InteractiveFly: GeneBrief

effete: Biological Overview | References

Gene name - effete

Synonyms - UbcD1

Cytological map position - 88D2-88D2

Function - ubiquitin conjugating enzyme

Keywords - protein degradation, ubiquitinization, germline stem cell maintenance, cell cycle, sensory neuron dendrite pruning, cell death

Symbol - eff

FlyBase ID: FBgn0011217

Genetic map position - 3R: 10,558,412..10,566,933 [-]

Classification - Ubiquitin-conjugating enzyme E2, catalytic (UBCc) domain

Cellular location - cytoplasmic

NCBI link: EntrezGene

eff orthologs: Biolitmine

Increasing evidence supports the idea that the regulation of stem cells requires both extrinsic and intrinsic mechanisms. However, much less is known about how intrinsic signals regulate the fate of stem cells. Studies on germline stem cells (GSCs) in the Drosophila ovary have provided novel insights into the regulatory mechanisms of stem cell maintenance. This study demonstrates that a ubiquitin-dependent pathway mediated by the Drosophila eff gene, which encodes the E2 ubiquitin-conjugating enzyme. Effete (Eff), plays an essential role in GSC maintenance. Eff both physically and genetically interacts with dAPC2, a key component of the anaphase-promoting complex (APC), which acts as a multisubunit E3 ligase and plays an essential role in targeting mitotic regulators for degradation during exit from mitosis. This interaction indicates that Eff regulates the APC/C-mediated proteolysis pathway in GSCs. Moreover, expression of a stable form of Cyclin A, but not full-length Cyclin A, results in GSC loss. Finally it was shown that, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Collectively, these data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Given that the regulation of mitotic Cyclins is evolutionarily conserved between flies and mammals, this study also implies that a similar mechanism may be conserved in mammals (Chen, 2009).

Germline stem cells (GSCs) of the Drosophila ovary provide an excellent model system for studying the molecular mechanisms of stem cell regulation in vivo. In adult Drosophila females, two to three GSCs are easily recognized by their molecular markers (either a spherical spectrosome, or an extending fusome when GSCs are dividing) and their location at the apical region of the germarium in close contact with surrounding somatic cells, the terminal filament and cap cells, which together generate a specific micro-environment, or niche, for GSC regulation. The GSC divisions take place along the anteroposterior axis of the ovary to produce an anterior GSC, which remains attached to the niche cells, and a posterior cystoblast (Cb). The Cb divides precisely four times by incomplete cytokinesis to generate 16 interconnected cells that form the germline cyst of the follicle and sustain oogenesis (Chen, 2009).

Genetic analyses have revealed that the stem cell state of GSCs is maintained by both extrinsic and intrinsic mechanisms that repress their differentiation. BMP ligands (Dpp, Gbb) from the niche cells maintain GSCs by suppressing Cb differentiation in the anteriormost cells. This is achieved by silencing the transcription of the bam gene, which encodes a GSC/Cb differentiation-promoting factor. In the GSCs, BMP signaling activates cytoplasmic Mad and Medea, the Drosophila Smads, and promotes their nuclear translocation. In the nucleus, the Smads complex physically interacts with both the bam silencer element and nuclear lamin-associated protein (Ote), resulting in bam transcriptional silencing. Thus, BMP/Dpp-dependent bam transcriptional control serves as the primary pathway for the regulation of GSC fate. Independent of the niche-based regulation of the bam silencing mechanism, the fate of GSCs is also intrinsically controlled by other GSC maintenance factors that repress their differentiation. It has been demonstrated that Pum/Nos-mediated and microRNA-mediated translational repression pathways are not required for bam silencing, suggesting that these pathways act either downstream of or parallel to bam action. Although it is proposed that the Pum/Nos-mediated and microRNA-mediated pathways repress the translation of key differentiation factors to prevent GSC differentiation, the targets of these translational pathways in GSCs are not identified. Thus, the intrinsic mechanisms that repress GSC differentiation are still poorly understood. In addition, as loss of function of the components in these translational repression pathways is not sufficient to completely cause bam mutant germ-cell differentiation, it is speculated that the repression of GSC differentiation may also be controlled by other unknown intrinsic mechanisms (Chen, 2009).

Ubiquitin-mediated protein degradation plays a variety of roles in the regulation of many developmental processes. The enzymatic reaction of protein ubiquitylation is a coordinated three-step process involving three classes of enzymes known as E1 (Uba1 -- FlyBase), E2 (UbcD4 -- FlyBase) and E3. Firstly, E1 (Ubiquitin activating enzyme 1) catalyzes the formation of a thiolester bond linkage between the active-site cysteine residue on E1 and the C terminus of ubiquitin. Secondly, the activated ubiquitin (E1-Ub) is then transferred to E2 (Ubiquitin conjugating enzyme 4) via formation of an E2-Ub thiolester. Thirdly, E3 (ubiquitin ligase) promotes the transfer of the ubiquitin from E2-Ub to a lysine residue of the target protein through an isopeptide bond. Repeated cycles of this reaction can result in polyubiquitylation of the target protein, which is finally targeted for degradation by the 26S proteasome. The Drosophila effete (eff) gene encodes a class I ubiquitin-conjugating enzyme that was first shown to be required for proper telomere behavior (Cenci, 1997). Early studies also showed that eff is required for proper cyst formation in ovary (Lilly, 2000). However, whether eff is involved in the regulation of GSC fate remains unknown (Chen, 2009). Mitotic cyclins, in concert with their Cyclin-dependent kinase (CDK) partners, play important roles in the promotion of mitosis, such as entry into mitosis and the alignment of chromosomes at metaphase. The degradation of mitotic cyclins by the anaphase-promoting complex/cyclosome (APC/C), a large multisubunit E3 ligase, leads to inactivation of CDKs during the metaphase-anaphase transition and is essential for the completion of mitosis in all eukaryotes. In Drosophila, it has been shown that induction of stable versions of the three mitotic cyclins, Cyclin A, B and B3, arrests mitosis with different phenotypes, suggesting that degradation of distinct mitotic cyclins is required for specific mitotic events. It has been shown that the asymmetric division of GSCs requires the activity of Cyclin B; however, it remains unknown whether the regulation of mitotic cyclins also contributes to fate determination in GSCs. This study demonstrates that the eff gene, which encodes an E2 ubiquitin-conjugating enzyme (Eff), is intrinsic to and essential for Drosophila GSC maintenance. Moreover, Eff both genetically and physically interacts with dAPC2, a key component of APC/C complexes. dCDC20/Fzy, a key regulator of APC/C complexes, is shown to be involved in GSC regulation. Finally, constant expression of a stable form of Cyclin A results in the loss of GSCs. Notably, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Thus, these data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of GSCs in Drosophila (Chen, 2009).

To identify new factors that regulate the self-renewal or differentiation of GSCs in the Drosophila ovary, female sterile lines, or weak fertile lines with P-element insertion, available from Bloomington Stock Center, were screened. The typical characteristic of GSC maintenance defects is a reduction in germ-cell number. This eventually results in an empty germarium lacking germ cells, and a decline in the production of egg chambers. Based on these criteria, a line with a P-element insertion in the third chromosome, P{PZ}eff8 was identified, that exhibits severe defects in germline development, including the loss of germ cells. To systematically study the behavior of GSCs and early germ cells in the eff8 mutant, anti-Vasa and anti-Hts antibodies were used to visualize germ cells and fusomes, respectively. In the tip of wild-type germarium, two or three GSCs were readily recognized by anti-Vasa antibody, and fusomes were morphologically spherical and anchored between the GSCs and cap cells. In addition, a normal germline lineage with sequentially differentiated cells marked by branched fusomes was also observed. However, in the 7-day-old ovaries from eff8 homozygous females, about 30% of mutant ovarioles contained either empty or abnormal germaria. Furthermore, the germ-cell defect phenotype became much more severe with age. These findings suggest that the loss of eff may affect the maintenance of GSCs. To determine whether the GSC maintenance defect associated with eff8 was indeed due to the loss of eff function rather than other genetic backgrounds, the phenotypes resulting from removal of eff were analyzed in several allelic combinations, eff8/effs1782, eff8/effmer1 and eff8/effmer4. The number of GSCs in the available eff allelic combinations were quantified at days 1, 7 and 14 after eclosion. Compared to wild type, the average number of GSCs in all eff mutants either rapidly or progressively declined during the testing period, indicating that the loss of eff resulted in the loss of GSCs. To further confirm this observation, a transgene, P{effP-eff}, was generated in which an eff cDNA was placed under the control of a 5.8 kb eff promoter. The GSC loss phenotype in different eff allelic backgrounds was fully rescued by the transgene line, P{effP-eff}. Taken together, these findings indicate that the eff gene plays an essential role in the maintenance of GSCs (Chen, 2009).

Attempts were made to understand the molecular mechanism underlying the action of Eff in GSCs by searching for Eff-interacting partners. Given that Eff functions as an E2 ubiquitin-conjugating enzyme, an E2/E3-based small-scale candidate screen was carried out by performing yeast two-hybrid experiments in which Eff was used as the bait to screen Eff-interacting E3. Notably, it was found that, among the candidates, dAPC2, encoded by the Drosophila morula (mr) gene, can strongly interact with Eff protein. To confirm this yeast two-hybrid interaction, whether Eff interacts with dAPC2 in Drosophila S2 cells was investigated by performing immunoprecipitation experiments. It was shown that Eff and dAPC2 can co-immunoprecipitate each other in transfected S2 cells, suggesting that Eff and dAPC2 are physically associated. To test whether dAPC2 physically associates with endogenous Eff in germ cells, a transgene, P{nosP-myc:dAPC2}, was generated. Results from co-immunoprecipitation showed that endogenous Eff physically associated with Myc:dAPC2, supporting further the argument that Eff interacts with dAPC2 in germ cells. In mitosis, the anaphase-promoting complex/cyclosome APC/C), a multisubunit complex that functions as an E3 ligase, plays important roles in ubiquitylating mitotic regulators such as mitotic cyclins and thus targets them for degradation by 26S proteasome. During this process, APC2, a cullin domain-containing protein, has been shown to function as a key mediator of APC/C complex activity. To test whether eff genetically interacts with mr (dAPC2) in the regulation of GSCs, the number of GSCs in both eff single-mutant and mr; eff double-mutant backgrounds were quantified at different time points. A weak allelic combination of mr (mr1/mr2) exhibited no apparent defect in GSC maintenance. However, mr; eff double-mutant ovaries showed more rapid GSC loss than the eff mutant alone, suggesting that dAPC2/mr enhances the phenotype of GSC loss in eff. Together, these results demonstrate that Eff interacts both physically and genetically with dAPC2 (Chen, 2009).

The ubiquitin-mediated proteolysis mechanism, which is evolutionarily conserved for the regulation of protein turnover, has been shown to play important roles in numerous biological processes, such as the cell cycle, pattern formation and tissue homeostasis. Drosophila Eff, which was initially identified as a class I E2 ubiquitin-conjugating enzyme encoded by the eff gene, has been shown to be involved in several cellular and developmental processes, including chromosome segregation, chromatin remodeling and protection against cell death (Cenci, 1997; Ryoo, 2002). This study found that loss of eff function results in the depletion of GSCs, revealing a new role for eff in the regulation of GSC fate. Using germline clonal analysis and rescue experiments, it was further defined that the eff gene is an intrinsic, rather than extrinsic, factor for the maintenance of GSCs. Previous studies have shown that Eff is involved in the rpr-induced apoptosis pathway through a physical interaction with DIAP1 that stimulates DIAP1 auto-ubiquitylation (Ryoo, 2002). Therefore, it is possible that loss of GSCs in eff mutants may be due to reduced viability of GSCs. The results clearly show that eff-/- GSCs undergo differentiation rather than apoptosis, thus supporting the idea that the role of eff is to repress the premature differentiation of GSCs (Chen, 2009).

In the ubiquitin pathway, E2 conjugating enzymes have much lower specificity compared with E3 ligases. Certain E2s are known to function together with distinct type E3 ligases for substrate ubiquitylation and degradation. Eff is involved in protein degradation mediated by various RING finger-containing E3 ligases [e.g., Sina, Neur and DIAP (Iap2 -- FlyBase)], and regulates several signaling transduction pathways (Kuo, 2006; Ryoo, 2002; Tang, 1997). Since Eff plays a role downstream of, or parallel to, bam function, it is important to know what biochemical functions Eff performs in the regulation of GSCs. This work provided biochemical evidence showing that Eff not only physically interacts with the dAPC2 protein, but is also crucial for the ubiquitylation and degradation of Cyclin A. Moreover, genetic analyses revealed that eff interacts with both dAPC2 and cyclin A with respect to the regulation of GSCs. In addition, this study shows that dCDC20/Fzy, a key regulator of APC/C complexes, is involved in GSC regulation. Together, these data strongly support a model in which Eff facilitates the E3 ligase function of APC/CDC20 to ensure the self-renewal of GSCs (Chen, 2009).

Early studies in Xenopus and clam extracts demonstrated that both UBC4, a homolog of Eff, and UBCx/E2-C equally supported APC-mediated ubiquitylation reactions in vitro. However, an in vivo study showed that these two classes of E2 are not functionally equivalent but exhibit distinct functions in mitotic cyclin degradation, suggesting that different E2 family members probably execute distinct functions (Seino, 2003). The Drosophila ortholog of UBCx/E2-C, Vihar E2, has been reported to be involved in Cyclin B degradation during the metaphase-anaphase transition (Mathe, 2004). This work presents both genetic and biochemical evidence that Eff, the Drosophila homolog of UBC4, is essential for Cyclin A degradation in GSCs. Because the mitosis-related ubiquitin-conjugating enzyme, Vihar E2, is involved in APC/C-mediated ubiquitylation that potentially regulates Cyclin A degradation, it would be interesting to determine whether and/or how different E2 family members (e.g. Eff and Vihar E2) coordinately support specific APC-mediated mitotic cyclin destruction with respect to GSC regulation (Chen, 2009).

It has been shown that APC/C activity is required for the asymmetric localization of Miranda and its cargo proteins during neuroblast division (Slack, 2007). In Drosophila ovary, previous studies have demonstrated that Cyclin B plays important roles in GSC division and is essential role for GSC maintenance (Hsu, 2008; Wang, 2005). However, it still remains unexplored whether the tight regulation of cyclins is also required for the fate determination of GSCs. The regulatory roles of mitotic cyclins at the cellular level during mitosis have been explored in detail (Parry, 2001). It has been reported that the sequential degradation of Cyclin A, Cyclin B and Cyclin B3 completes mitotic exit, which is mediated by APC/CDC20 in early M phase and by APC/Cdh1 during late M phase (Zachariae, 1998). Interestingly, the expression of stable forms of each cyclin leads to distinct mitosis defects, suggesting that the degradation of distinct mitotic cyclins is responsible for specific steps of mitosis. However, the biological basis for the control of the cyclin destruction remains elusive. Given that the APC-mediated pathway plays important roles in the proper cell mitosis, as loss of function of components in the pathway results in upregulation of mitotic cyclins that cause mitosis delay/or arrest, the question becomes whether the maintenance of GSCs requires the proper cell mitosis mediated by the regulation of mitotic cyclins. This study has shown that the forced expression of a stable form of Cyclin A leads to defects in GSC maintenance, suggesting that blocking mitotic progression may force germline stem cells to precociously differentiate, essentially altering their fate. Although the forced expression of a stable form of Cyclin B or Cyclin B3 does not give rise to any apparent defect in GSCs, one explanation is that stabilized Cyclin A may block cell-cycle progression more severely than the other stabilized Cyclins and prolonged M phase might be unfavorable for stem cell maintenance (Chen, 2009).

Taken together, these findings support a mechanism underlying the fate determination of stem cells that is linked to the control of the proper cell mitosis. Since the control of degradation of mitotic cyclins is evolutionarily conserved between flies and mammals, it would be interesting to also determine whether the control of proper cell mitosis is important for the maintenance of stem cells from other organisms, including mammals (Chen, 2009).

The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis

elfless (CG15150, FBgn0032660) maps to polytene region 36DE 5' (left) of reduced ocelli/Pray for Elves (PFE) on chromosome 2L and is predicted to encode a 187 amino acid RING finger E3 ubiquitin ligase that is putatively involved in programmed cell death (PCD, e.g., apoptosis). Several experimental approaches were used to characterize CG15150/elfless and test whether defects in this gene underlie the male sterile phenotype associated with overlapping chromosomal deficiencies of region 36DE. elfless expression is greatly enhanced in the testes and the expression pattern of UAS-elfless-EGFP driven by elfless-Gal4 is restricted to the tail cyst cell nuclei of the testes. Despite this, elfless transgenes failed to rescue the male sterile phenotype in Df/Df flies. Furthermore, null alleles of elfless, generated either by imprecise excision of an upstream P-element or by FLP-FRT deletion between two flanking piggyBac elements, are fertile. In a gain-of-function setting in the eye, it was found that elfless genetically interacts with key members of the apoptotic pathway including the initiator caspase Dronc and the ubiquitin conjugating enzyme UbcD1. DIAP1, but not UbcD1, protein levels are increased in heads of flies expressing Elfless-EGFP in the eye, and in testes of flies expressing elfless-Gal4 driven Elfless-EGFP. Based on these findings, it is speculated that Elfless may regulate tail cyst cell degradation to provide an advantageous, though not essential, function in the testis (Caldwell, 2009).

Emerging data are now elucidating the roles of key members of the apoptosome, including the caspases Dronc and Drice, in spermatid individualization in Drosophila. The molecules are thought to be inhibited by dBruce, a ubiquitin-conjugating enzyme with a BIR domain. In effect, dBruce may be acting in the spermatid cysts in a manner similar to another documented BIR-domain protein, DIAP1. This study examined the role of the E3 ubiquitin ligase, Elfless, and shows that, consistent with its prediction as a RING finger protein, Elfless interacts with key members of the apoptotic pathway (Caldwell, 2009).

It is proposed that Elfless acts to directly or indirectly regulate UbcD1 activity in the apoptotic pathway. w; GMR-Gal4; UAS-elfless in an otherwise wild type background produces pigment cell defects reminiscent of those of w; GMR-Gal4; UAS-Dronc. As previously shown, w; GMR-Gal4; UAS-Dronc in a ubcD1 heterozygous mutant background produces an eye phenotype slightly worse than w; GMR-Gal4; UAS-Dronc alone which suggests that Dronc may also be a target of UbcD1. Finally, inhibition of apoptosis through Dronc is effectively lost in w; GMR-Gal4; UAS-Dronc in a diap1/+ background; the eye defects exhibited in these lines are severe and flies die as pharate adults. w; GMR-Gal4; UAS-elfless flies in a ubcD1/+ mutant background similarly die as pharate adults and the eye phenotype is significantly worse than that of w; GMR-Gal4; UAS-elfless alone; pigmentation is absent in these flies and the size of the eye is reduced due to decreased UbcD1-mediated inhibition of Dronc and DIAP1. These data suggested that Elfless may be regulating UbcD1 activity (Caldwell, 2009).

While Elfless and UbcD1 had been shown to interact by yeast-two hybrid, it has not been possible to confirm a direct association between Elfless and either DIAP1 or UbcD1 by co-immunoprecipitation experiments. Nevertheless, it is clear from Western blot analysis that mis-expression of Elfless-EGFP in the eye or testes does not significantly change the level of UbcD1 protein but does increase DIAP1 protein levels. Thus, if Elfless is downregulating UbcD1 activity, it seems to be doing so without changing UbcD1 levels. Since DIAP1 auto-ubiquitination is UbcD1-dependent, Elfless-mediated downregulation of UbcD1 activity is consistent with reduced DIAP1 auto-ubiquitination and degradation, resulting in higher DIAP1. While on the one hand this would increase the anti-apoptotic activity of DIAP1, UbcD1 downregulation on the other hand would also increase the pro-apoptotic activity of Dronc, effectively circumventing DIAP1, and producing the somewhat mild eye phenotype evident in w; GMR-Gal4; UAS-elfless in an otherwise wild type background. Consistent with this model, in ubcD1 heterozygotes the eye phenotype is severely worsened and lethality is evident, while in Dronc heterozygotes, the eye phenotype is improved (Caldwell, 2009).

Although this study clearly demonstrates a genetic interaction of elfless with Dronc and ubcD1 in PCD and it is proposed that mis-expressed Elfless in the eye negatively regulates UbcD1 activity, no essential role in fertility could be ascribed to this locus, despite the promising role of this molecule in sperm development based on expression profiling and the Df/Df male sterility phenotype. Furthermore, an elfless transgene is not sufficient to rescue the male sterility associated with these deficiencies in polytene region 36DE. Despite the lack of a sterility phenotype when the RING finger protein encoded by elfless is deleted from the genome, the fact that elfless is functionally retained in the genome by selection indicates that elfless performs an important and advantageous, albeit redundant, function in the testes. Thus, while in laboratory vials, males are able to produce normal numbers of offspring, subtle functional differences can be quite significant in wild populations. What advantage could Elfess provide in the tail cyst cell? One possibility is that mobilization of Elfless in the nuclei of these cells facilitates tail cyst cell degeneration for more efficient resorption; sperm release from the cyst can still take place, but there may be a physiological burden on the testis during the life-time of the fly. The fact that Elfless is in the nucleus may suggest that it targets gene expression at this transitional stage of tail cyst cells. Full activation of apoptosis by Elfless in tail cyst cells is unlikely, as Elfless is nuclear, and no TUNEL staining in wildtype testes appears in the late stage cysts. Nevertheless, apoptosis signaling can be quite different in varying developmental contexts, and there is precedent for enlisting branches of the apoptotic pathway in different developmental processes. For example, Caspase-3 is activated in the cystic bulge of the developing spermatocyst, while the correct balance of DIAP1 levels is important for cellular mobilization of border cells and other cells during oogenesis. More incisive future approaches will be needed to discern the roles that Elfless and other subtle modulators play in mating and evolution (Caldwell, 2009).

Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing

Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila, the Imd pathway detects gram-negative bacterial infections through recognition of DAP-type peptidoglycan and activation of the NF-kappaB precursor Relish, which drives robust antimicrobial peptide (AMP) gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and K63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. This study demonstrates that Imd is rapidly K63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 (Effete) and Ubc13 (Bendless)-Uev1a, in conjunction with the E3 ligase Diap2. K63-ubiquitination activates the TGFβ-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of K63-chains and the addition of K48-polyubiquitin. This ubiquitin editing process results in the proteosomal degradation of Imd, which is proposed to function to restore homeostasis to the Drosophila immune response (Chen, 2017).

Previous work has demonstrated that Imd is cleaved, K63-polyubiquitinated and phosphorylated upon immune stimulation (Paquette, 2010). While this earlier study did not find K48- polyubiquitin chains, others have reported evidence of both K63- and K48-Imd modifications (Thevenon, 2009). However, the overall dynamics of and interconnections between these IMD post-translational modifications remained unclear. This study showed that peptidoglycan (PGN) from bacterial cell walls stimulation of S2 cells leads to five different Imd modifications: proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination, which leads to degradation of Imd through proteasome. These immune triggered signaling events are robust and incredibly rapid, with Imd cleavage and K63-polyubiquitination occurring as early as 2 minutes after PGN stimulation. While K63-modification peaks early and then steadily declines, K48-conjugation appears later, along with phosphorylation, and declines in proteasome-dependent manner. These kinetics argue that Imd is sequentially conjugated with K63 then K48 ubiquitin, so-called ubiquitin editing, as has been reported for IRAK1 and RIP1 in mammalian innate immune signaling pathways (Chen, 2017).

In addition to ubiquitination, two slow-migrating species of Imd were detected and shown to be phosphorylated forms. Judging by their size, these two phospho-forms appear to be derived from either full-length Imd (upper) or cleaved Imd (lower). Interestingly, persistence of phosphorylated Imd was observed in the proteasome-inhibited samples, suggesting that Imd is both K48-polyubiquitinated and phosphorylated before entering proteasome. Tak1 is required for these phosphorylation events as well as for ubiquitin editing, demonstrating key role for this MAP3K in a negative feedback loop (Chen, 2017).

Conjugation of ubiquitin usually occurs on lysine side chains of target proteins, and mass spectrometry of immuno-purified endogenously expressed Imd identified K137 and K153 as the sites of ubiquitin linkage. Note, the mass spec analysis includes 50% coverage of Imd, and a cluster of four lysine residues at the very C-terminus were not observed. Substitution of K137 and K153 residue with Arginine prevented signal- induced ubiquitination of Imd in S2 cells and reduced expression of the AMP gene Diptericin in both cells and flies. These results demonstrate that both lysine residues are required for K63- polyubiquitination and downstream signaling events. In S2 cells, mutation of single lysine residue led to a partial reduction of K63- ubiquitination and a partial reduction of AMP gene induction. Surprisingly, single lysine mutation did not correspondingly reduce Imd K48- polyubiquitination, while the double lysine mutation completely blocked it. These results suggest that even the reduced signal, mediated by a single K63-chain, is sufficient to trigger a robust feedback response with K48-chain formation. On the other hand, a complete block in K63-chains prevents Tak1 activation, which in turn fails to promote the ubiquitin editing of Imd. These findings are consistent with results observed with knockdown of Ubc5, Ubc13 and Uev1a. Ubc5 depletion prevented all K63 ubiquitination and signaling (as measured by Diptericin induction), and subsequent K48 modification was absent, while the Ubc13 and/or Uev1a knockdown showed residual K63 chains and greatly reduced Diptericin expression but robust K48- ubiquitination. These results also suggest that K48-polyubiquitination may occur on lysine residues beyond K137 and K153, although more detailed mass spectrometric analyses is required to map these sites more thoroughly (Chen, 2017).

On the other hand, only double lysine mutation leads to significant reduction of AMP expression in adult flies, and this is reduction is not as robust as in cultured cells. This pattern suggests that activation of the NF-κB protein Relish and its transcriptional targets do not solely rely on ubiquitination of Imd K137/153. Other possible ubiquitination targets include the upstream caspase Dredd, which has been shown to be critical for signaling (26), or the E3 ligase Diap2. This redundancy may represent multiple parallel mechanisms that contribute to the NF-κB activation. Furthermore, tissue specific immune differences may contribute to the discrepancy in Diptericin induction between macrophage-derived S2 cells and whole flies, in which the fat body rather than hemocytes is the major organ for inducible expression of AMPs (Chen, 2017).

Previous work has suggested that Diap2 is the E3 ligase for Imd ubiquitination. With the advantage of ubiquitin linkage specific antibodies, data presented in this study show that Diap2 is required for Imd K63-polyubiquitination and signaling, as measured by induction level of Diptericin. Moreover, the accumulation of cleaved but non-ubiquitinated Imd, in the Diap2 depleted cells and flies, provides further evidence that ubiquitination is downstream of Imd cleavage and highlights the role of Diap2 as the critical E3 in the K63- modification of Imd. In addition, Imd is no longer K48-modified when Diap2 is removed, suggesting that either Diap2 is also involved in K48- conjugation, or the failure of K63- polyubiquitination leads to the loss of K48- polyubiquitination. Given the role of Tak1 in this ubiquitin editing event, the latter hypothesis is favored, and another E3 is likely involved in the K48 conjugation (Chen, 2017).

In addition to E3 ligases, E2 ubiquitin conjugating enzymes are the other key factors in the ubiquitin conjugation reaction. Previously work has shown that Ubc5 and Ubc13-Uev1a are all involved in Imd ubiquitination. However, the mechanism by which these E2s collaborated was unclear. Results from in vitro reconstituted ubiquitination assays suggested a two-step reaction model for ubiquitin conjugation with different E2s. In particular, it was shown that some E2s, such as Ubc5, are effective at the initial ubiquitination of substrates but are ineffective at generating long chains, while other E2s, like Ubc13/Uev1a, are efficient at generating long ubiquitin chains but fail to conjugate substrate proteins. Thus, these two types of E2s can work together to generate long ubiquitin chains conjugated to target proteins. It is proposed that Imd undergoes ubiquitin chain initiation and elongation catalyzed by two separate E2s. Once cleaved, Imd interacts with Diap2 through its BIR repeats and is first modified by Ubc5-mediated substrate ubiquitination on lysines 137 and K153. Subsequently, the E2 complex of Ubc13-Uev1a pairs with an E3 (possibly Diap2 although another unidentifed E3 is not excluded) and switches the reaction to chain elongation mode, during which additional ubiquitin molecules are attached to the substrate-linked ubiquitin in a K63-specific manner. In the absence of Ubc13/Uev1a, Ubc5 alone is still able to elongate the polyubiquitin chains, but less efficiently and with unknown linkages (Chen, 2017).

Induction of Diptericin expression generally tracks with the K63-polyubiquitination signal (but not the total Ub signal) observed in various E2 knockdown cells. The one exception is the samples in which Ubc13 and Uev1a are both knocked down, and Ubc5 is still available. These samples display a similar K63 intensity as the single Ubc13 or Uev1a RNAi lanes, but Diptericin induction is lower, close to background levels. Ubc5 alone is known to conjugate ubiquitin without linkage specificity, a random polyubiquitin chain that consist of all seven types lysine linkage. Since the K63 antibody recognizes K63-linked diubiquitin, it is possible that Ubc5-mediated random ubiquitin chain elongation generates some K63 di-ubiquitin linkages which are detected by this antibody, but are unable support signaling due to their altered topology and limited amounts of K63-linkages. More detailed biochemical characterization of these Ubc5-catalyzed chains is required to confirm this hypothesis (Chen, 2017).

K48 modification of Imd shows a subtle difference relative to the K63 chains. Again, knock down of Ubc5 causes complete blocking of K48 conjugation, but depletion of Ubc13/Uev1a has no effect. The failure of K48 modification in the Ubc5 depleted cells may have two possible underlying causes. Firstly, the complete lack of K63 chains will fail to activate Tak1 and the subsequent ubiquitin editing feedback loop, while the Ubc13 and/or Uev1a RNAi display some residual K63 activity and thus can trigger Tak1 and the feedback response. Alternatively, Ubc5 might be directly required for Imd K48-polyubiquitination as shown in the degradation of proteins in multiple Drosophila pathways including eye development, maintenance of germline stem cells and apoptosis. These are not mutually exclusive possibilities (Chen, 2017).

Phosphorylation of Imd appears to be a major regulator of these ubiquitin-editing events. Knockdown of Tak1 prevents Imd phosphorylation in S2 cells and in adult flies. Moreover, immune-purified Tak1 can directly phosphorylate recombinant Imd in vitro, while neither JNKK nor IKK are required for phosphorylation of cleaved Imd, strongly arguing that Tak1 directly modifies Imd. RNAi-depletion or drug inhibition of Tak1 prevents K63-deubiquitination and the subsequent K48-polyubiquitination/proteasome-mediated degradation, leading to accumulation of cleaved but unphosphorylated Imd, presumably an intermediate during chain editing. From these results, it is inferred that Tak1-mediated phosphorylation of Imd is required for ubiquitin editing. Future studies will reveal the underlying mechanisms by which phosphorylation triggers K63-deubiquitination and K48 chain conjugation. Nonetheless, these results are consistent with earlier reports of Imd regulation by K63-deubiquitination and degradation (Chen, 2017).

Considering the results presented in this studdy together with earlier studies, the following model of Imd signal activation and subsequent down-regulation is proposed. One of the earliest events after PGN-stimulation is the rapid cleavage of Imd by the caspase-8 homolog DREDD at D30. Cleaved Imd then interacts with BIR-repeats of the E3 ligase Diap2 and is K63-polyubiquitinated through the sequential action of Ubc5, for substrate conjugation, and Ubc13-Uev1a, for catalyzing long K63 chains. These K63-polyubiquitin chains are then likely to activate the Tak1/Tab2 kinase complex through the conserved K63-binding motif in Tab2, which in turns signals through IKK complex to activate the NF-κB precursor Relish. Relish is central for the robust induction of AMP gene transcription. Meanwhile, Tak1 also mediates a retrograde signal that phosphorylates Imd and triggers ubiquitin editing, and leads to the degradation of Imd through proteasome. This regulatory interaction between Tak1 and Imd represents a novel homeostatic loop whereby the Drosophila immune response is rapidly activated but also quickly shutdown. Future studies are necessary to determine function of this feedback loop relative to other feedback mechanisms reported for the Imd pathway (Chen, 2017).

Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism

The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. This study shows that mutation or inhibition of Valosin-Containing Protein (VCP; termed TER94 by FlyBase), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, it was found that VCP inhibition causes an altered splicing pattern of the large pruning gene Molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). These data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 (Effete) and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, these results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism (Rumpf, 2014).

To achieve specific connections during development, neurons need to refine their axonal and dendritic arbors. This often involves the elimination of neuronal processes by regulated retraction or degeneration, processes known collectively as pruning. In the Drosophila, large-scale neuronal remodeling and pruning occur during metamorphosis. For example, the peripheral class IV dendritic arborization (da) neurons specifically prune their extensive larval dendritic arbors, whereas another class of da neurons, the class III da neurons, undergo ecdysone- and caspase-dependent cell death. Class IV da neuron dendrite pruning requires the steroid hormone ecdysone and its target gene SOX14, encoding an HMG box transcription factor. Class IV da neuron dendrites are first severed proximally from the soma by the action of enzymes like Katanin-p60L and Mical that sever microtubules and actin cables, respectively. Later, caspases are required for the fragmentation and phagocytic engulfment of the severed dendrite remnants . Another signaling cascade known to be required for pruning is the ubiquitin-proteasome system (UPS). Covalent modification with the small protein ubiquitin occurs by a thioester cascade involving the ubiquitin-activating enzyme Uba1 (E1), and subsequent transfer to ubiquitin-conjugating enzymes (E2s) and the specificity-determining E3 enzymes. Ubiquitylation of a protein usually leads to the degradation of the modified protein by the proteasome, a large cylindrical protease that consists of two large subunits, the 19S regulatory particle and the proteolytic 20S core particle. Several basal components of the ubiquitylation cascade—Uba1 and the E2 enzyme ubcD1—as well as several components of the 19S subunit of the proteasome have been shown to be required for pruning, as well as the ATPase associated with diverse cellular activities (AAA) ATPase Valosin-Containing Protein (VCP) (CDC48 in yeast, p97 in vertebrates, also known as TER94 in Drosophila), which acts as a chaperone for ubiquitylated proteins. Interestingly, autosomal dominant mutations in the human VCP gene cause hereditary forms of ubiquitin-positive frontotemporal dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). A hallmark of these diseases is the occurrence of both cytosolic and nuclear ubiquitin-positive neuronal aggregates that often contain the RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). It has been proposed that ubcD1 and VCP promote the activation of caspases during dendrite pruning via degradation of the caspase inhibitor DIAP1. However, mutation of ubcD1 or VCP inhibit the severing of class IV da neuron dendrites from the cell body, whereas in caspase mutants, dendrites are still severed from the cell body, but clearance of the severed fragments is affected. This indicates that the UPS must have additional, as yet unidentified, functions during pruning (Rumpf, 2014).

This study further investigated the role of UPS mutants in dendrite pruning. vcp mutation was shown to lead to a specific defect in ecdysone-dependent gene expression, as VCP is required for the functional expression and splicing of the large ecdysone target gene molecule interacting with CasL (MICAL). Concomitantly, mislocalization of Drosophila TDP-43 and up-regulation of other RNA-binding proteins were observed, and genetic evidence suggests that these alterations contribute to the observed pruning defects in VCP mutants. Defects in MICAL expression and TDP-43 localization are also induced by mutations in ubcD1 and in the 19S regulatory particle of the proteasome, but not by a mutation in the 20S core particle, despite the fact that proteasomal proteolysis is required for dendrite pruning, indicating the requirement for multiple UPS pathways during class IV da neuron dendrite pruning (Rumpf, 2014).

Class IV da neurons have long and branched dendrites at the third instar larval stage. In wild-type animals, these dendrites are completely pruned at 16-18 h after puparium formation (h APF). VCP mutant class IV da neurons were generated by the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique for clonal analysis. Mutant vcp26-8 class IV da neurons displayed strong pruning defects and retained long dendrites at 16 h APF. Expression of an ATPase-deficient dominant-negative VCP protein (VCP QQ) under the class IV da neuron-specific driver ppk-GAL4 recapitulated the pruning phenotype and also led to the retention of long and branched dendrites at 16 h APF. VCP inhibition also causes defects in class III da neuron apoptosis. This combination of defects in both pruning and apoptosis is reminiscent of the phenotypes caused by defects in ecdysone-dependent gene expression. Indeed, overexpression of the transcription factor Sox14, which induces pruning genes, led to a nearly complete suppression of the pruning phenotype caused by VCP QQ. This genetic interaction suggested that VCP might be required for the expression of one or several ecdysone target genes during pruning (Rumpf, 2014).

How could VCP be linked to Sox14? The suppression of the vcp mutant phenotype by Sox14 overexpression could be achieved in one of several ways. Sox14 could be epistatic to VCP -- that is, VCP could be required for functional Sox14 expression -- and this effect would be mitigated by Sox14 overexpression. However, VCP could also be required for the expression of one or several Sox14 target genes, and enhanced Sox14 expression could overcome this requirement either via enhanced induction of one or several particular targets or via enhanced induction of other pruning genes, in which case Sox14 would be a bypass suppressor of VCP QQ. To distinguish between these possibilities, the effects were assessed of VCP inhibition on the expression of known genes in the ecdysone cascade required for pruning in class IV da neurons. Class IV da neuron pruning is governed by the Ecdysone Receptor B1 (EcR-B1) isoform, which in turn directly activates the transcription of Sox14 and Headcase (Hdc), a pruning factor of unknown function. Sox14, on the other hand, activates the transcription of the MICAL gene encoding an actin-severing enzyme. In immunostaining experiments, VCP QQ did not affect the expression of EcR-B1, Sox14, or Hdc at the onset of the pupal phase. However, the expression of Mical was selectively abrogated in class IV da neurons expressing VCP QQ, or in vcp26-8 class IV da neuron MARCM clones . These data indicated that VCP might affect dendrite pruning by regulating the expression of the Sox14 target gene Mical, indicating that Sox14 might act as a bypass suppressor of VCP QQ (Rumpf, 2014).

How could VCP inhibition suppress Mical expression? To answer this question, whether Mical mRNA could still be detected in class IV da neurons expressing VCP QQ was assessed. To this end, enzymatic tissue digestion and FACS sorting were used to isolate class IV da neurons from early pupae (1-5 h APF). Total RNA was then extracted from the isolated neurons, and the presence of Mical mRNA expression was assessed by RT-PCR, using control samples or samples from animals expressing VCP QQ under ppk-GAL4. The Mical gene is large (~40 kb) and spans multiple exons that are transcribed to yield a ~15 kb mRNA. To detect Mical cDNA, primer pairs spanning several exons were used for two different regions of Mical mRNA, exons 14-16 and exons 8-12. [MICAL is on the (-) strand, but the exon numbering denoted by Flybase follows the direction of the (+) strand. Therefore, exons 14-16 are upstream of exons 8-12, and the latter are closer to the 3' end of MICAL mRNA.] MICAL mRNA was detectable upon VCP inhibition in these extracts with a primer pair spanning exons 14-16. The second primer pair spanning exons 8-12 also detected MICAL mRNA in both samples, but the RT-PCR product from the VCP QQ-expressing class IV da neurons had a larger molecular weight. Sequencing of the PCR products indicated that MICAL mRNA from VCP QQ-expressing class IV da neurons contained exon 11, which was not present in Mical mRNA from the control sample. Exon 11 is absent from all predicted MICAL splice isoforms except for a weakly supported isoform designated 'Mical-RM'. It introduces a stop codon into MICAL mRNA that would lead to the truncation of the C-terminal 1,611 amino acids from Mical protein. This portion of Mical protein contains several predicted protein interaction domains such as a proline-rich region, a coiled-coil region with similarity to Ezrin/Radixin/Moesin (ERM) domains, and a C-terminal PDZ-binding motif, and is required for the interaction between Mical and PlexinA. In addition, the truncated region contains the epitope for the antibody used in the immunofluorescence experiments, thus explaining the observed lack of Mical expression upon VCP inhibition. Given that a mutant of Mical with a smaller C-terminal truncation (compared with the one induced by VCP inhibition) was not sufficient to rescue the class IV da neuron dendrite pruning defect of mical mutants, disruption of VCP function likely results in expression of a truncated Mical protein without pruning activity. Taken together, these data suggest that the observed defect in MICAL mRNA splicing contributes significantly to the pruning defects of VCP mutants (Rumpf, 2014).

How is VCP linked to alternative splicing of MICAL mRNA? A plausible mechanism for the control of an alternative splicing event would be the modulation of specific (pre)mRNA-binding proteins. VCP has recently been linked to several RNA-binding proteins: human autosomal dominant VCP mutations cause frontotemporal dementia or ALS with inclusion bodies that contain aggregated human TDP-43; a genetic screen in Drosophila identified the RNA-binding proteins Drosophila TDP-43, HRP48, and x16 as weak genetic interactors of the dominant effects of VCP disease mutants; and HuR (a human homolog of the neuronal Drosophila RNA-binding protein elav) was recently shown to bind human VCP. Of these, TDP-43 and also elav have been linked to alternative splicing in various model systems, including Drosophila. Therefore this study used available specific antibodies to assess the levels and distribution of Drosophila TDP-43 (hereafter referred to as TDP-43) and elav. TDP-43 has previously been shown to localize to the nucleus in Drosophila motoneurons and mushroom body Kenyon cells. Surprisingly, TDP-43 was largely localized to the cytoplasm in class IV da neurons, where it was enriched in a punctate pattern around the nucleus, with only a small fraction also detectable in the nucleus, a localization pattern that could be reproduced with transgenic N-terminally HA-tagged TDP-43. Elav is a known nuclear marker for Drosophila neurons; in class IV da neurons, it was somewhat enriched in nuclear punctae. The effects of VCP inhibition on these two RNA-binding proteins was assessed. Elav localization did not change notably upon VCP QQ expression. Strikingly, TDP-43 became depleted from the cytoplasm of class IV da neurons and relocalized to the nucleus upon VCP QQ expression. Closer inspection revealed that TDP-43 in VCP-inhibited neurons was now enriched in nuclear dots that often also exhibited increased elav staining. The relocalization of TDP-43 from the cytoplasm to the nucleus was also observed in vcp26-8 mutant class IV da neuron MARCM clones. Importantly, quantification and normalization of TDP-43 levels showed that VCP inhibition did not alter the absolute levels of TDP-43, suggesting that the observed effects were not a consequence of a defect in TDP-43 degradation. In fact, the only manipulation that resulted in a mild but significant increase in TDP-43 levels -- but without a change in localization -- was the expression of an RNAi directed against the autophagy factor ATG7, perhaps reflecting the degradation of cytoplasmic RNA granules through the autophagy pathway (Rumpf, 2014).

It was next asked whether manipulation of TDP-43 would affect class IV da neuron dendrite pruning. A previously characterized TDP-43 mutant, TDP-43 Q367X (28-128">28), did not display pruning defects, but overexpression of TDP-43 led to strong dendrite pruning defects at 16 h APF. In support of the hypothesis that TDP-43 acts in the same or a similar pathway as VCP during dendrite pruning, it was also found that a more weakly expressed TDP-43 transgene (UAS-TDP-43weak) and VCP A229E, a weakly dominant-active VCP allele corresponding to a human VCP disease mutation, exhibited a synergistic inhibition of pruning when coexpressed. Interestingly, manipulation of elav gave very similar results as with TDP-43: elav down-regulation by RNAi did not affect pruning, but elav overexpression led to highly penetrant pruning defects (Rumpf, 2014).

To exclude the possibility that the pruning defects induced by TDP-43 or elav overexpression were due to long-term expression and aggregation of RNA-binding proteins, TDP-43 and elav overexpression was also induced acutely (24 h before the onset of pupariation). Pruning was still inhibited in these cases. Also, overexpression of several other RNA-binding proteins did not cause pruning defects, with two exceptions: a UAS-carrying P-element in the promotor of the adjacent x16 and HRP48 genes caused a strong pruning defect when expression was induced in class IV da neurons, and levels of a GFP protein trap insertion into the x16 gene were also markedly increased in class IV da neurons expressing VCP QQ, possibly indicating a role for VCP in x16 degradation. In further support of an involvement of VCP with RNA-binding proteins during neuronal pruning processes, it was also found that VCP is required for mushroom body γ neuron axon pruning and induces the accumulation of Boule, an RNA-binding protein that had previously been shown to inhibit γ neuron axon pruning when overexpressed. Thus, the data suggest that VCP regulates a specific subset of RNA-binding proteins and that this regulatory role of VCP is associated with its role in pruning (Rumpf, 2014).

As VCP is an integral component of the UPS, it was next asked whether the role of VCP in MICAL regulation and TDP-43 localization was also dependent on ubiquitylation and/or the proteasome. To address this question, Mical levels and TDP-43 distribution was assessed in UPS mutants with known pruning defects. An ubiquitylation enzyme known to be required for pruning is the E2 enzyme ubcD1. When TDP-43 localization was assessed in larval ubcD1D73 mutant class IV da neurons, TDP-43 was again localized to the nucleus in these cells. Furthermore, a pronounced reduction of Mical expression in ubcD1D73 mutant class IV da neurons was noted during the early pupal stage, indicating that ubiquitylation through ubcD1 is involved in the regulation of TDP-43 localization and Mical expression (Rumpf, 2014).

TDP-43 localization and Mical expression were assessed in proteasome mutants. A previously characterized mutant in the Mov34 gene encoding the 19S subunit Rpn8 was used. TDP-43 was again relocalized to the nucleus in Mov34 mutant class IV da neurons, and Mical expression was absent from Mov34 mutant class IV da neurons at 2 h APF. To rigorously address whether proteasomal proteolysis was also required for TDP-43 localization and Mical expression, the effect was assessed of Pros261, a previously characterized mutation in the 20S core particle subunit Prosβ6. In contrast to Mov34 mutant class IV da neurons, Pros261 mutant class IV da neurons displayed cytoplasmic TDP-43 localization, and robust Mical expression was detected in these neurons at 2 h APF. Thus, although ubiquitylation and the 19S proteasome are both required for Mical expression and normal TDP-43 localization, proteolysis through the 20S core particle of the proteasome is not. Importantly, Pros261 MARCM class IV da neurons showed strong dendrite pruning defects at 16 h APF, as did expression of RNAi constructs directed against subunits of the 20S core particle (Rumpf, 2014).

These data indicate that there must be several ubiquitin- and proteasome-dependent pathways that are required for dendrite pruning: one pathway requires ubcD1, VCP, and the 19S regulatory particle of the proteasome, but not the 20S core particle. This pathway regulates MICAL expression. A second UPS pruning pathway does depend on proteolysis through the 20S core. In an E3 ubiquitin ligase candidate screen, cul-1/lin19 was identified as a pruning mutant. Cul-1 encodes cullin-1, a core component of a class of multisubunit ubiquitin ligases known as SCF (for Skp1/Cullin/F-box) ligases. Class IV da neurons mutant for cul-1 or class IV da neurons expressing an RNAi construct directed against cul-1 had not pruned their dendrites at 16 h APF. However, unlike with VCP, ubcD1, and Mov34, cul-1 mutation did not affect Mical expression at 2 h APF, indicating that cullin-1 is not a component of the VCP-dependent UPS pathway involved in splicing and might thus be a component of a proteolytic UPS pathway. In support of this notion, a recent report independently identified cul-1 as a pruning mutant and associated it with protein degradation (Rumpf, 2014).

It has been proposed that the E2 enzyme ubcD1 and VCP would act to activate caspases during pruning. However, the dendrite pruning defects caused by those UPS mutants are much stronger than the phenotypes caused by caspase inactivation, which mostly causes a delay in the phagocytic uptake of severed dendrites by the epidermis. Although it cannot be excluded that ubcD1 and VCP contribute to caspase activation during pruning, the new mechanism proposed in this study -- control of RNA-binding proteins and MICAL expression -- likely makes a much stronger contribution to the drastic pruning phenotypes of UPS mutants (Rumpf, 2014).

How precisely do VCP, ubcD1, and the 19S proteasome contribute to MICAL expression? The data indicate that VCP inhibition causes missplicing of MICAL mRNA that likely leads to the expression of an inactive Mical protein variant. At the same time, VCP inhibition leads to the mislocalization of TDP-43, and possibly the dysregulation of a number of other RNA-binding proteins. The fact that these phenotypes correlate in the vcp, ubcD1, and Mov34 mutants gives a strong indication that they are related. TDP-43 had previously been identified as a suppressor of the toxicity induced by a weak VCP disease allele in the Drosophila eye. In class IV da neurons, reducing the amounts of TDP-43 (with a deficiency) or elav (by RNAi) did not ameliorate the pruning defect induced by VCP inhibition. Therefore, the possibility cannot be excluded that the two proteins act in parallel rather than in an epistatic fashion. As VCP has been shown to remodel protein complexes that contain ubiquitylated proteins and is structurally similar to the 19S cap, it is interesting to speculate that VCP and the 19S cap might alter the subunit composition of ubiquitylated TDP-43-containing complexes of RNA-binding proteins, and that this activity—rather than a direct action on TDP-43 (or maybe also elav) alone—might lead to both MICAL missplicing and TDP-43 mislocalization (Rumpf, 2014).

Interestingly, autosomal dominant mutations in human VCP cause frontotemporal dementia and ALS, a hallmark of which is the formation of aggregates that contain TDP-43. Most of these aggregates are cytoplasmic (and contain TDP-43 that has relocalized from the nucleus to the cytoplasm), but VCP mutations also induce TDP-43 aggregation in the nucleus, a situation that might be similar to the situation caused by VCP inhibition in class IV da neurons. Although human VCP disease mutations have been proposed to act as dominant-active versions of VCP with enhanced ATPase activity, both the disease allele and the dominant-negative ATPase-dead VCP QQ mutant cause class IV da neuron pruning defects and TDP-43 relocalization to the nucleus of class IV da neurons and therefore act in the same direction. It is thought that VCP can only bind substrates when bound to ATP, and will release bound substrates upon ATP hydrolysis. Thus, it is conceivable that the phenotypic outcome of inhibiting the ATPase (no substrate release) should be similar to that of ATPase overactivation (reduced substrate binding or premature substrate release): in both cases, a substrate protein complex would not be properly remodeled (Rumpf, 2014).

Taken together, these results indicate the existence of a nonproteolytic function of VCP and the UPS in RNA metabolism and highlight its importance during neuronal development (Rumpf, 2014).

Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, an E2/E3 ubiquitinating enzyme mutant screen was conducted, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme encoding Effete, resulted in retention of C4da neuron dendrites during metamorphosis. Further, UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling (Kuo, 2006).

The ubiquitin-proteasome system (UPS), evolutionarily conserved for the regulation of protein turnover, targets proteins for degradation via a complex, temporally regulated process that results in proteasome-mediated destruction of polyubiquitinated proteins. There are two distinct steps involved: first, the covalent conjugation of ubiquitin polypeptide to the protein substrates, and second, the destruction of tagged proteins in the proteasome complex. The transfer of ubiquitin to a target molecule slated for degradation involves at least three enzymatic modifications: ubiquitin is first activated by the ubiquitin-activating enzyme E1; ubiquitin is then transferred to a carrier protein, a ubiquitin-conjugating enzyme E2, and finally, ubiquitin is transferred to a protein substrate bound by a ubiquitin ligase E3. There are minor variations to this enzymatic cascade, but overall, these highly specific protein-protein interactions ensure ubiquitin targeting specificity and regulate many aspects of housekeeping protein turnover and cellular maintenance. However, with the multiple regulatory layers, different parts of this complex machinery can break down. Mutations in the UPS pathway causing accumulation of nondegraded proteins have been implicated in a variety of human diseases (Kuo, 2006).

In the nervous system, aberrations in the UPS pathway have been implicated in disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. One of the common pathological features of neurodegenerative diseases, besides neuronal loss, is local axon degeneration. For example, in the case of Wallerian degeneration in vertebrates, distal parts of a severed axon remain viable and conduct action potentials in vivo for some time before a rapid dismantling of cytoskeletal proteins and axon degeneration, and the initiation of this rapid axon degeneration involves the UPS pathway. It is thought that UPS activation can lead to microtubule depolymerization and subsequent neurofilament degradation, possibly acting in conjunction with the Ca2+-dependent protease calpain. Moreover, inhibiting UPS activity in neurons prior to severing their axons can dramatically retard degradation of the severed axons. These results suggest that a cell-intrinsic UPS pathway regulates axon stability and that pharmaceutical inactivation of the UPS may prevent axonal degeneration in disease states (Kuo, 2006 and references therein).

In Drosophila, the remodeling of neuronal processes during normal development closely resembles the pathological phenotypes in Wallerian degeneration. In the mushroom body γ neurons, extensive pruning of larval axons occurs during metamorphosis in a process regulated by glia engulfment and neuron-intrinsic UPS activity. Similarly, in the fly peripheral nervous system, the class IV dendritic arborization (C4da) neurons undergo complete pruning of their extensive larval dendrites during metamorphosis, in a process that is also regulated by UPS activity (Kuo, 2005). In both of these examples, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration, all phenotypes resembling Wallerian degeneration. Therefore, these fly neurons represent excellent systems in which to understand the roles of the UPS in regulating neuronal axon/dendrite integrity, given the rather limited knowledge of how the UPS participates in the degradation of neuronal processes. It is not known which specific E2 ubiquitin-conjugating enzyme(s) and E3 ubiquitin ligase(s) are involved in UPS-mediated remodeling/degradation of neuronal processes, or their specific downstream target(s) (Kuo, 2006).

It has been shown that mutations in the fly ubiquitin activation enzyme (uba1) and the proteasome complex (mov34) can prevent efficient pruning of C4da neuron larval dendrites during metamorphosis (Kuo, 2005). To further investigate the role of UPS in C4da neuron dendrite remodeling, a candidate gene screen was conducted to identify the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase required for this process. Analysis of genetic mutants showed that UPS activation in C4da neurons likely results in UbcD1 (an E2 ubiquitin-conjugating enzyme) mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1), an E3 ligase that antagonizes caspase activity. Degradation of DIAP1 leads to activation of caspase Dronc, which results in local caspase activation and cleavage of proximal dendrites in C4da neurons during metamorphosis. In addition to the identification of a set of E2/E3 ubiquitinating enzymes for C4da neuron dendrite remodeling (with the surprising finding that the UPS mediates degradation of the potent protease inhibitor DIAP1) this study also establishes a mechanistic link between the UPS and caspase pathways in regulating C4da neuron dendrite pruning (Kuo, 2006).

To identify the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase mediating dendrite pruning of C4da neurons during metamorphosis, a candidate gene approach was taken to systematically test the roles of known E2/E3 ubiquitinating enzymes in Drosophila. A set of putative E2/E3 ubiquitinating enzyme mutations was assembled, and live imaging was used to visualize C4da neurons carrying the E2/E3 mutation via the pickpocket(ppk)-EGFP marker, which specifically labels C4da neurons during Drosophila development. Those mutants with an early lethal phase were characterized by generating mosaic analysis with a repressible cell marker (MARCM) mutant neuronal clones. Since wild-type (wt) C4da neurons during metamorphosis do not retain any larval dendrites following head eversion, as imaged 18-20 hr after puparium formation (APF), mutations that caused larval dendrite retention in C4da neurons at this stage were sought. The candidate genes tested mostly showed no defects in dendrite pruning or neuronal cell death. However, one candidate, ubcD1, showed a modest level of larval dendrite retention at 18 hr APF (Kuo, 2006).

Live imaging of wt C4da neuron MARCM clones at the start of pupariation (white pupae, WP) showed primary and secondary dendritic branching patterns typical of C4da neurons. Consistent with previous reports (Kuo, 2005; Williams, 2005), wt C4da neurons sever their larval dendrites during early metamorphosis and by 18 hr APF are devoid of any dendrites. The ubcD1 mutant C4da MARCM clones showed similar dendritic morphology to the wt clones at the onset of metamorphosis. However, at 18 hr APF, the mutant clones consistently retained intact, nonsevered larval dendrites. Thus, the UbcD1 E2 ubiquitin-conjugating enzyme is required for proper UPS-mediated dendrite pruning in C4da neurons during metamorphosis (Kuo, 2006).

UbcD1, encoded by the gene effete, regulates UPS-mediated degradation of the antiapoptotic protein DIAP1 (Treier, 1992; Wang, 1999; Ryoo, 2002). In protecting cells from apoptosis, the DIAP1 E3 ubiquitin ligase antagonizes Dronc caspase activity by regulating ubiquination and degradation of the Dronc protein. Following apoptotic stimuli, UbcD1 mediates self-ubiquination and degradation of DIAP1, allowing for subsequent Dronc caspase activation. The biochemical and genetic interactions between these molecules are well established. The baculovirus p35, which is commonly used to inhibit caspase activity in Drosophila, and does not block C4da neuron dendrite pruning (Kuo, 2005). This may seem to make the involvement of caspases in this process unlikely; however, p35 has only limited activity against the caspase Dronc. To study the effects of dronc mutation on C4da neuron dendrite pruning, two null alleles of Dronc, dronc51 and dronc11, were used. MARCM analysis of dronc mutant clones revealed that the dendrites of mutant C4da neurons appeared normal at larval stages. However, unlike wt clones, without Dronc these neurons failed to properly prune their larval dendrites during metamorphosis, and most showed relatively intact primary and secondary larval dendritic arbors at 18 hr APF. These results show that severing of primary larval dendrites from C4da neurons during early metamorphosis requires the Dronc caspase (Kuo, 2006).

During apoptosis, Dronc activation requires the degradation of the antiapoptotic/anticaspase protein DIAP1, which is downstream of UbcD1. The requirement of UbcD1 for C4da neuron larval dendrite pruning during metamorphosis, together with the finding that Dronc caspase activity is also essential, raised the question of whether UPS-mediated DIAP1 degradation is a key step that allows for the severing of larval dendrites. Because loss of DIAP1 function causes C4da neuron cell death prior to the onset of metamorphosis, this question was approached using a gain-of-function allele of diap1, diap16-3s, which has a single amino acid mutation that makes DIAP1 an inefficient substrate for UPS-mediated degradation. ppk-EGFP was crossed into the gain-of-function mutant background and live imaging was used to follow C4da neuron dendrite pruning during metamorphosis. The diap16-3s mutation did not significantly affect the ability of C4da neurons to elaborate larval dendrites. However, unlike wt C4da neurons that completely pruned their larval dendrites by 18 hr APF, C4da neurons in the diap16-3s gain-of-function mutants failed to efficiently sever larval dendrites at 18 hr APF. These results suggest that the degradation of DIAP1 during early metamorphosis is required for proper C4da neuron larval dendrite pruning. Quantitatively, mutations in the UPS pathway that modulate Dronc activity (diap16-3s and ubcD1) resulted in less severe dendrite pruning defects than dronc mutants, both in terms of total number of large dendrites attached to soma and in the length of the longest attached dendrite at 18 hr APF (Kuo, 2006).

The UbcD1-DIAP1-Dronc pathway in apoptosis is well established. Thus, it may be necessary for C4da neurons to restrict the action of this pathway to specific cellular locations in order to prune unwanted dendrites without triggering apoptosis. To address this possibility, the subcellular distribution of DIAP1 and Dronc proteins was examined in ppk-EGFP C4da neurons. During the transition from third instar larvae to white pupae at the onset of metamorphosis, as well as 2 hr APF, there was a consistent induction of nuclear DIAP1 in GFP-labeled C4da neurons. During the same period a concurrent decrease was detected in Dronc staining in the soma of C4da neurons, unlike those from the neighboring cells at 2 hr APF. These results are consistent with previous observations that C4da neurons survive through this stage of metamorphosis. However, the level of antibody staining made it difficult to monitor the distribution of DIAP1 and Dronc within the dendritic structures of the C4da neurons. Because overexpression of Dronc caused C4da neuron to undergo apoptosis prior to metamorphosis, it was not possible to use GFP-tagged Dronc to examine its distribution in these neurons during pupariation. It was therefore necessary to search for alternative means to visualize activated Dronc or its downstream caspases (Kuo, 2006).

An antibody generated against activated mammalian caspase 3 has been shown to be effective in recognizing activated caspases in Drosophila. Whereas this antibody reportedly recognizes the Drosophila effector caspase Drice, it may also cross react with other activated Drosophila caspases such as Dronc during tissue staining, because of similarities in the sequences of these caspases in the region corresponding to the peptide used to generate this antibody. Therefore this antibody was used to determine whether activated caspase is localized to the dendrites of C4da neurons during the initial severing event. At 4 hr APF, just prior to dendrite severing, antibody staining for activated caspase was consistently observed within the proximal larval dendrites of C4da neurons, especially within dendritic swellings. In the diap16-3s gain-of-function mutant that inhibits Dronc activity, as well as in ubcD1 and dronc mutant MARCM clones, C4da neurons did not show dendritic swellings or activated caspase staining in dendrites during early metamorphosis. Consistent with previous observation that C4da neurons do not remodel their axons during concurrent dendrite pruning, no activated caspase staining was seen within the axons of C4da neurons during dendrite severing. Since overexpression of p35 in these neurons did not block dendrite pruning (Kuo, 2005), it is believed this antibody staining likely recognizes activated Dronc directly or recognizes a p35-resistant caspase that is activated by Dronc. These results show that, concurrent with the nuclear upregulation of DIAP1 in C4da neurons that prevents apoptosis, there is a local activation of caspases in the dendrites, likely as a result of UPS-mediated degradation of DIAP1. The spatially restricted activation of caspases then allows the severing of proximal larval dendrites from the soma (Kuo, 2006).

This study has shown that the UPS regulates pruning of larval dendrites from C4da neurons in a cell-intrinsic manner. To better understand the molecular pathways regulating UPS-mediated pruning, a candidate E2/E3 ubiquitinating enzyme screen was conducted. In this screen an E2 ubiquitin-conjugating enzyme mutation in was uncovered ubcD1, causing dendrite pruning defects. Taken together with the extensive biochemical characterization of interactions between UbcD1, DIAP1, and Dronc, this study suggests that in C4da neurons, UPS activation leads to UbcD1-mediated degradation of E3 ubiquitin ligase DIAP1, thereby allowing Dronc caspase activation and the subsequent cleavage of larval dendrites. This work not only identifies a set of E2/E3 ubiquitinating enzymes regulating neuronal process remodeling, it also links the UPS to a hitherto unappreciated mechanism for local caspase activation in dendrites during Drosophila metamorphosis (Kuo, 2006).

The mechanistic link between the UPS and caspase activity in regulating C4da neuron dendrite pruning is unexpected. Although the UPS is known to regulate remodeling and degradation of neuronal processes, it is generally believed that this process is accomplished by degradation of cellular proteins (such as microtubules and neurofilaments) that are required to keep dendrites and axons intact. However, it was found that the UPS in C4da neurons is in fact causing the degradation of an E3 ligase, DIAP1, thereby allowing for subsequent dendrite pruning. In this case, UPS-mediated degradation of a protein does not in and of itself lead to a structural compromise in dendrites, but rather it leads to the activation of another protease that executes dendrite pruning. This two-step activation cascade, which involves both the UPS and the apoptotic machinery, may provide an additional level of control and flexibility that would not be possible if UPS alone regulated the pruning program. After all, these C4da neurons, which are specified during fly embryogenesis, maintain a highly elaborate dendritic field to receive sensory inputs throughout larval development, which lasts for several days. The maintenance of these dendrites over time requires a network of finely tuned cell-intrinsic and -extrinsic pathways. Just as important, the dendritic pruning program enables dramatic neuronal remodeling in response to profound environmental changes during metamorphosis. It is conceivable that C4da neurons evolved this dual control mechanism to prevent any accidental triggering of dendrite pruning prior to metamorphosis. Initiation of C4da neuron dendrite pruning requires cell-intrinsic ecdysone signaling, and ecdysone receptors have been shown to regulate Dronc expression. It will be of interest to determine how this UPS/caspase dendritic pruning pathway is related to the ecdysone signaling cascade (Kuo, 2006).

During metamorphosis, C4da neurons upregulate DIAP1 expression in the nucleus, which is consistent with this class of neurons surviving early stages of the metamorphosis (only one of the three C4da neurons per hemisegment, the ventral neuron, is lost at a later stage of pupariation). Remarkably, there are activated caspases within the dendrites prior to severing, and a gain-of-function diap1 mutation can block dendrite pruning, strongly implicating a local dendritic program that can activate caspases without causing apoptosis of the neuron. Although mutations in both the Dronc caspase and the UPS pathway that modulate Dronc activity (UbcD1 and DIAP1) result in retention of larval dendrites, their dendrite pruning defects differed somewhat quantitatively. Compared to dronc mutants, diap1 gain-of-function and especially ubcD1 mutants showed less retention of larval dendrites during metamorphosis. This is not surprising for diap1 gain-of-function, as it is an effective Dronc inhibitor but unlikely to be 100% efficient. UbcD1, as an E2 ubiquitin-conjugating enzyme, has wider substrate specificity than E3 ligases. Previous study showed that UbcD1 is involved in mushroom body neuroblast proliferation, so it may be involved in other UPS-mediated pathways during dendrite pruning. It is also conceivable that in the absence of UbcD1 another E2 may trigger a low level of DIAP1 degradation, allowing residual Dronc activation which results in a milder dendrite pruning phenotype in ubcD1 mutants. It is currently unclear whether UbcD1 is also required during DIAP1-mediated degradation of Dronc. However, pruning defects in the ubcD1 mutants suggest that it may not be absolutely required, since undegraded DIAP1 continues to inhibit Dronc, presumably via interaction with another E2 protein (Kuo, 2006).

How is the specificity of dendrite pruning achieved? Several possible mechanisms are proposed: first, C4da neurons do not change their axonal projections during dendrite pruning, so there could be dendrite-specific trafficking of components of the UPS, such as UbcD1, and/or the caspase Dronc. Of the known proteins that are preferentially trafficked to dendrites, these molecules have not been implicated but warrant further investigation. Second, it is also possible that activated Dronc, or another p35-resistant protease activated by Dronc, could cleave a dendrite-specific substrate. Examples are now emerging from other cellular systems, such as in sperm formation and border cell migration, in which caspases can participate in cleavage of proteins not resulting in apoptosis. Third, the dendritic pruning program takes place during drastic environmental changes that include concurrent degradation and regrowth of the overlying epidermis, activation of extracellular matrix metalloproteases, and blood phagocytes. These environmental cues likely complement the neuronal intrinsic pruning programs, but their exact relationships are not known. Experiments addressing these and other possible mechanisms should provide a greater insight into how the large-scale remodeling of C4da neuron dendrites is achieved (Kuo, 2006).

In vertebrates, the UPS pathway has been implicated in Wallerian degeneration of severed axons. In the fly, mushroom body γ neurons undergo extensive remodeling of their processes during metamorphosis. The initial stages of axon pruning in these mushroom body neurons closely resemble Wallerian degeneration, and the UPS again plays a critical role. To date, the specific ubiquitin-conjugating enzymes and ligases that mediate target protein degradation have not been identified in these systems. It will be interesting to see whether the UbcD1-DIAP1-Dronc pathway implicated in C4da neuron dendrite pruning also participates in remodeling/degradation of neuronal processes in other systems. It seems likely that more than one pathway would be employed in remodeling different neurons; a previous study excluded UbcD1 as a possible ubiquitin-conjugating enzyme regulating mushroom body γ neuron remodeling, and normal remodeling of mushroom body neuron processes in is seen dronc mutant MARCM clones during metamorphosis (Kuo, 2006).

A multilayered regulatory machinery for remodeling neurons, as uncovered in this study for C4da neurons, offers versatility and flexibility. It is conceivable that another ubiquitin ligase/caspase pair may function in an analogous UPS pathway during mushroom body neuron remodeling, potentially affording differential regulation of neuronal remodeling. Although pharmacological inhibition of mammalian caspases showed no effect on Wallerian degeneration, it would be important to assess the in vivo effectiveness of the inhibitors against a comprehensive panel of caspases. Moreover, a dual control mechanism, similar to what is proposed for C4da neuron remodeling, may coordinately regulate UPS and another protease that executes axon degradation. Conceivably, instead of having the target of the UPS directly involved in maintaining dendrite/axon stability, the executor of neuronal process degradation may involve a different protease: in the case of C4da neurons it is the caspase Dronc, and in Wallerian degeneration the relevant protease might be the Ca2+-responsive calpain. Future experiments along these lines of thinking may accelerate the identification of specific ubiquitinating enzymes involved in other areas of developmental neuronal remodeling and in diseases where the UPS pathway has been implicated. As target-specific E3 ligases are excellent candidates for pharmaceutical intervention, this approach may also help to find effective treatments for developmental and neurodegenerative diseases that involve degeneration of neuronal processes (Kuo, 2006).

Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It was not clear that how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. This study found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in the reconstitution assay. However Grim did not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2 (Yoo, 2005).

Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif. IAPs bind to caspases and inhibit their activity, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Drosophila IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. Rpr, but not Hid (Head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, ubcD1 was found to suppresses rpr-induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro. These results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting DIAP1 degradation (Ryoo, 2002).

To investigate how DIAP1 is regulated, a polyclonal antibody against recombinant DIAP1 protein was generated that detects a single band on western blots. In whole-mount staining of larval imaginal discs, it was noted that the amount of DIAP1 varied between some tissues. Notably, in wing imaginal discs, a strip of cells showed increased anti-DIAP1 labelling. These cells constitute the dorso-ventral (D-V) boundary, as demonstrated by double labelling with the wingless (wg)-lacZ marker. Consistent with the anti-DIAP1 antibody labelling pattern, imaginal discs from thj5c8 animals, which have a P[lacZ] insertion in the 5' untranslated region of the diap1 transcription unit, showed stronger anti-β-galactosidase labelling along the D-V boundary. It is concluded that in the wing imaginal disc, thj5c8 P[lacZ] reporter expression reflects diap1 transcription (Ryoo, 2002).

To determine if DIAP1 degradation is also regulated in Drosophila, rpr and the baculovirus p35 caspase inhibitor were co-expressed in the wing imaginal disc posterior compartment using the engrailed (en)-Gal4 driver. Expression of p35 prevented rpr-expressing cells from dying, and the resulting 'undead' cells enabled observation of cellular changes downstream of rpr. Whereas animals expressing rpr alone died as embryos, those co-expressing rpr and p35 survived to adulthood with largely normal wings. rpr-induced cell death was completely blocked by p35, as assayed by TUNEL staining of wing imaginal discs. Wing imaginal discs were also labelled with the CM1 antibody, which detects activated caspase-3 in humans and cross-reacts with activated caspases in Drosophila. In the control anterior compartment, sporadic apoptotic cells were intensely labelled with CM1. In contrast, all undead cells of the posterior compartment showed low but consistent levels of CM1 labelling. Whereas in apoptotic cells, CM1 labelling was detected throughout the cell body, in undead cells, CM1 labelling was detected only in the cytoplasm, indicating that the nuclear membrane was intact and cells were not dying (Ryoo, 2002).

Interestingly, cells expressing both rpr and p35 had reduced anti-DIAP1 labelling, compared to the wild type cells of the anterior compartment. Expression of p35 alone did not affect the amount of diap1 expression. Unlike rpr, co-expression of hid and p35 did not cause obvious DIAP1 downregulation. The different effects of hid and rpr on DIAP1 downregulation might partly account for different cell killing properties (Ryoo, 2002).

The reduction in DIAP1 levels by Rpr was not caused by reduced transcription; diap1-lacZ expression in thj5c8 mutants actually increased in these cells. As Rpr can bind directly to DIAP1, the result suggests that Rpr-DIAP1 complex formation triggers DIAP1 downregulation through a post-transcriptional mechanism and that this occurs independently of caspase activity (Ryoo, 2002).

A number of diap1 mutant alleles exist that disrupt the RING domain of DIAP1. Two such diap1 mutant alleles, diap133-1S and diap122-8S, were used to address the requirement of the RING domain for rpr-dependent DIAP1 degradation (Ryoo, 2002).

Homozygous or trans-heterozygous combinations of these alleles are embryonic lethal, indicating that the RING domain provides an essential function for DIAP1. Thus, the effect of the RING finger mutations on rpr-dependent DIAP1 degradation was measured in embryos. The protein distribution of DIAP1 was analysed in embryos that express rpr under the prd-Gal4 driver. In wild type embryos, DIAP1 was homogenously distributed. However, if rpr expression was induced in a prd-like expression pattern, DIAP1 protein levels decreased in exactly those domains where rpr was expressed. Co-expression of p35 did not affect the decrease of DIAP1 protein levels, suggesting that it occurs upstream and independently of caspase activation (Ryoo, 2002).

To address the importance of the RING domain for the decrease in DIAP1 levels, rpr was expressed in a RING domain mutant background using the diap133-1S and diap122-8S alleles. Under this experimental condition, rpr was no longer able to downregulate DIAP1. These observations suggest that the reduction of DIAP1 by Rpr is caused by auto-ubiquitination-mediated protein degradation (Ryoo, 2002).

GMR-hid and GMR-rpr animals ectopically express hid or rpr in the developing eye discs. Such expression triggers cell killing and is sensitive to the dosage of other cell-death-related genes, such as diap1. To identify additional factors that regulate DIAP1 auto-ubiquitination, animals were sought that were heterozygous for mutations in the ubiquitin pathway that enhanced or reduced GMR-hid or GMR-rpr induced cell killing. Several dominant enhancers of GMR-rpr, including Pros26, a proteasome subunit, fat facet, a de-ubiquitinating enzyme and Drosophila bruce, encoding a ubiquitin-conjugating enzyme. None of these mutations enhanced GMR-hid-induced apoptosis (Ryoo, 2002).

Mutations in genes that are required for Rpr-mediated DIAP1 degradation are expected to dominantly suppress GMR-rpr. Inly one such suppressor, ubcD1, also known as effete, was found. ubcD1 encodes a 147-amino-acid ubiquitin-conjugating enzyme that is similar to the mammalian ubcH5 class. The dominant suppression of GMR-hid and GMR-rpr was observed with two independent alleles, ubcD1598 and ubcD1D73. Interestingly, ubcD1 was previously identified as a genetic interactor of sina, a gene encoding a RING domain with high sequence similarity to the DIAP1 RING domain (Neufeld, 1998). To test if ubcD1 interacted genetically with the RING domain of diap1, ubcD1 flies were crossed to GMR-diap1-RING flies, which overexpress the carboxy-terminal part of DIAP1, including the RING domain. GMR-diap1-RING induces cell death and results in small eyes that lack pigment cells. In ubcD1-/+ animals, the eye phenotype caused by GMR-diap1-RING was suppressed and more pigment cells survived (Ryoo, 2002).

The dominant suppression of GMR-rpr, GMR-hid and GMR-diap1-RING seems to be specific to ubcD1, as mutations in lesswright (lwr; a ubc9 homologue) and ubcD2 (a ubc4 homologue) had no effect on GMR-rpr, GMR-hid or GMR-diap1-RING animals. ubcD2 has high sequence similarity to ubcD1, and both genes have been shown to functionally substitute for ubc4 in yeast. Therefore, the results indicate a striking level of specificity among similar ubiquitin-conjugating enzymes in vivo (Ryoo, 2002).

hid and rpr induce apoptosis through dronc, a Drosophila caspase-9 homologue. GMR-dronc induces apoptosis during late pupal stages, resulting in a loss of pigment cells within normal-sized eyes. ubcD1 did not dominantly suppress GMR-dronc activity, indicating that it functions downstream of rpr, but upstream of dronc. In fact, ubcD1 slightly enhanced GMR-dronc activity, raising the possibility that Dronc may be an additional target for ubcD1-mediated ubiquitination and degradation. Taken together, these genetic interaction assays suggest that UBCD1 is an E2 ubiquitin ligase for DIAP1 (Ryoo, 2002).

Ubiquitin ligases physically interact with their cognate ubiquitin-conjugating enzymes to transfer ubiquitin to their substrates. To test if UBCD1 directly interacts with DIAP1, a glutathione S-transferase (GST) pull-down assay was performed with 35S-methionine-labelled DIAP1. Consistent with a function for UBCD1 as an E2 ubiquitin ligase for DIAP1, 35S-DIAP1 was retained by UBCD1-GST. Interestingly, UBCD2, a ubiquitin-conjugating enzyme with high sequence similarity to UBCD1, did not bind DIAP1 in this assay. These results demonstrate that the UBCD1-DIAP1 interaction is specific, and consistent with the observation where ubcD1, but not ubcD2, genetically interacts with GMR-rpr (Ryoo, 2002).

The observed affinity between UBCD1-GST and DIAP1 was comparable to the reported interaction between DIAP1 and Hid. RprδN-GST, which lacks the RHG motif at the N terminus, bound poorly to DIAP1. It is concluded that DIAP1 interacts specifically with Rpr, Hid and UBCD1 in vitro (Ryoo, 2002).

To determine if DIAP1 auto-ubiquitination is directly mediated by UBCD1, the ubiquitination reaction was reconstituted in vitro using recombinant E1, UBCD1 and 35S-labelled DIAP1. Ubiquitination of DIAP1 was assayed by the appearance of higher molecular weight bands on SDS-polyacrylamide gel electrophoresis (PAGE) gels. Minor ubiquitination of 35S-DIAP1 was observed in the absence of UBCD1, caused by basal ubiquitin-conjugating enzyme activity in reticulocyte lysates. The addition of UBCD1 moderately stimulated polyubiquitin chain formation on 35S-DIAP1. When in vitro translated Rpr-GST was added to the reaction in increments, stronger DIAP1 ubiquitinating activity was observed. Such stimulation was not caused by any background activity in the S30 extract itself, where Rpr-GST was translated (Ryoo, 2002).

To examine if DIAP1 itself was the source of the ubiquitin ligase activity, the reaction was repeated with 35S-DIAP121-4S, which has an inactivating mutation in the RING domain. 35S-DIAP121-4S was not ubiquitinated by UBCD1 and Rpr. These results demonstrate that UBCD1 physically and functionally interacts with the DIAP1 RING domain for DIAP1 auto-ubiquitination, and Rpr stimulates this DIAP1 auto-ubiquitinating activity. Whereas many proteins stimulate ubiquitination by bringing ubiquitin ligases in proximity to the substrates, the ability of Rpr to stimulate the ubiquitin ligase activity itself represents a novel mechanism to regulate ubiquitination (Ryoo, 2002).

Compared to UBCD1, UBCD2 was significantly less efficient in ubiquitinating DIAP1 in vitro. When the ubiquitinated DIAP1 bands were quantified by phosphorimager, it was found that DIAP1 was ubiquitinated four to sixfold less with UBCD2. Likewise, UBCD2 was less efficient in mediating DIAP1 ubiquitination in the presence of Rpr. These results indicate that UBCD1 shows specificity in promoting DIAP1 ubiquitination in vitro, and is consistent with the results from the genetic and physical interaction assays (Ryoo, 2002).

Whether Hid might have a similar stimulating activity for DIAP1 auto-ubiquitination was examined. Whereas purified Rpr-GST stimulated DIAP1 ubiquitination, purified Hid-GST, which binds DIAP1 with high affinity, was not able to stimulate DIAP1 auto-ubiquitination in vitro. In fact, addition of Hid-GST inhibited the background DIAP1 ubiquitinating activity. This indicates that the stimulation of DIAP1 auto-ubiquitination is not a simple consequence of RHG proteins binding to DIAP1 BIR domain. Furthermore, this in vitro assay is consistent with the observation that hid does not promote DIAP1 downregulation in wing imaginal discs (Ryoo, 2002).

To examine the function of ubcD1 in DIAP1 degradation in vivo, mosaic animals were generated with ubcD1-/- cells in the context of a largely ubcD1-/+ animal by using the Flipase/FRT system. Flipase driven with the eyeless promoter (ey-flp) created small ubcD1-/- clones in eye imaginal discs. A subset of ubcD1-/- cells labelled more strongly with the anti-DIAP1 antibody, whereas elevated levels of DIAP1 were never detected in wild type cells. This is consistent with a function of ubcD1 in promoting DIAP1 degradation (Ryoo, 2002).

Each Drosophila sensory hair (macrochaete) represents a sensory neuron. Drosophila adults have an invariant pattern of sensory cells in the thorax, with four macrochaetes in the scutellum. Therefore, the number of macrochaetes has been used as an indicator for defects in cell death or survival. The control of apoptosis partly contributes to the regulation of the number of macrochaetes, as expression of p35 with pannier (pnr)-Gal4 driver results in extra macrochaetes in 30% of animals. UAS-bcl-2 flies crossed with pnr-Gal4 produced similar results. Neither of the two parent lines, pnr-Gal4 nor uas-p35, had extra macrochaetes (Ryoo, 2002).

Viable adults were obtained with a trans-hetero-allelic combination of ubcD1D73/ubcD1D112, albeit with low penetrance. Interestingly, 21% of the surviving adults had extra macrochaete, similar to UAS-p35; pnr-Gal4 adults. Examination of ubcD1598/ubcD1D112 adults gave similar results. These results demonstrate that ubcD1 affects the number of macrochaetes in adults, and are consistent with a function for ubcD1 in apoptosis (Ryoo, 2002).

To test if overexpression of ubcD1 is sufficient to promote DIAP1 degradation, ubcd1 was overexpressed in en-Gal4/UAS-ubcD1 animals. No developmental abnormalities were observed in these larvae, as examined during late third instar larval stage. DIAP1 protein level remained unchanged in cells expressing ubcd1, as compared to its neighbouring control cells of the anterior compartment. It is concluded that ubcD1 is not sufficient to promote DIAP1 degradation when expressed on its own. It is speculated that additional factors, such as Rpr, may be required to ubiquitinate DIAP1 in vivo (Ryoo, 2002).

Loss-of-function diap1 mutants have been shown to undergo massive apoptosis, whereas overexpression of diap1 suppresses cell death. Furthermore, a 50% reduction of DIAP1 dosage in diap1-/+ animals strongly sensitizes cells to apoptosis, indicating that the amount of DIAP1 in a cell determines the threshold to cell death. This study has demonstrated that the amount of DIAP1 is actively regulated during Drosophila development and apoptosis. Transcriptional regulation of diap1 was observed during development and post-transcriptional regulation of DIAP1 during the course of apoptosis. This study focused on the post-transcriptional regulation of DIAP1, since it has important conceptual and physiological significance. As apoptosis culminates in morphological changes, including DNA fragmentation, transcriptional regulation would not be an effective means of controlling apoptosis after caspase activation. Furthermore, degradation of IAPs would ensure the irreversibility of the apoptotic process, as it would preclude residual IAP binding to activated caspases. This is especially important in metazoans, where cell survival after DNA fragmentation can result in the propagation of genetic aberrations that are harmful to the organism (Ryoo, 2002).

This study found that rpr promotes DIAP1 degradation. Rpr-mediated DIAP1 degradation is likely to be upstream of caspase activation, since blocking caspases with p35 maintained the ability of rpr to downregulate DIAP. The following pieces of evidence indicate that rpr degrades DIAP1 by ubiquitin-mediated protein degradation. First, whereas DIAP1 is downregulated, the transcription of diap1-lacZ is upregulated, precluding the possibility of a transcriptional downregulation. Second, in diap1 RING mutant embryos, DIAP1 is not degraded by rpr. Third, it was possible to reconstitute the DIAP1 auto-ubiquitinating assay in vitro. In this assay, the DIAP1 RING domain and E2 ubiquitin ligase activity of UBCD1 is essential for DIAP1 auto-ubiquitination. Interestingly, Rpr is able to stimulate the auto-ubiquitinating activity of DIAP1 in vitro. Such stimulating activity of a ubiquitin ligase has not been reported to date and provides a novel mechanism to regulate ubiquitination. Fourthly, UBCD1 is an E2 ubiquitin-conjugating enzyme that genetically and physically interacts with DIAP1. The DIAP1-UBCD1 interaction is specific, as similar ubiquitin-conjugating enzymes, such as UBCD2, do show a genetic or physical interaction with DIAP1. Surprisingly, ectopic hid expression produced only a minor effect on DIAP1 levels. Instead, the subcellular distribution of DIAP1 seemed to be modified by hid, but remains to be characterized in detail. Consistently, Hid does not stimulate DIAP1 auto-ubiquitination in vitro. Taken together, in addition to its DIAP1 binding activity, Rpr has an additional function of stimulating DIAP1 auto-ubiquitination and thereby degrading DIAP1 during apoptosis. As Hid does not stimulate DIAP1 degradation, it is likely that stimulating DIAP1 ubiquitination is not a simple consequence of binding to the DIAP1 BIR domains. Rather, it is likely that Rpr contains a specific motif that mediates its E3-stimulating activity (Ryoo, 2002).

Genetic studies on Drosophila diap1 have previously identified alleles with inactivating mutations in the diap1 RING domain, which is predicted to specifically abolish its ubiquitin ligase activity. These alleles enhanced GMR-rpr-induced eye ablation, whereas they suppressed GMR-hid. Superficially, these reports seem to be in conflict with a pro-apoptotic function of the DIAP1 RING domain in auto-ubiquitination and self-degradation in response to Rpr. It is believed that the reason for this apparent discrepancy is probably based on the fact that DIAP1 targets multiple apoptotic regulators for ubiquitination, both pro- and anti-apoptotic, and the genetic interaction assays reflect a mere net effect under a given experimental condition. An accompanying paper, shows that the caspase Dronc is also ubiquitinated by DIAP1, and this seems to contribute to the RING domain mutant phenotype (Wilson, 2002). This can account for the partial loss-of-function phenotype of diap1 RING alleles, which show excessive apoptosis and die as advanced embryos. Under such conditions, the ability of DIAP1 to ubiquitinate pro-apoptotic proteins seems to override the potential to self-destruct. Nevertheless, this study demonstrates that the ability of DIAP1 to self-degrade in cells that are doomed to die seems to be an important and integral part of apoptosis regulation in Drosophila. It is proposed that Rpr-mediated DIAP1 degradation facilitates apoptosis by removing the 'brake on death', and that it might be possible to exploit this mechanism for therapeutic purposes (Ryoo, 2002).

It is interesting to note the parallels between the nature of mammalian thymocyte apoptosis and Drosophila metamorphosis. In mammalian thymocytes, IAP degradation can be triggered by glucocorticoids, but the genes that mediate glucocorticoid-induced IAP degradation are not known. During Drosophila metamorphosis, the steroid hormone ecdysone triggers larval tissues to undergo apoptosis. Ecdysone receptors activate, among other genes, rpr and hid, accounting for the cell killing activity of ecdysone. Based on such similarity between mammals and Drosophila, it is speculated that, in thymocytes, glucocorticoids might activate a Rpr-like protein before IAP degradation and apoptosis. It remains to be determined whether Smac/DIABLO or HtrA2, the only known mammalian RHG proteins to date, promote IAP degradation (Ryoo, 2002).

Significantly, the implications from this study may improve strategies against human pathological conditions, such as cancer. It has been shown that most cancer cells express high levels of IAPs, such as XIAP. At least in some cases, this seems to account for the increased survival ability of tumour cells. It is suggested that molecules that combine BIR-binding and E3-ligase-stimulating activities will be useful for the selective reduction of IAP protein levels and may have the potential to selectively kill cancer cells. Although the feasibility of generating small-molecule drugs with such properties remains to be seen, the finding that the 65-amino-acid protein Rpr can stimulate degradation of DIAP1 in Drosophila provides a conceptual framework for designing a novel class of anti-cancer drugs (Ryoo, 2002).

Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2n cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. The cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. These observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production (Lilly, 2000).

The fusome occupies a key location within growing cysts for mediating synchrony. By the onset of mitosis at each round of cyst division, the fusome has completed its cycle of growth and new segments have fused into a single continuous structure. The branches of the G2 fusome pass uninterrupted through the ring canals that interconnect the cystocytes, providing a single internal interface between all cyst cells. Synchronous cell cycles occur only while the fusome is present. Cell-cell junctions alone are insufficient to guarantee synchrony, since ring canals remain present in older cysts when the cells cycle asynchronously. Also, genetic data support a role for the fusome in cystocyte synchrony. Mutations that disrupt the fusome disrupt synchrony, as indicated by the production of cysts that no longer contain 2n cells (Lilly, 2000).

These studies suggest that the fusome affects cystoctye synchrony through an interaction with CycA. CycA associates with the fusome only in synchronously cycling cystocytes, and not in 16-cell cysts. CycA-fusome association begins in G2 when CycA levels on the fusome rise above those observed in the rest of the cyst. By early prophase CycA levels on the fusome are much higher than in the surrounding cytoplasm. As mitosis progresses CycA is seen at high levels throughout the cyst before being destroyed at approximately the metaphase-to-anaphase transition. The accumulation of CycA on the fusome prior to the activation of CycA/Cdk1 at the G2/M transition suggests that CycA association with the fusome synchronizes the entry of all the interconnected cystocytes within a cyst into mitosis (Lilly, 2000).

How might CycA effect mitotic synchrony through its association with the fusome? The fusome most likely acts in some way to spatially equalize the activation of CycA/ Cdk1 activity in all cystocytes during late G2. In yeast, Cyc/Cdk activation is controlled by changes in the levels of stimulatory and inhibitory phosphorylation at specific target sites. The phosphatases and kinases carrying out these changes are themselves subject to control by cdk-mediated phosphorylation to generate a strong positive feedback loop. It is proposed that enzymes responsible for CycA/Cdk1 activation are present in the fusome and are subject to similar positive feedback control. Activation of CycA/Cdk1 activity in any portion of the fusome might spread to adjacent molecules, causing a wave of activation to spread rapidly throughout the fusome. The ability of the fusome to propagate an active state of CycA/Cdk1 would synchronize the mitotic entry of all cystocytes that retained an intact fusome (Lilly, 2000).

A genetic screen to identify components of the sina signaling pathway in Drosophila eye development

Specification of the R7 photoreceptor cell in the developing Drosophila eye requires the seven in absentia (sina) gene. Ectopic expression of sina in all cells behind the morphogenetic furrow disrupts normal eye development during pupation, resulting in a severely disorganized adult eye. Thirteen independent sina transformant lines were isolated, each of which displayed eyes with abnormal exteriors, ranging from a slight roughening of the ommatidial lattice to a gross disruption of normal eye morphology. This range in phenotype is most likely due to variances in expression among the lines, since making each line homozygous results in a stronger phenotype. Eyes from strong sina overexpressing lines are notably smaller and less pigmented than wild-type, and a fusion of ommatidial surfaces results in a glazed cuticle covering the eye. Microscopic examination of sections through such eyes reveals corresponding abnormalities in the underlying retinal cells. In these lines, retinal patterning appears severely disrupted, and no normal ommatidia were identified. However, no cell types appeared to be lacking, as judged by the presence of pigment granules (pigment cells), lens structures (cone cells), rhabdomeres (photoreceptor cells) and bristles. Sections through eyes from the weaker GMR-sina lines with mild exterior phenotypes display more subtle defects, including defective ommatidial rotation, lattice disorganization, and occasional missing photoreceptors. In no case are extra R7 photoreceptor cells observed, indicating that misexpression of sina in uncommitted cells in the eye disc is insufficient to direct them into a neuronal program of development (Neufeld, 1998).

A genetic screen for dominant enhancers and suppressors of this phenotype has identified mutations in a number of genes required for normal eye development, including UbcD1, which encodes a ubiquitin conjugating enzyme; SR3-4a, a gene previously implicated in signaling downstream of Ras1, and a Drosophila homolog of the Sin3A transcriptional repressor (see Drosophila Sin3A). The genetic interaction between sina and UbcD1 presented here, as well as the demonstration of physical interaction between Sina and UBCD1, provides a molecular framework for beginning to understand how Sina may regulate the stability of proteins such as Ttk. Members of the Sin3 class of transcriptional corepressors serve as requisite components of the Mad-Max repressor complex. A Drosophila member of this family, Sin3A, interacts genetically with sina. At present, the role played by Sin3A during development is unclear. Clones of cells homozygous for Sin3A mutations could not be recovered, suggesting that this gene is required for cell proliferation or survival. Identification of mutations in Drosophila Sin3A should contribute to an understanding of this important class of transcriptional regulators (Neufeld, 1998).

UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior

The end-to-end association of chromosomes through their telomeres has been observed in normal cells of certain organisms, as well as in senescent and tumor cells. The molecular mechanisms underlying this phenomenon are currently unknown. This study shows that five independent mutant alleles in the Drosophila UbcD1 gene cause frequent telomere-telomere attachments during both mitosis and male meiosis that are not seen in wild type. These telomeric associations involve all the telomeres of the D. melanogaster chromosome complement, albeit with different frequencies. The pattern of telomeric associations observed in UbcD1 mutants suggests strongly that the interphase chromosomes of wild-type larval brain cells maintain a Rab1 orientation within the nucleus, with the telomeres and centromeres segregated to opposite sides of the nucleus. The UbcD1 gene encodes a class I ubiquitin-conjugating (E2) enzyme. This indicates that ubiquitin-mediated proteolysis is normally needed to ensure proper telomere behavior during Drosophila cell division. It is therefore suggested that at least one of the targets of UbcD1 ubiquitination is a telomere-associated polypeptide that may help maintain proper chromosomal orientation during interphase (Cenci, 1997).

Selective protein degradation by the ubiquitin-proteasome pathway has a crucial role in many cellular regulatory mechanisms, including those that govern the cell cycle. Ubiquitin-mediated proteolysis is a multistep process. Ubiquitin is first linked by a thiol ester bond to a ubiquitin-activating, or El, enzyme. Activated ubiquitin is then transferred to a ubiquitin-conjugating, or E2, enzyme. Finally, this enzyme, often in cooperation with the accessory factor ubiquitin ligase, or E3, donates ubiquitin to a substrate protein. Repeated ubiquitination cycles lead to multiubiquitinated proteins that are degraded rapidly by the 26S proteasome. Most cells have several E2 enzymes and an unknown number of E3 factors, leading to the suggestion that the E2 and E3 factors may interact combinatorially to determine substrate specificity (Cenci, 1997).

The UbcD1 gene encodes a class I E2 enzyme. Class I E2 proteins, such as the UbcDl protein, contain only the I6-kD UBC domain common to E2 proteins. Class I E2 enzymes function poorly in vitro in the absence of an E3 factor. Other E2 enzymes contain additional sequences at either the carboxyl terminus (class II) or amino terminus (class III) and often carry out efficient ubiquitin conjugation without the addition of E3s (Cenci, 1997).

The UbcDl protein exhibits a strong homology with the S. ceievisiae E2 enzymes UBC4 and UBC5, which perform overlapping functions along with UBC I. Deletion of the gene for one of these enzymes results in no evident phenotype; deletion of two of the three renders yeast sensitive to a variety of stresses; deletion of all three is lethal. The Drosophila UbcDl-coding sequence has been transformed into yeast under the control of the yeast UBC4 promoter. This construct rescues a number of defects of yeast UBC4 UBC5 double mutants, restoring ubiquitin-mediated proteolysis of abnormal polypeptides. It is therefore quite likely that UbcDl mediates ubiquitin-dependent proteolysis in flies (Cenci, 1997).

Little is known about the substrates of class I E2 enzymes. UBC4 and UBC5 act synergistically with two other E2 enzymes to target the yeast MATa2 protein for degradation. Either the human UbcHSB and UbcHSC enzymes, or the related A. thaliana E2 enzyme AtUBCS, can in the presence of E6- E6AP complex (which acts as an E3 factor), mediate p53 ubiquitination. Cytological observations of phenotypes associated with five independent UbcDl mutant alleles show clearly that UbcDl is required for proper telomere behavior. However, at least in the male germ line, UbcDl is likely to have additional functions. The leaky mutants effl, eff3, and eff8 do not exhibit abnormal telomere behavior during male meiosis, but are nonetheless sterile. This suggests that UbcDl is also needed for ubiquitination of proteins required in postmeiotic stages of spermatogenesis (Cenci, 1997).

Larval brain metaphases exhibit two types of telomere-telomere associations, double telomere associations (DTAs in which 2 sister telomeres fuse with another pair of sister telomeres) and single telomere associations (STAs; involving a single telomere that associates with either its sister or a nonsister telomere). Neither kind of chromosomal configuration results from chromosome or chromatid exchanges because they are never accompanied by acentric fragments. Nevertheless, dicentric and ring DTAs are structurally similar to mutagen-induced chromosome exchanges generated during G1, whereas STAs have the same appearance as chromatid exchanges produced during S-G2. This suggests that DTAs arise from telomere-telomere fusions that occurred during G1 and that are replicated and maintained subsequently in S-G2. STAs are likely to be the consequence of associations between telomeres of chromosomes that have replicated already (Cenci, 1997).

The formation of both DTAs and STAs requires the physical proximity of telomeres during interphase. Telomeres would be clustered at one side of the nucleus if chromosomes maintained their anaphase configuration in the subsequent interphase, as suggested by more than a hundred year ago by Rabl. In D. melanogaster, a Rabl orientation of chromosomes is evident in both polytene salivary gland nuclei and embryonic nuclei, though not in polytene chromosomes from the larval midgut, in imaginal discs, and neuroblast nuclei. To explain these findings it has been suggested that during the short embryonic interphases, chromosomes do not have sufficient time to relax from their anaphase configuration and, therefore, maintain a Rabl orientation throughout interphase. However, in later stages of development, when interphase is much longer, the chromosomes would have the time to assume a more compact arrangement within the nucleus (Cenci, 1997).

Observations on UbcDl mutants suggest that the chromosomes of actively dividing brain cells maintain a residual Rabl conformation during early interphase (i.e., the G1 phase), but progressively lose it as these cells proceed through the cycle. That chromosomes continue to be arranged in a Rabl configuration for at least part of the interphase is suggested by the finding that, in both males and females, XL is involved in DTAs with a higher frequency than XR. Because XL is nearly the same length as the arms of the large metacentric autosomes, whereas XR is extremely short, in a Rabl configuration one would expect the tip of XL to be closer to the telomeres of the major autosomes than would be the tip of XR. Moreover, two findings suggest that chromosomes relax from the Rabl orientation as brain cells proceed through the cycle. The observation that DTAs are more frequent than STAs can be interpreted as indicating that chromosome ends disperse when cells pass from G1 to S-G2. Similarly, the higher frequency of DTA rings compared with the STA rings may reflect cell-cycle-dependent variations in the Rabl conformation. During G1 the opposite telomeres of the same chromosome would be sufficiently close to interact to form DTA rings. However, the proximity of opposite telomeres would be reduced later in the cell cycle, preventing STA ring formation (Cenci, 1997).

Although the results show that telomeric associations must occur during both meiotic divisions, it is believed that these are restricted only to certain kinds of interactions. No interactions were detected between telomeres of chromosomes in different bivalents during meiosis I, nor were DTAs detected in meiotic metaphases II. The absence of telomeric associations between chromosomes in different meiotic bivalents is anticipated, because as early as prophase I, the bivalents are physically separated, which would prevent interactions between heterologous chromosomes (Cenci, 1997).

The failure of mutant metaphase II figures to show the presence of DTAs may reflect the fact that after the first meiotic division, secondary spermatocytes do not go through an S phase and are therefore unlikely to generate DTAs (Cenci, 1997).

Although most telomere-telomere associations in larval brain cells are resolved during anaphase, telomeric associations in male meiotic chromosomes fail to be resolved and give rise to chromosome breakage during anaphase. One explanation for this difference is that telomeres of meiotic chromosomes are connected more tightly than those of mitotic chromosomes. Alternatively, these cells may differ in the mechanics or speed of anaphase chromosome movement and therefore the application of forces across telomere associations during anaphase A. This is conceivable because the meiotic spindle is much larger than the mitotic spindle and the maximum distance between the separating sets of anaphase chromosomes is higher in meiosis than in mitosis. Additional support for the latter hypothesis is the finding that although ~60% of mutant meiotic anaphases exhibit lagging acentric fragments, only 2% of these cells display chromatin bridges. This suggests that meiotic anaphase bridges persist for a shorter time than those found in somatic cells, implying that meiotic anaphase A is more rapid than mitotic anaphase in brain cells. Interestingly, no evidence waa found of chromosome loss and nondisjunction during meiosis of UbcDl mutant females. One interpretation of these results is that telomere-telomere associations do occur in female meiosis but they are resolved before or during anaphase, as is the case for mitosis. Alternatively, female meiotic chromosomes might not undergo end-to-end associations. Ultrastructural studies on chromosome organization in Drosophila female pachytene nuclei support this second alternative. In these cells the pericentric regions of the chromosomes lie at one side of the nucleus but the telomeres are neither clustered in the typical bouquet arrangement nor obviously associated with the nuclear envelope (Cenci, 1997).

Although the findings demonstrate that UbcDl is required for proper telomere behavior, they do not prove that this enzyme interacts directly with telomeric proteins. For example, one could argue that telomeric associations might be the consequence of changes in cell cycle timing, which cause chromosome end tangling (Cenci, 1997).

This is clearly not the case. The cytological phenotype of UbcDl mutants is unique, in that none of the many Drosophila mitotic mutants, including those that cause more or less severe delays in the cell cycle, exhibit telomeric associations. In addition, although telomeric associations have been observed in senescent and cancer cells, no one has reported that they can be induced by impairing proper progression of the mammalian cell cycle. Finally, and perhaps most importantly, the SIR4 protein of yeast telomeres has be found to bind the deubiquitinating enzyme UBP3, suggesting that telomeric protein ubiquitination is a general phenomenon (Cenci, 1997).

Therefore, it is proposed that the telomeric associations observed in UbcDl mutant mitoses and male meioses result from failure to degrade one or more telomere- associated proteins. The identity of these putative polypeptide targets of UbcDl are not known. Telomeres in Drosophila are unique in their absence of short repeats and appear to be maintained by transpositions of particular retrotransposons to the chromosome ends. Unfortunately, little is currently known about proteins that bind to these HeT-A and TART retrotransposons, or to components of subtelomeric heterochromatin (Cenci, 1997).

There are three ways in which proteins could mediate telomere-telomere associations. The most straightforward is that dimers or other multimers of a telomere-binding protein might be able to associate simultaneously with two telomeres. This model has been proposed to explain protease-sensitive telomeric associations in ciliate macronuclei. Another possibility is that proteins may link telomeres indirectly, through a third element such as a component of the nuclear envelope. Finally, it is conceivable that telomere- associated proteins might facilitate DNA-DNA interactions between telomeres. The p subunit of the Oxytrichia telomere protein and the RAPl protein of yeast have the ability to help fold or stabilize simple repeat telomeric DNA into G-quartet structures that mediate telomere-telomere associations in vitro (for review, see Henderson 1995). Although G quartets are unlikely to exist in Drosophila telomeres, protein-stabilized DNA-DNA interactions between either retrotransposons or other components of subtelomeric heterochromatin might explain the nucleic acid-containing fibers that link telomeres in polytene chromosomes (Cenci, 1997).

Observations in many different kinds of cells indicate that the clustering and association of telomeres is a widespread aspect of nuclear architecture. It is therefore believed that the most straightforward explanation of the current results is that during at least some part of interphase in wild-type Drosophila cells, telomeres are normally associated directly or indirectly through UbcDl targets. Action of the UbcDl enzyme disrupts this association by prophase, where telomere- telomere interactions are not observed normally. Therefore, the metaphase and anaphase telomeric associations observed in UbcDl mutants would be the consequence of the failure to degrade these protein targets of the ubiquitin pathway (Cenci, 1997).


Search PubMed for articles about Drosophila Effete

Caldwell, J. C., Joiner, M. L., Sivan-Loukianova, E. and Eberl, D. F. (2009). The role of the RING-finger protein Elfless in Drosophila spermatogenesis and apoptosis. Fly (Austin) 2(6): 269-79. PubMed ID: 19077536

Cenci G., et al. (1997). UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior. Genes Dev. 11: 863-875. PubMed ID: 9106658

Chen, D., et al. (2009). Effete-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Development 136(24): 4133-42. PubMed ID: 19906849

Chen, L., Paquette, N., Mamoor, S., Rus, F., Nandy, A., Leszyk, J., Shaffer, S. A. and Silverman, N. (2017). Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing. J Biol Chem [Epub ahead of print]. PubMed ID: 28377500

Henderson, E. (1995). Telomere DNA structure. In Telomeres (ed. E.H. Blackburn and C.W. Greider), pp. 11-34. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Hsu H. J., LaFever L. and Drummond-Barbosa D. (2008). Diet controls normal and tumorous germline stem cells via insulin-dependent and-independent mechanisms in Drosophila. Dev. Biol. 313: 700-712. PubMed ID: 18068153

Kuo, C. T., Jan, L. Y. and Jan, Y. N. (2005). Dendrite-specific remodeling of Drosophila sensory neurons requires matrix metalloproteases, ubiquitin-proteasome, and ecdysone signaling, Proc. Natl. Acad. Sci. 102: 15230-15235. PubMed ID: 16210248

Kuo C. T., Zhu S., Younger S., Jan L. Y. and Jan Y. N. (2006). Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning. Neuron 51: 283-290. PubMed ID: 16880123

Lilly M. A., de Cuevas M. and Spradling A. C. (2000). Cyclin A associates with the fusome during germline cyst formation in the Drosophila ovary. Dev. Biol. 218: 53-63. PubMed ID: 10644410

Mathe, E., Kraft, C., Giet, R., Deak, P., Peters, J. M. and Glover, D. M. (2004). The E2-C vihar is required for the correct spatiotemporal proteolysis of cyclin B and itself undergoes cyclical degradation. Curr. Biol. 14(19): 1723-33. PubMed ID: 15458643

Neufeld, T. P., Tang, A. H. and Rubin G. M. (1998). A genetic screen to identify components of the sina signaling pathway in Drosophila eye development. Genetics 148: 277-286. PubMed ID: 9475739

Paquette, N., Broemer, M., Aggarwal, K., Chen, L., Husson, M., Erturk-Hasdemir, D., Reichhart, J. M., Meier, P., and Silverman, N. (2010) Caspase-mediated cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF- kappaB signaling. Mol Cell 37: 172-182. PubMed ID: 20122400

Parry D. H. and O'Farrell P. H. (2001). The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis. Curr. Biol. 11: 671-683. PubMed ID: 11369230

Rumpf, S., Bagley, J. A., Thompson-Peer, K. L., Zhu, S., Gorczyca, D., Beckstead, R. B., Jan, L. Y. and Jan, Y. N. (2014). Drosophila Valosin-Containing Protein is required for dendrite pruning through a regulatory role in mRNA metabolism. Proc Natl Acad Sci U S A 111(20):7331-6. PubMed ID: 24799714

Ryoo H. D., et al. (2002). Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat. Cell Biol. 4: 432-438. PubMed ID: 12021769

Seino H., Kishi T., Nishitani H., and Yamao F. (2003). Two ubiquitin-conjugating enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, have distinct functions for ubiquitination of mitotic cyclin. Mol. Cell. Biol. 23: 3497-3505. PubMed ID: 12724408

Slack C., Overton P., Tuxworth R. and Chia W. (2007). Asymmetric localisation of Miranda and its cargo proteins during neuroblast division requires the anaphase-promoting complex/cyclosome. Development 134: 3781-3787. PubMed ID: 17933789

Tang Z., et al. (2001). APC2 Cullin protein and APC11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex. Mol. Biol. Cell 12: 3839-3851. PubMed ID: 11739784

Thevenon, D., Engel, E., Avet-Rochex, A., Gottar, M., Bergeret, E., Tricoire, H., Benaud, C., Baudier, J., Taillebourg, E., and Fauvarque, M. O. (2009) The Drosophila ubiquitin- specific protease dUSP36/Scny targets IMD to prevent constitutive immune signaling. Cell Host Microbe 6: 309-320. PubMed ID: 19837371

Treier, M. Seufert, W. and Jentsch, S. (1992). Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation. EMBO J. 11: 367-372. PubMed ID: 1310935

Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H.-A. J., and Hay, B. A. (1999). The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98: 453-463. PubMed ID: 10481910

Wang Z. and Lin H. (2005). The division of Drosophila germline stem cells and their precursors requires a specific cyclin. Curr. Biol. 15: 328-333. PubMed ID: 15723793

Williams, D. W. and Truman, J. W. (2005). Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons. Development 132(16): 3631-42. PubMed ID: 16033801

Wilson, P. et al. The RING finger of DIAP1 is essential for regulating apoptosis. Nature Cell Biol.4(6): 445-50. PubMed ID: 12021771

Yoo, S. J. (2005). Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1. Mol. Cells 20(3): 446-51. PubMed ID: 16404163

Zaccai M. and Lipshitz H. D. (1996). Differential distributions of two adducin-like protein isoforms in the Drosophila ovary and early embryo. Zygote 4: 159-166. PubMed ID: 8913030

Zachariae, W., Schwab, M., Nasmyth, K. and Seufert, W. (1998). Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282(5394): 1721-4. PubMed ID: 9831566

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date revised: 30 December 2014

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