InteractiveFly: GeneBrief

no poles: Biological Overview | References

BIOLOGICAL OVERVIEW

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) (Lee, 1997). Mammalian TRIP was recently demonstrated to have RING-dependent E3 ubiquitin ligase activity in an auto-ubiquitination assay (Besse, 2007). 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 (Lee, 1997). 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 (Lee, 1997). 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 (Park, 2007). 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 (Nguyen, 1999). 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 (Andersen, 2005). 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).

Traip controls mushroom body size by suppressing mitotic defects

Microcephaly is a failure to develop proper brain size and neuron number. Mutations in diverse genes are linked to microcephaly, including several with DNA damage repair (DDR) functions; however, it is not well understood how these DDR gene mutations limit brain size. One such gene is TRAIP, which has multiple functions in DDR. This study characterized the Drosophila TRAIP homolog nopo, hereafter traip, and found that traip mutants (traip^-) have a brain-specific defect in the mushroom body (MB). traip^- MBs were smaller and contained fewer neurons, but no neurodegeneration, consistent with human primary microcephaly. Reduced neuron numbers in traip^- were explained by premature loss of MB neuroblasts (MB-NBs), in part via caspase-dependent cell death. Many traip^- MB-NBs had prominent chromosome bridges in anaphase, along with polyploidy, aneuploidy or micronuclei. Traip localization during mitosis is sufficient for MB development, suggesting that Traip can repair chromosome bridges during mitosis if necessary. The results suggest that proper brain size is ensured by the recently described role for TRAIP in unloading stalled replication forks in mitosis, which suppresses DNA bridges and premature neural stem cell loss to promote proper neuron number (O'Neill, 2022).

This study in Drosophila shows that traip^- shares several characteristics with human microcephaly mutants. First, the traip^- phenotype is highly brain specific, with body defects being rare. Second, the traip^- MB phenotype is developmental rather than neurodegenerative, reflecting a primary rather than secondary microcephaly-like disorder. Finally, as with many human microcephaly genes, traip functions to promote NPC proliferation and survival. Thus, traip^- represents a powerful new disease model for understanding the etiological mechanisms underlying microcephaly (O'Neill, 2022).

TDespite their ubiquitous expression, mutations in microcephaly genes primarily affect the cerebral cortex in humans. Similarly, both traip and the DDR microcephaly gene MCPH1 are ubiquitously expressed in Drosophila, yet the MB is the only adult structure affected in their mutants. Although many tissues can make up for lost cells via compensatory proliferation, no such process appears to exist for replacing lost NPCs. Additionally, whereas most NBs have a limited window of proliferation, MB-NBs divide continuously from embryogenesis into late pupal stages, potentially allowing more accumulation of rare or small effects over many cell cycles. Thus, it is speculated that mutations in microcephaly genes likely affect all CB-NBs to some degree, but the MB-NBs are especially sensitive to these mutations as a consequence of their relatively prolonged period of proliferation. It is speculated that a similar explanation, including a prolonged period of rapid proliferation and lack of compensatory proliferation, may account for the sensitivity of the human cortex to microcephaly gene mutation (O'Neill, 2022).

This work provides the first link between a known function of Traip and proper brain development. Interphase nuclear localization is not required for Traip function, suggesting that Traip interphase functions are dispensable for MB-NB survival under normal conditions. Instead, it was discovered the presence of mitotic DNA bridges, sensitivity to inter-strand crosslinking agents, and RING domain dependence, consistent with the well-established role of TRAIP in unloading stalled forks to initiate repair. Furthermore, GFP::TraipΔNLS rescue experiments suggest either that Traip primarily performs this unloading function during mitosis, or else that Traip normally functions during interphase but is able to unload stalled forks during mitosis if necessary. Alternatively, it cannot be ruled out that there may be residual GFP::TraipΔNLS in the nucleus that allows continued function during interphase, or else that nuclear localization of Traip is not required for an interphase function. It is surmised that traip^- MB-NBs have stalled replication machinery that remains loaded throughout mitosis, preventing mitotic DNA synthesis repair and proper sister chromatid segregation. As anaphase proceeds, attached sister chromatids are pulled to opposite poles and they form UFBs as the under-replicated DNA is stretched out between them. These bridges could be physically broken, leading to chromosome fragmentation, generating aneuploidy or micronuclei and causing nuclear deformations in daughter cells. Alternatively, persistence of DNA bridges at the cytokinetic furrow could induce mitotic exit and furrow regression, leading to multiple nuclei or polyploidy, which likely prevent further proliferation (O'Neill, 2022).

Under normal conditions, MB-NBs are lost at the end of pupal development via caspase-dependent apoptosis. This study found that traip^- MB-NBs are lost prematurely, in part via caspase-dependent cell death, and thus fail to generate proper KC numbers. However, the caspase-inhibition experiments did not fully suppress traip^- MB phenotypes, suggesting that additional redundant mechanisms may play a role in MB-NB loss. For example, when caspase-dependent apoptosis is inhibited, MB-NBs are primarily lost via autophagy. Alternatively, the irregular, crenellated nuclear envelope morphology of some traip^- MB-NBs ( could point to non-apoptotic cell death pathways. Finally, aneuploidy-induced cell cycle exit in traip^- MB-NBs could lead to loss via premature differentiation . Furthermore, it is likely that loss of KCs and/or GMCs also contributes some to traip^- MB size defects (O'Neill, 2022).

TDNA bridge-induced defects likely feed into premature cell loss, but further work is required to dissect the pathways connecting them. In Drosophila, polyploid NBs can accumulate significant DNA damage as they enter mitosis, and chromosome breakage during mitosis in traip^- could induce death through DNA-damage signaling. Drosophila embryos laid by traip^- mothers do not survive, with extensive chromosome bridging and Chk2-dependent cell death, suggesting that DNA damage accumulation leads to cell loss in the rapidly dividing cells of the early embryo. In mammalian NPCs, polyploidy and binucleation can cause G1 arrest and apoptosis. In Drosophila, neurons can become polyploid in response to DNA damage, and NBs can become massively polyploid in some mutants , suggesting that, even though polyploidy may be better tolerated in flies, polyploid NBs are unlikely to complete additional mitoses successfully. The existence is inferred of traip^- aneuploid MB-NBs, which produce a wide range of daughter KC numbers, suggesting that traip^- generates some aneuploidies that are well tolerated and others that are highly lethal. Similarly, one recent study found that, although many karyotypes are permitted in NBs, loss of both copies of any of the three major Drosophila chromosomes resulted in a failure to proliferate and likely elimination. This parallels the situation in mammals, in which aneuploid NPCs and neurons are common, but also sensitive to G1 arrest, cell cycle exit, and apoptosis. Thus, both polyploidy and aneuploidy could stop further proliferation in traip^- MB-NBs by preventing proper mitosis or inducing G1 arrest and cell cycle exit, eventually triggering cell loss via various mechanisms (O'Neill, 2022).

This study identified centrosome, spindle and cytokinetic furrow localizations for Traip that are important for function. One possibility is that the dynamic movement of Traip on the mitotic spindle and cytokinetic furrow promotes encounters with unresolved DNA bridges. GFP::Traip was never observed on bridges. However, as a single TRAIP protein is probably sufficient to unload each replisome, fluorescence detection may be unlikely. Interestingly, centrosome localization is a common aspect of microcephaly-linked proteins, including MCPH1, which also functions in DDR. Similar to Traip, MCPH1 has mitotic functions required for proper chromosome segregation, and mutations in MCPH1 lead to lagging chromosomes, DNA bridges and micronuclei. Mutations in microcephaly genes with centrosome-associated functions, such as CEP135 and CDK5RAP2, cause dysregulation of centrosome numbers, which also lead to chromosome segregation errors and aneuploidy . Thus, mitotic roles, ensuring proper chromosome segregation, and suppressing aneuploidy are common features of microcephaly-linked proteins. Future work seeking to understand these shared defects better may reveal a deeper etiological connection across microcephaly disorders (O'Neill, 2022).

Search PubMed for articles about Drosophila Nopo

Andersen, P. L., Zhou, H., Pastushok, L., Moraes, T., McKenna, S., Ziola, B., Ellison, M. J., Dixit, V. M. and Xiao, W. (2005). Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J. Cell Biol. 170: 745-755. PubMed ID: 16129784

Besse, A., Campos, A. D., Webster, W. K. and Darnay, B. G. (2007). TRAF-interacting protein (TRIP) is a RING-dependent ubiquitin ligase. Biochem. Biophys. Res. Commun. 359: 660-664. PubMed ID: 17544371

Lee, S. Y., Lee, S. Y. and Choi, Y. (1997). TRAF-interacting protein (TRIP): a novel component of the tumor necrosis factor receptor (TNFR)- and CD30-TRAF signaling complexes that inhibits TRAF2-mediated NF-kappaB activation. J. Exp. Med. 185: 1275-1285. PubMed ID: 9104814

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 ID: 19141674

Nguyen, L. T., Duncan, G. S., Mirtsos, C., Ng, M., Speiser, D. E., Shahinian, A., Marino, M. W., Mak, T. W., Ohashi, P. S. and Yeh, W. C. (1999). TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses. Immunity 11: 379-389. PubMed ID: 10514016

O'Neill, R. S. and Rusan, N. M. (2022). Traip controls mushroom body size by suppressing mitotic defects. Development 149(7). PubMed ID: 35297981

Park, E. S., Choi, S., Kim, J. M., Jeong, Y., Choe, J., Park, C. S., Choi, Y. and Rho, J. (2007). Early embryonic lethality caused by targeted disruption of the TRAF-interacting protein (TRIP) gene. Biochem. Biophys. Res. Commun. 363: 971-977. PubMed ID: 17927961

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 ID: 17895362

date revised: 20 September 2009

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