SCCRO (DCUN1D1) is an essential component of the E3 complex for neddylation

Covalent modification of cullins by the ubiquitin-like protein NEDD8 (neddylation) regulates protein ubiquitination by promoting the assembly of cullin-RING ligase E3 complexes. Like ubiquitination, neddylation results from an enzymatic cascade involving the sequential activity of a dedicated E1 (APPBP1/Uba3), E2 (Ubc12), and an ill-defined E3. We show that SCCRO (also known as DCUN1D1) binds to the components of the neddylation pathway (Cullin-ROC1, Ubc12, and CAND1) and augments but is not required for cullin neddylation in reactions using purified recombinant proteins. We also show that SCCRO recruits Ubc12 approximately NEDD8 to the CAND1-Cul1-ROC1 complex but that this is not sufficient to dissociate or overcome the inhibitory effects of CAND1 on cullin neddylation in purified protein assays. In contrast to findings in cellular systems where no binding is seen, we show that SCCRO and CAND1 can bind to the neddylated Cul1-ROC1 complex in assays using purified recombinant proteins. Although neddylated (not unneddylated) Cul1-ROC1 is released from CAND1 upon incubation with testis lysate from SCCRO+/+ mice, the addition of recombinant SCCRO is required to achieve the same results in lysate from SCCRO(-/-) mice. Combined, these results suggest that SCCRO is an important component of the neddylation E3 complex that functions to recruit charged E2 and is involved in the release of inhibitory effects of CAND1 on cullin-RING ligase E3 complex assembly and activity.     

Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

Fbxw8 is essential for Cul1-Cul7 complex formation and for placental development

Cullin-based ubiquitin ligases (E3s) constitute one of the largest E3 families. Fbxw8 (also known as Fbw6 or Fbx29) is an F-box protein that is assembled with Cul7 in an SCF-like E3 complex. Here we show that Cul7 forms a heterodimeric complex with Cul1 in a manner dependent on Fbxw8. We generated mice deficient in Fbxw8 and found that Cul7 did not associate with Cul1 in cells of these mice. Two-thirds of Fbxw8-/- embryos die in utero, whereas the remaining one-third are born alive and grow to adulthood. Fbxw8-/- embryos show intrauterine growth retardation and abnormal development of the placenta, characterized by both a reduced thickness of the spongiotrophoblast layer and abnormal vessel structure in the labyrinth layer. Although the placental phenotype of Fbxw8-/- mice resembles that of Cul7-/- mice, other abnormalities of Cul7-/- mice are not apparent in Fbxw8-/- mice. These results suggest that the Cul7-based SCF-like E3 complex has both Fbxw8-dependent and Fbxw8-independent functions.     

The Drosophila homolog of Aut1 is essential for autophagy and development

The Drosophila homolog of yeast Aut1, CG6877/Draut1, is a ubiquitously expressed cytosolic protein. Draut1 loss of function was achieved by expression of an inverted repeat transgene inducing RNA interference. The effect is temperature-dependent and resembles an allelic series as described by Fortier, E. and Belote, J.M. (Genesis 26 (2000) 240-244). Draut1 loss of function larvae are unable to induce autophagy and heterophagy in fat body cells before pupariation and die during metamorphosis. To our knowledge, this is the first report of a multicellular animal lacking the function of a gene participating in the protein conjugation systems of autophagy.     

Interaction of Par-6 and Crumbs complexes is essential for photoreceptor morphogenesis in Drosophila

Apicobasal cell polarity is crucial for morphogenesis of photoreceptor rhabdomeres and adherens junctions (AJs) in the Drosophila eye. Crumbs (Crb) is specifically localized to the apical membrane of photoreceptors, providing a positional cue for the organization of rhabdomeres and AJs. We show that the Crb complex consisting of Crb, Stardust (Sdt) and Discs-lost (Dlt) colocalizes with another protein complex containing Par-6 and atypical protein kinase C (aPKC) in the rhabdomere stalk of photoreceptors. Loss of each component of the Crb complex causes age-dependent mislocalization of Par-6 complex proteins, and ectopic expression of Crb intracellular domain is sufficient to recruit the Par-6 complex. We also show that the absence of Par-6 complex proteins results in severe mislocalization and loss of Crb complex. We further demonstrate that Dlt directly binds to Par-6, providing a molecular basis for the mutual dependence of the two complexes. These results suggest that the interaction of Crb and Par-6 complexes is required for the organization and maintenance of apical membranes and AJs of photoreceptors.     

Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation

Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide Nedd8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities. Deneddylation, which removes the Nedd8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN). Here we show that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. We further show that neddylated Cul1 and Cul3 are unstable - as suggested by the evidence that Nedd8 promotes the instability of both cullins - and that the unneddylatable forms of cullins are stable. The protein stability of Nedd8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that Nedd8-conjugated cullins are degraded en bloc. We propose that while Nedd8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo.     

Talin is essential for integrin function in Drosophila

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.     

Drosophila Pax-6/eyeless is essential for normal adult brain structure and function

A role for the Pax-6 homologue eyeless in adult Drosophila brain development and function is described. eyeless expression is detected in neurons, but not glial cells, of the mushroom bodies, the medullar cortex, the lateral horn, and the pars intercerebralis. Furthermore, severe defects in adult brain structures essential for vision, olfaction, and for the coordination of locomotion are provoked by two newly isolated mutations of Pax-6/eyeless that result in truncated proteins. Consistent with the morphological lesions, we observe defective walking behavior for these eyeless mutants. The implications of these data for understanding postembryonic brain development and function in Drosophila are discussed.     

Rab11 is essential for fertility in Drosophila

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P-insertion line, Rab11(mo), was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11(mo) males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4-Rab11/+; Rab11(mo)/Rab11(mo) egg chambers, confirming the requirement of Rab11 in these events.     

The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID.

Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1. Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly. Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity. Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated.     

DEAF-1 function is essential for the early embryonic development of Drosophila

The Drosophila protein DEAF-1 is a sequence-specific DNA binding protein that was isolated as a putative cofactor of the Hox protein Deformed (Dfd). In this study, we analyze the effects of loss or gain of DEAF-1 function on Drosophila development. Maternal/zygotic mutations of DEAF-1 largely result in early embryonic arrest prior to the expression of zygotic segmentation genes, although a few embryos develop into larvae with segmentation defects of variable severity. Overexpression of DEAF-1 protein in embryos can induce defects in migration/closure of the dorsal epidermis, and overexpression in adult primordia can strongly disrupt the development of eye or wing. The DEAF-1 protein associates with many discrete sites on polytene chromosomes, suggesting that DEAF-1 is a rather general regulator of gene expression.     

The Drosophila orthologue of xPlkk1 is not essential for Polo activation and is necessary for proper contractile ring formation

Polo-like kinases (Plks) are essential for progression through mitosis. The activity of these kinases peak during M phase and this activation has been attributed to phosphorylation. Kinases capable of activating Plks in vitro have been previously identified both in mammalian cells and in Xenopus laevis oocytes (SLK and xPlkk1, respectively), although an in vivo correlation has not been clearly established. In order to study the regulation of Polo activity, we identified and cloned a Drosophila melanogaster kinase belonging to the ste20 ser/thr family that presents a close sequence homology with xPlkk1 and SLK. We termed this kinase dPlkk and showed that dPlkk associates with and phosphorylates Polo in vitro, resulting in the activation of the latter. On the other hand, when dPlkk is depleted from S2 cells, Polo activation does not seem to be impaired, suggesting that other kinases are involved in regulating Polo activity in vivo. Additionally, we found that a percentage of dPlkk-depleted cells fail to form a proper actin ring at the end of mitosis, leading to a failure in the assembly of the cleavage furrow and to the formation of binucleated cells. The detected accumulation of dPlkk in the contractile ring late in anaphase reinforces the idea that this kinase plays a role in cytokinesis.     

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.     

CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory

DNA synthesis during S-phase and upon DNA repair is accompanied by chromatin assembly. The chromatin assembly factor CAF-1 has been biochemically well-characterized to deposit histones onto newly synthesized DNA. To gain insights into the in vivo functions of CAF-1 in Drosophila, we generated null mutants of the largest subunit of dCAF-1, dCAF-1-p180. We show that, unlike CAF-1 mutant yeast, dCAF-1-p180 mutant flies are hemizygous lethal. Removal of maternal dCAF-1-p180 activity by germline clones blocks oogenesis. Tissue-specific deletion of dCAF-1-p180 in the eye primordia disrupts eye development. In addition, reduction of dCAF-1-p180 activity suppresses gene silencing at heterochromatin and antagonizes Polycomb-mediated cell fate determination. Furthermore, heterozygous dCAF-1-p180 mutant flies display an increased sensitivity to γ-irradiation and a reduced efficiency in recombinational double strand break (DSB) repair. Our experiments also show that human hCAF-1-p150 can rescue the dCAF-1-p180 mutant flies, demonstrating a functional conservation of eukaryotic CAF-1 activities in vivo. Together, our results establish that dCAF-1-p180 is an essential gene for Drosophila development and further underscore the importance of dCAF-1 in regulating gene expression and DNA repair in vivo.     

Drosophila BubR1 is essential for meiotic sister-chromatid cohesion and maintenance of synaptonemal complex

The partially conserved Mad3/BubR1 protein is required during mitosis for the spindle assembly checkpoint (SAC). In meiosis, depletion causes an accelerated transit through prophase I and missegregation of achiasmate chromosomes in yeast [1], whereas in mice, reduced dosage leads to severe chromosome missegregation [2]. These observations indicate a meiotic requirement for BubR1, but its mechanism of action remains unknown. We identified a viable bubR1 allele in Drosophila resulting from a point mutation in the kinase domain that retains mitotic SAC activity. In males, we demonstrate a dose-sensitive requirement for BubR1 in maintaining sister-chromatid cohesion at anaphase I, whereas the mutant BubR1 protein localizes correctly. In bubR1 mutant females, we find that both achiasmate and chiasmate chromosomes nondisjoin mostly equationally consistent with a defect in sister-chromatid cohesion at late anaphase I or meiosis II. Moreover, mutations in bubR1 cause a consistent increase in pericentric heterochromatin exchange frequency, and although the synaptonemal complex is set up properly during transit through the germarium, it is disassembled prematurely in prophase by stage 1. Our results demonstrate that BubR1 is essential to maintain sister-chromatid cohesion during meiotic progression in both sexes and for normal maintenance of SC in females.