrinsic cues must be integrated with signals from the surrounding environment to ensure appropriate temporal and spatial regulation of tissue regeneration. Identifying the signaling pathways that control these events will not only provide insights into a fascinating biological phenomenon but may also yield new molecular targets for use in regenerative medicine. Among classical models to study regeneration, freshwater planarians represent an attractive system in which to investigate the signals that regulate cell proliferation and differentiation, as well as the proper patterning of the structures being regenerated. Recent studies in planarians have begun to define the role of conserved signaling pathways during regeneration. Here, we extend these analyses to the epidermal growth factor (EGF) receptor pathway. We report the characterization of three epidermal growth factor (EGF) receptors in the planarian Schmidtea mediterranea. Silencing of these genes by RNA interference (RNAi) yielded multiple defects in intact and regenerating planarians. Smed-egfr-1(RNAi) resulted in decreased differentiation of eye pigment cells, abnormal pharynx regeneration and maintenance, and the development of dorsal outgrowths. In contrast, Smed-egfr-3(RNAi) animals produced smaller blastemas associated with abnormal differentiation of certain cell types. Our results suggest important roles for the EGFR signaling in controlling cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.