InteractiveFly: GeneBrief

bendless: Biological Overview | References

The signaling mechanisms that allow the conversion of a growth cone into a mature and stable synapse are yet to be completely understood. Ubiquitination plays key regulatory roles in synaptic development and may be involved in this process. Previous studies identified the Drosophila ubiquitin conjugase bendless (ben) as important for central synapse formation, but the precise role it plays has not been elucidated. These studies indicate that Ben plays a pivotal role in synaptic growth and maturation. An incipient synapse is present with a high penetrance in ben mutants, suggesting that Ben is required for a developmental step after target recognition. Cell-autonomous rescue experiments were used to show that Ben has a presynaptic role in synapse growth. The TARGET system was harnessed to transiently express UAS-ben in a ben mutant background and a well defined critical period for Ben function was identified in establishing a full-grown, mature synaptic terminal. The protein must be present at a time point before but not during the actual growth process. Phenotypic evidence is provided demonstrating that Ben is not a part of the signal transduction pathway involving the well characterized ubiquitin ligase Highwire. It is concluded that Bendless functions as a novel developmental switch that permits the transition from axonal growth and incipient synapse formation to synaptic growth and maturation in the CNS (Uthaman, 2008).

The results from this study have given new insights into how ubiquitin system components establish functional synaptic connections. The temporal analysis of Bendless has been critical in illustrating its role as a developmental 'switch' in converting a growth cone into a mature synapse. As mentioned previously, the ben mutation is the result of a single amino acid change in the conserved catalytic core of the conjugase domain of the protein. This highlights the fact that the conjugase activity of the protein is necessary for the observed synaptic phenotype. Analysis of synaptic growth in Drosophila has primarily been done at the peripheral synapse of the NMJ. Components of the ubiquitin system, such as the ubiquitin ligase hiw, the deubiquitinating protease faf, and the synapse-associated E3 ligase PDZRN3, are known to play important roles in the growth and function of the fly NMJ. Significant studies have been performed with particular regard to the conserved family of hiw ubiquitin ligases. In Drosophila, hiw functions as a negative regulator of synapse development as mutants exhibit dramatic synaptic overgrowth at the larval NMJ. In Caenorhabditis elegans, loss of function of the hiw homolog rpm-1 results in multiple phenotypes at the NMJ as well as in the CNS. At the NMJ, some NMJs exhibit enlarged presynaptic terminals containing multiple active zones, whereas others contain underdeveloped or absent presynaptic terminals. In the worm mechanosensory circuit, the sensory neurons were found to retract synaptic branches, extend ectopic axons, and fail to accumulate synaptic vesicles, whereas some of the motor neurons exhibited phenotypes such as altered synaptic organization, branching, and overgrowth. Downstream signaling components have been isolated for both hiw and rpm-1 in Drosophila and C. elegans, respectively, and a number of conserved elements have been identified. Mutations in homologs of hiw in zebrafish and mice are also known to cause a variety of synaptic disruptions (Uthaman, 2008).

Ben and Hiw play distinct roles in synapse growth. This study has analyzed the novel roles played by these ubiquitin system components at the giant fiber system (GFS) central synapse. ben and hiw loss of function result in very different phenotypes, with ben specimens exhibiting synaptic undergrowth and hiw specimens exhibiting synaptic overgrowth. Ben function does not involve JNK, a well characterized downstream signaling partner identified for Hiw in Drosophila. It is also interesting to compare and contrast the role Hiw plays at a peripheral synapse with the role Ben plays at a central synapse. hiw mutants exhibit a presynaptic overgrowth phenotype at the NMJ, whereas ben mutants exhibit a reduction in presynaptic growth in the CNS. Also, Hiw does not localize to the nucleus and was found to regulate synaptic growth throughout development, whereas Ben has nuclear as well as cytosolic localization and only functions in a critical time period. Finally, Hiw activity is associated with the bone morphogenetic protein (BMP) retrograde signaling pathway that is known to be dependent on the retrograde motor. No evidence was found that Bendless function is dependent on the retrograde motor. All these data underline the fact that there are distinct targets for the ubiquitination cascades involving Ben and Hiw (Uthaman, 2008).

Functional neuronal circuits are established through a series of events: neurite outgrowth, axon guidance, target recognition, synapse formation, and synaptic growth and maturation. When the bendless mutant was originally characterized, Ben was thought to play an important role in either axon guidance or target recognition (Muralidhar, 1993; Oh, 1994). Analysis of the ben mutant clearly shows that Ben has an important role in synaptic growth. A number of specimens exhibit dye coupling between the GF and the motorneuron dendrite demonstrating that an incipient synapse is still formed and that the mutant phenotype arises from a failure of this immature connection to grow into a mature synapse. In addition, both gap junctional and chemical components are present at ben mutant terminals with synaptic vesicle marker localization as well as ultrastructural analyses (Uthaman, 2008).

The current view of synapse formation is that a nascent synapse can be rapidly assembled from material present in a growth cone in prepackaged vesicles and packets. After this primary rapid assembly of a nascent synapse, a secondary slower growth and maturation process takes place to result in a stable mature synapse. An insightful study on the Drosophila kinesin immaculate connections (imac) has shown it to be a permissive regulator of presynaptic maturation at the larval NMJ. Imac was found to be involved in the anterograde transport of synaptic vesicle precursors to the tip of the growth cone, an initial stage of synaptogenesis. In ben specimens, synaptic vesicles are still transported all the way down to the tip of the truncated terminal as evidenced by the localization of GFP-tagged synaptotagmin and synaptobrevin. The data strongly suggest that the ben mutant phenotype is resultant at a point after synaptic vesicular transport. Hence, it is concluded that the bendless terminal is an incipient synapse that fails to grow and mature and that Ben is a permissive regulator whose function is required for the initiation of a secondary process in presynaptic growth (Uthaman, 2008).

It is counterintuitive that, although Bendless is required for synaptic growth and maturation, the data show that it is not required during the growth process. This highlights the important role Ben plays as a developmental switch. Transient expression of UAS-ben before the growth of the presynaptic terminal was sufficient to rescue the ben phenotype anatomically and physiologically, but expression during the growth period had no effect. This suggests that Bendless is not involved in the actual growth process but rather has to be present in advance to alter signaling and initiate changes that allow growth to take place. Here it is essential to differentiate between axonal and synaptic growth, because it has been determined previously that axonal growth is unaffected in ben mutants (Muralidhar, 1993). Hence, Ben function is required to permit axonal growth to switch to synaptic growth (Uthaman, 2008).

The molecules in the signaling pathway of this novel mechanism remain to be further investigated. In conclusion, tight spatial and temporal control of synaptic connectivity in the nervous system is undoubtedly crucial to normal function. Determining how exactly Bendless regulates the formation of a mature synapse will give future novel insights into this phenomenon (Uthaman, 2008).

no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila

In a screen for cell-cycle regulators, a Drosophila maternal effect-lethal mutant was identified named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, was identified as the nopo gene. A conserved residue in the RING domain is altered in the EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. Mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. Bendless (Ben), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support the model that an E2-E3 ubiquitination complex consisting of Ben-Uev1A (E2 heterodimer) and Nopo (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis (Merkle, 2009).

To ensure faithful transmission of the genome upon cell division, eukaryotic cells have developed checkpoints, regulatory pathways that delay cell-cycle progression until completion of prior events. The DNA damage/replication checkpoint plays a crucial role in preserving genomic integrity. Upon detection of DNA defects, the kinases ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3-related) are recruited to sites of damage and activated. ATM and ATR substrates include checkpoint kinases CHK1 and CHK2, which phosphorylate proteins that mediate cell-cycle arrest. The ensuing delay, resulting from engagement of this checkpoint, presumably allows cells time to correct defects (Merkle, 2009).

Research over the past decade has highlighted major roles for protein ubiquitination in regulating cellular responses to DNA damage. This post-translational modification, which involves covalent linkage of one or more ubiquitin molecules to another protein, regulates many fundamental cellular processes. Ubiquitination may alter the fate of a protein in numerous ways, such as targeting it for destruction by the 26S proteasome, changing its subcellular location, or changing its protein-protein interactions (Merkle, 2009).

Ubiquitination is a highly dynamic, multi-step process that requires three components: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2 or Ubc) and ubiquitin ligase (E3). E3s can be divided into two main classes: HECT and RING domain-containing proteins. RING-type E3 ubiquitin ligases contain a specialized motif of 40 to 60 residues that binds two zinc atoms. Many RING-type E3s bind to partnering E2 conjugating enzymes via their RING domains. Database searches of the Drosophila genome predict that it contains one E1, 36 E2s and ~130 E3s, which represents ~40% of the ubiquitination machinery in humans (Merkle, 2009).

Significant insights into the roles of many cell-cycle regulators have come from studying their functions in Drosophila. Drosophila is well suited for studying cell-cycle regulation during the formation of a multicellular organism, in large part because of its developmental use of cell cycles that differ in structure from canonical G1-S-G2-M cycles and the availability of genetic tools. The first thirteen cell cycles of Drosophila embryogenesis involve nearly synchronous nuclear divisions driven by stockpiles of maternally expressed mRNA and protein. These rapid cycles (~10 minutes in length) consist of oscillating S-M (DNA replication-mitosis) phases without intervening gap phases or cytokinesis. Minimal gene transcription occurs during this developmental stage, so cell cycles are regulated by post-transcriptional mechanisms. At cycle 14, the embryo cellularizes and initiates zygotic transcription at the midblastula transition (MBT) (Merkle, 2009).

This study reports the identification and characterization of a Drosophila maternal-effect lethal mutant 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with acentrosomal, barrel-shaped spindles during syncytial divisions. The results indicate that this arrest is secondary to the activation of a CHK2-mediated DNA checkpoint in early embryos. Nopo, a predicted E3 ubiquitin ligase, interacts with an E2 component, Ben. ben females are sterile, producing embryos with nopo-like defects. It is proposed that Ben-Uev1A and Nopo function together as an E2-E3 complex required for genomic integrity during Drosophila embryogenesis (Merkle, 2009).

nopo encodes a predicted protein of 435 amino acids containing an N-terminal RING domain. The putative mammalian homolog of Nopo was named 'TRAF-interacting protein' (TRIP) based on its ability to bind tumor necrosis factor (TNF) receptor-associated factors (TRAFs). Mammalian TRIP was recently demonstrated to have RING-dependent E3 ubiquitin ligase activity in an auto-ubiquitination assay. Drosophila Nopo and human TRIP are 20% identical and 34% similar overall, with 47% identity and 65% similarity in their RING domains. Importantly, nopo^Z1447 causes a glutamic acid to lysine change in the RING domain at position 11 of the predicted protein, a residue that is invariantly negatively charged across species (Merkle, 2009).

A model is proposed in which Nopo interacts with the Ben-Uev1A heterodimer to form a functional E2-E3 ubiquitin ligase complex required during syncytial embryogenesis for genomic integrity, cell-cycle progression, and the continuation of development. In the absence of Nopo, a lack of ubiquitination of, as yet unidentified, Nopo targets results in the truncation of S-phase and/or spontaneous DNA damage. Mitotic entry with unreplicated and/or damaged DNA triggers the activation of a CHK2-mediated checkpoint that leads to changes in spindle morphology, mitotic arrest and failure of nopo-derived embryos to develop to cellularization (Merkle, 2009).

The idea is favored thatNopo regulates the timing of S-M transitions in syncytial embryos to ensure that S-phase is of sufficient length to allow the completion of DNA replication prior to mitotic entry. The inhibition of DNA replication in syncytial embryos (e.g. via aphidicolin injection) leads to chromatin bridging in subsequent mitoses and CHK2 activation, both of which occur in nopo-derived embryos, presumably because of mitotic entry with unreplicated chromosomes. The mechanism by which Nopo coordinates S-M transitions is unknown. The data suggest that nopo may alter the timing of these transitions independently of CDK1-Cyclin B, although localized changes in the levels and/or activities of these regulators not detectable by immunoblotting of whole-embryo lysates could play a crucial role. It is unclear why the MEI-41/GRP-dependent checkpoint, which appears to be functional in nopo-derived embryos, is not sufficient to slow mitotic entry (Merkle, 2009).

The punctate nuclear localization observed for Nopo and its human homolog, TRIP, expressed in HeLa cells may indicate a direct role for these proteins in the regulation of chromatin structure. Furthermore, the G2 phase-specific localization that was observe for Nopo/TRIP in transfected HeLa cells may be consistent with a role for Nopo in slowing S-M transitions in syncytial embryos; in the absence of nopo, embryos that enter mitosis prematurely would probably do so without finishing DNA replication because of a lack of gap phases (Merkle, 2009).

An alternative explanation for CHK2 activation in nopo-derived embryos is that they might incur elevated levels of spontaneous DNA damage. Syncytial embryos are considered to be unusual in that they activate CHK2 but not CHK1 in response to DNA-damaging agents. Thus, spontaneous DNA damage would not be predicted to elicit the MEI-41/GRP-mediated replication checkpoint but would cause CHK2-dependent centrosomal inactivation during mitosis. Such a model would be consistent with the apparent lack of activation of the MEI-41/GRP-dependent checkpoint in nopo-derived embryos, although it would not explain why interphase 11 is shortened (Merkle, 2009).

Syncytial embryos from microcephalin (mcph1) mutant females undergo mitotic arrest with a phenotype similar to that described for nopo (Rickmyre, 2007). Like nopo, CHK2-mediated centrosomal inactivation causes mitotic arrest in embryos lacking mcph1. nopo and mcph1 are unique among maternal-effect lethal mutants in which CHK2-mediated centrosomal inactivation has been reported (e.g., grp, mei-41, wee1) in that their phenotypes appear to be more severe: centrosomes typically detach from spindles, and mitotic arrest occurs earlier, during precortical syncytial divisions. The underlying defects in nopo and mcph1 mutants may be distinct, however, because mnk mcph1-derived embryos (referring to maternal nuclear kinase (mnk), also known as loki, which encodes Drosophila CHK2) exhibit normal cycle 11 interphase length, which is truncated in mnk nopo-derived embryos (Rickmyre, 2007). Furthermore, no genetic interaction was detected between nopo and mcph1 (Merkle, 2009).

Mammalian TRIP was identified in a yeast two-hybrid screen for tumor necrosis factor (TNF) receptor-associated factor (TRAF) interactors. TRAFs transduce signals from members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily, which elicit diverse cellular responses in the immune and inflammatory systems. TRIP has been reported to inhibit TRAF2-mediated NFkappaB activation; the RING domain of TRIP, however, was not required for inhibition. By contrast, the current analysis of nopo^Z1447 indicates that this motif is essential for Nopo function in Drosophila embryogenesis, probably by mediating its interactions with E2 components, as has been shown for other E3 ligases. Drosophila Eiger (TNF ligand) and Wengen (TNF receptor) play roles in dorsal closure, neuroblast divisions, and the response to fungal pathogens. A role for TNF signaling in early Drosophila embryogenesis has not been reported (Merkle, 2009).

TRIP was recently reported to be an essential factor in mice. TRIP-deficient mice die soon after implantation as a result of defects in early embryonic development. Compared with wild-type littermates, TRIP^-/- embryos are smaller in size with a reduced cell number. TRAF2 does not appear to be required until later in development, suggesting that TRIP has TRAF2-independent roles in early embryos. It will be interesting to see whether mammalian TRIP, by analogy to Drosophila Nopo, is required for genomic integrity during embryonic development (Merkle, 2009).

The data support a model in which Nopo ubiquitin ligase acts in concert with Ben-Uev1A heterodimers to regulate Drosophila syncytial embryogenesis. The yeast two-hybrid interaction and co-localization of Nopo and Ben led to the identification of an unanticipated role for Ben in early embryogenesis and additional roles for Nopo in synapse formation and innate immunity. Although the spindle defects of ben-derived embryos are strikingly similar to those of nopo mutants, they typically arrest earlier in syncytial development, suggesting that another E3 ligase that requires Ben may function in parallel with Nopo. Although nopo egg chambers appear normal, a possible requirement for Ben-Uev1A-Nopo complexes during oogenesis has not been ruled out; some defects in nopo- and ben-derived embryos could be a secondary consequence of previous defects during oogenesis (Merkle, 2009).

K63-linked ubiquitin chains are thought to act as non-proteolytic signals (e.g. affecting protein localization and/or interactions), whereas K48-linked ubiquitin chains have established roles in targeting proteins for proteasome-mediated degradation. Ben-Uev1A E2 homologs in budding yeast (Ubc13-Mms2p) mediate K63-linked polyubiquitination of PCNA during postreplicative repair. In mammalian cells, the E2 heterodimer Ubc13-Mms2 mediates DNA damage repair, while Ubc13-Uev1A promotes NFkappaB activation; both E2 complexes regulate these processes by mediating K63 ubiquitin chain assembly on target proteins. It is proposed that Ben-Uev1A-Nopo (E2-E3) complexes mediate the assembly of K63-linked ubiquitin chains on proteins that preserve genomic integrity in early Drosophila embryogenesis (Merkle, 2009).

The role of ubiquitination in Drosophila innate immunity: Signaling upstream of Relish

Infection of Drosophila by Gram-negative bacteria triggers a signal transduction pathway (the IMD pathway) culminating in the expression of genes encoding antimicrobial peptides. A key component in this pathway is a Drosophila IkappaB kinase (DmIKK) complex, which stimulates the cleavage and activation of the NF-kappaB transcription factor Relish. Activation of the DmIKK complex requires the MAP3K dTAK1, but the mechanism of dTAK1 activation is not understood. In human cells, the activation of TAK1 and IKK requires the human ubiquitin-conjugating enzymes Ubc13 and UEV1a. This study demonstrates that the Drosophila homologs of Ubc13, (Bendless) and UEV1a, are similarly required for the activation of dTAK1 and the DmIKK complex. Surprisingly, the Drosophila caspase DREDD and its partner dFADD are required for the activation of DmIKK and JNK, in addition to their role in Relish cleavage. These studies reveal an evolutionarily conserved role of ubiquitination in IKK activation, and provide new insights into the hierarchy of signaling components in the Drosophila antibacterial immunity pathway (Zhou, 2005).

A cell culture system was established to study the IMD and Toll signaling pathways in S2 cells. The IMD pathway is activated by treating the cells with Gram-negative peptidoglycan (which is present in crude preparations of lipopolysaccharides), whereas activation of the Toll pathway is achieved by treating the cells with the Spätzle ligand. Active Spätzle is produced from a cell line stably transfected with a plasmid containing the copper-inducible metallothionein promoter driving the expression of active Spätzle C-106. When these cells are treated with copper, active SPZ is secreted into the medium, and this conditioned media can be used to activate naïve cells. Using the RNAi-mediated gene inactivation method, it was found that SPZ-induced Drosomycin gene activation in S2 cells requires the Drosophila Rel proteins Dif and Dorsal, as well as Toll, dMyD88, Tube, Pelle, and Slimb, as expected. In sharp contrast, these dsRNAs do not block the expression of antimicrobial peptide genes induced by peptidoglycan. Instead, RNAi studies demonstrate that peptidoglycan-induced gene expression requires all known components of the IMD pathway. Therefore this RNAi approach was used to determine the roles of candidate signaling components in the IMD and Toll signaling pathways (Zhou, 2005).

Previous studies have shown that activation of the mammalian IKK complex requires a ubiquitination step. In particular, TRAF6-mediated IKK activation was shown to require a dimeric ubiquitin-conjugating enzyme composed of the Ubc13 and UEV1a proteins. Bendless and dUEV1a are the Drosophila homologs of Ubc13 and UEV1a, respectively. Bendless and dUEV1a, like their mammalian counterparts, associate with each other in vivo. To investigate whether Bendless and dUEV1a are required for antibacterial gene expression in response to peptidoglycan, the RNAi-mediated gene inactivation method was used. S2 cells were transfected with various dsRNAs. After 48 h, cells were first treated with 20-hydroxy-ecdysone for 24 h to enhance their competence to induce antimicrobial genes in response to immune challenge, and then treated with peptidoglycan or SPZ to activate the IMD or Toll signaling pathways, respectively. Total RNA was isolated from these cells and subjected to Northern blotting analysis using cDNA fragments corresponding to Diptericin or Drosomycin as probes, to examine the activation of the IMD and Toll pathways, respectively. Both Bendless and dUEV1a are required for maximal levels of antibacterial peptide gene expression in response to peptidoglycan treatment. In fact, when both Bendless and dUEV1a are both targeted by RNAi, Diptericin induction is reduced to near background levels. (The partial effect of Bendless or dUEV1a RNAi alone is likely because of the fact that RNAi often does not generate a complete null phenotype.) By contrast, the induction of Drosomycin by Toll activation is unaffected by the RNAi-mediated knock-down of Bendless and dUEV1a. Note that the same cells were stimulated with either peptidoglycan or SPZ in. As a control, S2 cells treated with DmIKKgamma or Toll dsRNA showed significantly reduced peptidoglycan-induced Diptericin or SPZ-induced Drosomycin gene expression, respectively. As a control, mRNA and/or protein levels of targeted genes were examined by RT-PCR and/or Western blotting to confirm the effectiveness of RNAi (Zhou, 2005).

To provide further evidence that Bendless is involved in the IMD pathway, a dominant negative mutant Bendless was used to determine whether peptidoglycan-induced antibacterial gene activation can be blocked. Stable S2 cell lines were generated that express either wild-type or C87A Bendless under the control of the metallothionein promoter. The cysteine to alanine mutation at position 87 creates a dominant-negative mutant because this residue, located in the catalytic pocket in ubiquitin-conjugating enzymes, is crucial for the catalytic activity of E2s. These cells were then treated with various combinations of peptidoglycan and copper, and Northern blotting was employed to examine the expression of antibacterial genes including Attacin, Cecropin and Diptericin. Overexpression of wild-type Bendless has no effect on peptidoglycan-induced antibacterial gene activation, since similar levels of antibacterial peptide gene expression were detected in cells treated with or without copper. In contrast, overexpression of Bendless C87A leads to a significant reduction in peptidoglycan-activated expression of antibacterial peptide genes (Zhou, 2005). Bendless flies have been identified which carry a proline to serine substitution at position 97 within the strictly conserved active site region of E2s. In order to determine whether bendless flies are defective in response to Gram-negative bacterial infection, both wild-type and bendless flies were subjected to E. coli infection and Diptericin gene activation was examined by Northern blotting. bendless flies display significantly weaker Diptericin gene activation compared with wild-type flies. These results indicate that the Bendless-dUEV1a E2 complex is required for signaling by the IMD pathway (Zhou, 2005).

Experiments were carried out to determine whether Bendless and dUEV1a are required for peptidoglycan-induced activation of the DmIKK complex. Previous studies have shown that peptidoglycan treatment induces the kinase activity of the endogenous DmIKK complex in S2 cells. Cells were transfected with dsRNAs corresponding to various mRNAs. After 48 h, these cells were treated with peptidoglycan for 15 min, and the endogenous DmIKK complex was immunoprecipitated and subjected to in vitro kinase assays using recombinant Relish protein as substrate. Bendless or dUEV1a dsRNA treatment leads to a significant decrease in peptidoglycan-induced DmIKK kinase activity, suggesting that Bendless and dUEV1a are required for peptidoglycan-induced DmIKK activation. As a control, DmIKKgamma dsRNA treatment completely abolished peptidoglycan-induced DmIKK activation. It is concluded that the ubiquitin-conjugating enzymes Bendless and dUEV1a are specifically involved in the Drosophila IMD pathway, and they play a role upstream of the DmIKK complex (Zhou, 2005).

Overexpression of IMD in Drosophila results in the activation of antibacterial genes in the absence of bacterial infection. IMD overexpression, under control of the copper-inducible metallothionein promoter, can also strongly activate expression of the Diptericin gene in S2 cells. This stable cell line therefore provides a useful tool to perform an epistatic analysis to determine the position of Bendless/dUEV1a complex relative to IMD in the Drosophila antibacterial signaling pathway. The IMD stable cells were first transfected with dsRNAs derived from various genes and then stimulated with copper or peptidoglycan, and IMD- and peptidoglycan-induced Diptericin gene activation was examined. Overexpression of IMD, via the addition of copper, leads to strong activation of the Diptericin gene. In fact, copper-induced IMD expression is as potent as peptidoglycan in driving diptericin expression. Cells transfected with LacZ dsRNA show a similar Diptericin expression profile compared with cells that were mock-treated. Consistent with the observation that the DmIKK complex functions downstream of IMD, cells transfected with DmIKKgamma dsRNA are severely defective in both IMD- and peptidoglycan-induced Diptericin expression. Furthermore, cells treated with dsRNAs derived from Bendless and dUEV1a genes display a significant reduction in both peptidoglycan- and IMD-mediated Diptericin gene activation. These results indicate that the ubiquitin conjugating enzymes Bendless and dUEV1a function downstream of IMD in this signaling pathway (Zhou, 2005).

Thus, by differentially activating the IMD and Toll signaling pathways in Drosophila S2 cells, this study shows that Bendless (dUbc13) and dUEV1a are required for the Drosophila IMD signaling pathway. Using RNAi to target Bendless and/or dUEV1a significantly reduces the levels of peptidoglycan-induced antibacterial peptide gene expression and activation of the Drosophila IKK complex. This mechanism of IKK activation is highly conserved; in mammals Ubc13 and UEV1a are required for TNFalpha-, IL-1β-, and TCR-mediated IKK and NF-kappaB activation (Zhou, 2005).

This ubiquitin-dependent kinase activation does not involve proteasome-mediated degradation. Proteasome inhibitors do not block IKK activation, in flies or humans. Moreover, ubiquitination without degradation has been shown to activate the human IKK complex, and a similar Drosophila activity has been identified, The primary sequence of the mammalian Ubc13/UEV1a and those of the Drosophila Bendless/dUEV1a are highly conserved (90% similarity for Ubc13, 79% for UEV1a). The crystal structures of the yeast and human Ubc13/UEV1a (Mms2) have shown clearly that this E2 complex can make only K63-linked polyubiquitin chains. Thus, it is likely that the Drosophila Bendless/dUEV1a E2 catalyzes the formation of K63-linked ubiquitin chains (Zhou, 2005).

In a cell-free system, human TRAF6 was shown to be an E3 ligase that auto-ubiquitinates in conjunction with Ubc13/UEV1a. This results in the activation of TRAF6 and, in turn, the activation of TAK1. Activated TAK1 phosphorylates key serine residues in the activation loop of IKKβ, resulting in the activation of IKKβ. It is suspected that similar mechanisms are involved in the Drosophila IMD pathway. This study demonstrates that the Drosophila TAK1 homolog functions downstream of Bendless and dUEV1a. Furthermore, a Drosophila homolog of TAB2/TAB3 is also required for the IMD pathway. Interestingly, the C-terminal zinc finger domain of TAB2, which is conserved in the Drosophila protein, has recently been shown to bind specifically to K63-polyubiquitin chains. Strikingly, it has found that a galere/dTAB2 mutant, which is defective in the IMD pathway, carries a mutation in this zinc finger domain (Zhou, 2005).

The Drosophila E3 ligase, analogous to human TRAF2 or TRAF6, which functions with Bendless and dUEV1a in the activation of dTAK1 and DmIKK remains to be identified. In Drosophila, the dTRAF2 protein is the closest homolog of mammalian TRAF6, and it is the only Drosophila TRAF protein that contains the RING domain, typical of E3 ligases. However, RNAi knockdown and dominant-negative studies suggest that dTRAF2 is not involved in either the IMD or the Toll signaling pathways in S2 cells. In fact this gene is expressed at undetectably low levels in S2 cells. In one previous study dTRAF2 was reported to interact physically and functionally with Pelle, a key signaling component in the Toll signaling pathway that controls the antifungal immune response. However, these studies were based on overexpression experiments and in vitro binding assays, which might not reflect the physiological role of dTRAF2. In contrast, a recent publication demonstrated that dTRAF2 mutants are not fully able to induce antimicrobial peptide genes following E. coli infection. However, these studies did not clearly determine whether dTRAF2 is involved in the Toll or IMD pathways. The data suggest that dTRAF2 is not a critical component of the IMD pathway in S2 cells. Further studies, in cells and in flies, are necessary to elucidate the role of dTRAF2 in Drosophila immunity (Zhou, 2005).

The possibility is considered that other Drosophila RING-containing proteins might be involved in Drosophila immunity. However, RNAi knockdown studies with 10 different RING domain-encoding genes failed to block either the Toll or IMD pathways. Finally, since the structure of the RING domain of Rad5 has been successfully modeled to fit into the structure of the dimeric ubiquitin-conjugating enzyme complex Ubc13/UEV1a, it was reasoned that the potential ubiquitin ligase involved in the IMD pathway might physically interact with Bendless and dUEV1a. Therefore yeast two-hybrid screens were performed using Bendless and dUEV1a as baits in an effort to identify their protein interaction partners. A Drosophila RING protein, CG14435, was identified in such screens. CG14435 interacts robustly with both Bendless and dUEV1a in yeast two-hybrid assays. Furthermore, the CG14435-Bendless and CG14435-dUEV1a interaction was confirmed by co-immunoprecipitation of overexpressed proteins in S2 cells. However, RNAi-based studies suggest that CG14435 is not involved in the Drosophila innate immunity signaling pathways. It has been shown that bendless flies display defective synaptic connectivity and abnormal morphology within the visual system, suggesting Bendless functions in a variety of developmental processes. In addition, the Saccharomyces cerevisiae homologs of Bendless and dUEV1a have been implicated in DNA damage repair. Therefore it is possible that CG14435 is involved in some cellular processes other than immunity which require Bendless and dUEV1a. Further studies are necessary to elucidate the physiological role of CG14435 and to identify the ubiquitin ligase activity required for ubiquitination-dependent DmIKK activation (Zhou, 2005).

As in the TRAF6 pathway, dTRAF2 and/or other E3 ligases that function with Bendless and dUEV1a in the IMD pathway may be the target(s) of K63 polyubiquitination. Another possible target of Bendless/dUEV1a-mediated ubiquitination is the Drosophila IKKgamma subunit (also known as NEMO in mammals) of the IKK complex. In mammals, it has recently been shown that NEMO is K63 polyubiquitinated by the Ubc13/UEV1a complex in response to Bcl10 expression or T-cell activation. Other possible targets of ubiquitination by Bendless and dUEV1a in the IMD pathway include the Drosophila TAB2 homolog and IMD. Recently, it was shown that the two mammalian homologs of TAB2 and TAB3 were ubiquitinated or associated with other ubiquitinated proteins. In addition, the mammalian RIP1, which is homologous to IMD protein especially in its death domain, has recently been shown to be K63 polyubiquitinated and associated with TAB2 in a TNFalpha-dependent manner. In any case, K63 polyubiquitin chains likely function to recruit the Drosophila TAK1/TAB2 complex, via the TAB2 K63 polyubiquitin binding domain, to either (or both) the upstream activators, such as IMD, and/or the downstream target of dTAK1 kinase activity, the Drosophila IKK complex (Zhou, 2005).

As expected, the epistatic analyses presented in this study demonstrate that IMD functions upstream of all other components in the pathway except the receptor PGRP-LC, and is required for IKK activation. Moreover, Bendless and dUEV1a function downstream of IMD and upstream of dTAK1, as predicted from the model for Ubc13 and UEV1a in mammals. dTAK1 is required for activation of the Drosophila IKK complex and likely functions as the IKK kinase (Zhou, 2005).

Although it is established that dFADD and DREDD are required for the IMD pathway, previous experiments have suggested that they function downstream of the DmIKK complex. For example, DREDD overexpression in flies leads to Diptericin gene expression in the absence of Gram-negative bacterial infection, and DREDD-mediated Diptericin gene activation requires neither the DmIKK complex nor dFADD. Also, recent studies have shown that DREDD interacts with Relish, and that a caspase-cleavage site within Relish is required for peptidoglycan-induced Relish activation. Therefore, it was speculated that DREDD functions downstream of the DmIKK complex by directly cleaving DmIKK-phosphorylated Relish. This possibility is consistent with the observation that DREDD and dFADD are required for dTAK1Delta-mediated Diptericin gene activation. Surprisingly, dFADD and DREDD are required for peptidoglycan-induced DmIKK activation, arguing that DREDD and dFADD function upstream in the pathway. In addition, DREDD is also required for peptidoglycan-induced JNK activation, but neither DREDD nor dFADD are required for dTAK1- or dTAK1Delta-mediated DmIKK activation, suggesting that dFADD and DREDD act at a step upstream of dTAK1 in response to peptidoglycan. Based on these observations, it is proposed that dFADD and DREDD play dual roles in the Drosophila antibacterial signaling pathway. On the one hand, dFADD transduces signals from IMD to DREDD, resulting in DREDD activation and enabling Relish cleavage; in contrast, dFADD and DREDD contribute to peptidoglycan-induced DmIKK activation through a mechanism that remains to be elucidated. DREDD may function similarly to human Caspase-8, which is a DED-containing apical caspase similar to DREDD. Caspase-8 has recently been shown to be required for NF-kappaB activation in response TCR-signaling. This role of Caspase-8 requires the enzymatic activity of full-length protein Caspase-8 and is involved in recruiting the IKK complex to the upstream signaling complex of CARMA1, Bcl10, and MALT1. Interestingly MALT1 is also a caspase-like gene (sometimes referred to as a paracaspase), and it is thought to function as an E3-ligase accessory factor with Ubc13 and UEV1a in TCR-mediated NF-kappaB activation. DREDD may similarly function as E3-ligase accessory factors with Bendless and dUEV1a as the E2, in the IMD pathway (Zhou, 2005).

The following scheme is proposed for the Drosophila antibacterial signaling pathway. Peptidoglycan treatment or Gram-negative bacterial infection leads to the activation of the membrane-bound peptidoglycan-recognition protein, PGRP-LC. Activated PGRP-LC in turn transduces signal to IMD. IMD, in turn, interacts with dFADD and subsequently DREDD. It is proposed that IMD, dFADD and DREDD form a complex that contributes to dTAK1 activation, perhaps as part of an E3 ligase. This complex is likely to function in conjunction with Bendless/dUEV1a to activate dTAK1 and then DmIKK. Once activated, the Drosophila IKK complex phosphorylates Relish, which is subsequently cleaved. The N-terminal Relish cleavage product, an NF-kappaB transcription factor, then translocates to the nucleus where it activates antimicrobial peptide gene expression. In addition to their role in IKK activation, DREDD and dFADD are also proposed to function downstream in this pathway, in the signal-induced cleavage of phospho-Relish (Zhou, 2005).

bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog

The Drosophila bendless (ben) gene was originally isolated as a mutation affecting the escape jump response (Thomas, 1982; Thomas, 1984). This behavioral defect was ascribed to a single lesion affecting the connectivity between the giant fiber and the tergotrochanter motor neuron. A closer examination of the ben phenotype suggests that ben activity is broader and affects a variety of other neurons including photoreceptor cells and their axons. Mosaic analysis indicates that the focus of ben activity is presynaptic. This study reports the cloning of the ben gene through a chromosomal walk, and shows that it is homologous to a class of ubiquitin-conjugating enzymes. The major role of ubiquitination in the protein degradative pathway suggests that ben regulates neural developmental processes such as growth cone guidance by targeting specific proteins for degradation (Oh, 1994).

The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes

The bendless (ben) mutation of Drosophila has been shown to alter synaptic connectivity between a subset of CNS neurons. ben also causes morphological abnormalities within the visual system, suggesting that ben functions in a number of different developmental processes. This study shows that the ben gene encodes a protein which is closely related to ubiquitin-conjugating enzymes and that a missense mutation in the highly conserved active site region is associated with the ben mutation. High levels of ben expression are restricted to the nervous system during development. These results suggest a role for ubiquitin-mediated protein modification in nervous system development, including, but not exclusive to, the regulation of synaptic connectivity (Muralidhar, 1993).

Bendless alters thoracic musculature in Drosophila

bendless (ben) is an X chromosome mutation in Drosophila melanogaster, known to alter patterns of connections in the CNS and thus modify behavior. This study reports that in addition to its CNS effects, ben^- has pleiotropic phenotypes affecting thoracic muscle patterning, pupal mortality, and post-eclosional mobility. The tergal depressor of the trochanter (TDT) normally attaches ventrally to an apodeme on the trochanter and dorsally to the lateral scutum just posterior to the intrascutal suture. In ben^- individuals, TDT may attach anywhere within the boundaries of the attachment areas for TDT and dorsoventral muscles I (DVM I) and II (DVM II). Furthermore, TDT may completely lack a dorsal attachment, although it still maintains a ventral attachment. DVMs may also attach abnormally to dorsal sites normally occupied by an adjacent DVM, or may be entirely eliminated. DVM loss occurs independently of the position or presence of TDT dorsal attachment. The cytology of ben^- TDT is altered. Muscles may have fibers that are swollen and stain abnormally. Other fibers may have large, axially aligned holes. ben^- flies have an increased likelihood of failing to eclose and, upon eclosion, show impaired mobility. Several possible mechanisms are described for the ben^- developmental defects (Edgecomb, 1993).

Mutations altering synaptic connectivity between identified neurons in Drosophila

By studying the effects of mutations on a simple circuit of identified neurons in Drosophila, genes were found whose proper functioning is necessary to produce normal synaptic connections between the neurons. These neurons comprise the giant fiber (GF) system; the GFs are command neurons activated by a light-off stimulus and evoke a stereotyped pattern of activity in the thoracic muscles producing an escape jump. Each GF monosynaptically drives a motor neuron innervating the tergotrochanteral muscle (jump muscle, TTM). Each GF also disynaptically drives the motor neurons innervating the dorsal longitudinal flight muscle (DLM) via the peripherally synapsing interneuron (PSI). A search was made for mutations affecting these identified synapses. Fifty thousand mutagenized flies were screened for nonjumping behavior to the light-off stimulus. Fifty-seven nonjumping mutant lines were established from individuals selected in the screen. Members of the lines were then tested for abnormal GF motor output to the TTM and DLM. From these lines, four X-linked mutations (representing three complementation groups) were isolated which affect the circuit. The mutations differentially disrupt specific synapses within the GF system. One mutation, bendless, disrupts synaptic transmission between the GF and the TTM motor neuron. Another, gfA, disrupts the synaptic connections of the PSI, and a third mutation, passover, disrupts transmission in both pathways (Thomas, 1984).

Search PubMed for articles about Drosophila Bendless

Edgecomb, R. S., Ghetti, C. and Schneiderman, A. M. (1993). Bendless alters thoracic musculature in Drosophila. J. Neurogenet. 8(4): 201-19. PubMed Citation: 8320599

Merkle, J. A., et al. (2009). no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila. Development 136(3): 449-59. PubMed Citation: 19141674

Muralidhar, M. G. and Thomas, J. B. (1993). The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 11(2): 253-66. PubMed Citation: 8394720

Rickmyre, J. L., Dasgupta, S., Ooi, D. L., Keel, J., Lee, E., Kirschner, M. W., Waddell, S. and Lee, L. A. (2007). The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo. J. Cell Sci. 120: 3565-3577. PubMed Citation: 17895362

Oh, C. E., McMahon, R., Benzer, S. and Tanouye, M. A. (1994). bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog. J. Neurosci. 14(5 Pt 2): 3166-79. PubMed Citation: 8182464

Thomas, J. B. and Wyman, R. J. (1982). A mutation in Drosophila alters normal connectivity between two identified neurons. Nature 298: 650-651. PubMed Citation: 6808394

Thomas, J. B. and Wyman, R. J. (1984). Mutations altering synaptic connectivity between identified neurons in Drosophila. J. Neurosci. 4: 530-538. PubMed Citation: 6699687

Uthaman, S. B., Godenschwege, T. A. and Murphey, R. K. (2008). A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation. J. Neurosci. 28(34): 8615-23. PubMed Citation: 18716220

Zhou, R., Silverman, N., Hong, M., Liao, D. S., Chung, Y., Chen, Z. J. and Maniatis, T. (2005). The role of ubiquitination in Drosophila innate immunity. J. Biol. Chem. 280: 34048-34055. PubMed Citation: 16081424

date revised: 20 October 2009

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