The closest Drosophila homolog of the vertebrate mib gene is the predicted gene CG5841, D-mib. A P-element inserted into the 5' untranslated region of the D-mib gene was recently isolated. Insertion of this P-element confers late pupal lethality. Lethality was reverted by precise excision of the P-element, suggesting that insertion of this P-element is a D-mib mutation, referred to as D-mib1. A 13.6-kb deletion that removes the entire D-mib coding region was selected by imprecise excision of this P-element. This deletion represents a null allele of D-mib and was named D-mib2. This deletion also deletes the 3' flanking RpS31 gene. The D-mib1 and D-mib2 mutant alleles do not complement the l(3)72CdaJ12 and l(3)72CdaI5 lethal mutations that have been mapped to the same cytological interval as the D-mib gene. This indicates that these two lethal mutations are D-mib mutant alleles, and they were therefore renamed D-mib3 and D-mib4, respectively. The D-mib1 and D-mib3 mutations behave as genetic null alleles. In contrast, D-mib4 is a partial loss-of-function allele because flies trans-heterozygous for D-mib4 and any other D-mib null alleles are viable (Le Borgne, 2005).
These four mutations identify the CG5841 gene as D-mib by the following evidence: (1) lethality of homozygous D-mib1 pupae is associated with the insertion of a P-element into the 5' UTR of the D-mib gene; (2) genomic sequencing of the D-mib3 allele revealed the presence of a stop codon at position 258. This allele is therefore predicted to produce a truncated protein devoid of the catalytic RING domain, consistent with D-mib3 being a null allele. Genomic sequencing of the D-mib4 allele showed that this mutation is associated with a valine-to-methionine substitution at a conserved position in the second Mib repeat. (3) Western blot analysis showed that the D-mib protein was not detectable in imaginal disc and brain complex extracts prepared from homozygous D-mib1 and D-mib1/D-mib2 larvae. (4) The leaky, GAL4-independent expression of a UAS-D-mib transgene fully rescues the lethality of D-mib1/D-mib2 flies. Thus, this analysis identified both complete and partial D-mib loss-of-function alleles (Le Borgne, 2005).
Complete loss of zygotic D-mib activity in homozygous D-mib1 and trans-heterozygous D-mib2/D-mib3, D-mib1/D-mib3 and D-mib1/D-mib2 individuals leads to late pupal lethality. Mutant pupae die as pharate adults showing ectopic macrochaetes, increased microchaete density on the dorsal thorax, short legs lacking tarsal segmentation, and nearly complete loss of eye and wing tissues. Tissue losses are associated with a dramatic reduction in size of the eye field and of the wing pouch in mutant discs of third instar larvae. Hypomorphic D-mib2/D-mib4 mutant flies showed ectopic sensory organs, rough eyes, small wings, and thickened veins(Le Borgne, 2005).
All these phenotypes may result from reduced N signaling. More specifically, the bristle and leg phenotypes are likely to result from reduced signaling by Dl (and not by Ser). Indeed, a reduction in Dl-mediated lateral inhibition can result in ectopic sensory organs and increased bristle density on the body surface. In contrast, a complete loss of Ser signaling had no effect on bristle density. Likewise, loss of Dl signaling has been shown to result in short unsegmented legs, similar to the ones seen in the absence of D-mib activity, whereas a complete loss of Ser activity led to the formation of elongated unsegmented legs. Finally, the vein phenotype seen in D-mib hypomorphic flies is similar to the one seen in Dlts mutant flies. Together, these observations suggest that D-mib regulates Dl signaling in several developmental contexts. Consistent with this conclusion, D-mib has been shown to bind Dl and promote Dl signaling, and overexpression of D-mib down-regulates the accumulation of Dl at the cell surface (Le Borgne, 2005).
The function of D-mib during wing development was studied in more detail. Growth of the wing pouch depends on the activity of an organizing center located at the dorsal-ventral (D-V) boundary. This boundary is established in first instar larvae and is defined by the apterous expression boundary. Apterous activates the expression of the Ser and fringe genes in dorsal cells. High levels of Ser in dorsal cells activate N in trans in ventral cells and suppress N activation in cis in dorsal cells, whereas Fringe modifies N in dorsal cells such that dorsal cells located at the D-V boundary respond to Dl. Thus, composite signaling by Ser and Dl leads to symmetric N activation in margin cells located along the D-V boundary. N then regulates the expression of the vestigial and wingless (wg) genes that cooperate to promote growth of the wing pouch. N also regulates expression of the cut gene in margin cells. Thus, loss of N signaling results in a reduction in size of the wing pouch accompanied by the loss of cut and wg expression along the D-V boundary (Le Borgne, 2005).
A complete loss of Cut and Wg accumulation and wg-lacZ expression was observed in the central region of third instar D-mib mutant wing discs. Thus, the D-mib wing phenotype may result from defective N inductive signaling at the D-V boundary. It is concluded that the activity of the D-mib gene is required for the specification of the wing margin and, hence, growth of the wing pouch. Interestingly, wing margin formation and expression of Cut are not affected by the complete loss of neur activity. Similarly, loss of neur activity has no detectable effect on leg segmentation and vein determination, two processes shown here to depend on D-mib gene activity. It is therefore concluded that D-mib and neur have distinct and complementary functions in Drosophila (Le Borgne, 2005).
A functional assay was then used to genetically position the requirement for the D-mib gene activity relative to Ser and N. Expression of an activated version of N, Ncdc10, led to the activation of Cut and promoted growth in dorsal cells of D-mib2/D-mib3 mutant discs. This indicates that D-mib acts at a step upstream of N activation. By contrast, elevated levels of Ser expression failed to restore Cut expression and growth of the wing pouch in D-mib2/D-mib3 mutant larvae. This confirms that Ser signaling requires the activity of the D-mib gene, i.e., that D-mib acts downstream of Ser (Le Borgne, 2005).
The different requirements for neur and D-mib gene activity may suggest that Neur and D-mib have distinct molecular activities. Alternatively, this difference may reflect a difference in gene expression. Consistent with the latter hypothesis, the neur gene is not expressed in wing pouch and wing margin cells, where it is not required, and appears to be expressed only in sensory cells, where it is required. By contrast, D-mib appears to be uniformly expressed in imaginal discs. To test this hypothesis, whether the forced ubiquitous expression of the neur gene can suppress the D-mib loss-of-function phenotype was examined. Expression of Neur, using actin-GAL4, restores growth of the wing pouch and formation of the wing margin. Moreover, expression of Neur in dorsal cells, using Ser-GAL4, is sufficient to rescue growth of the wing pouch as well as the expression of Cut in margin cells in D-mib mutant discs. It is concluded that ectopic expression of Neur compensates for the loss of D-mib activity (Le Borgne, 2005).
In a converse experiment, it was found that the neur-driven expression of D-mib, using neurPGAL4, does not rescue the cuticular neurogenic phenotype of neurPGAL4/neur1F65 embryos. Three UAS-D-mib transgenic lines were tested, and none showed detectable rescue whereas the two UAS-neur lines used as positive controls either fully or partially rescued the cuticular neurogenic phenotype of neurPGAL4/neur1F65 embryos. This indicates that a key function of Neur in the embryo cannot be provided by D-mib. It is therefore suggested that Neur and D-mib functions overlap but are not strictly identical (Le Borgne, 2005).
The receptor Notch and its ligands of the Delta/Serrate/LAG2 (DSL) family are the central components in the Notch pathway, a fundamental cell signaling system that regulates pattern formation during animal development. Delta is directly ubiquitinated by Drosophila and Xenopus Neuralized, and by zebrafish Mind-bomb, two unrelated RING-type E3 ubiquitin ligases with common abilities to promote Delta endocytosis and signaling activity. Although orthologs of both Neuralized and Mind-bomb are found in most metazoan organisms, their relative contributions to Notch signaling in any single organism have not yet been assessed. A Drosophila ortholog of Mind-bomb (D-mib) has been shown in this study to be a positive component of Notch signaling that is required for multiple Neuralized-independent, Notch-dependent developmental processes. Furthermore, D-mib associates physically and functionally with both Serrate and Delta. D-mib uses its ubiquitin ligase activity to promote DSL ligand activity, an activity that is correlated with its ability to induce the endocytosis and degradation of both Delta and Serrate. D-mib can functionally replace Neuralized in multiple cell fate decisions that absolutely require endogenous Neuralized, a testament to the highly similar activities of these two unrelated ubiquitin ligases in regulating Notch signaling. It is concluded that ubiquitination of Delta and Serrate by Neuralized and D-mib is an obligate feature of DSL ligand activation throughout Drosophila development (Lai, 2005).
Mind-bomb was originally characterized in zebrafish through forward genetic studies of a novel locus that was absolutely required for Notch-mediated lateral inhibition of neural precursors. The presence of a clear Drosophila ortholog of Mind-bomb was somewhat of a surprise then, given that: (1) almost without exception, different species display similar functional requirements for evolutionarily conserved components of the Notch pathway; (2) this locus never emerged from any of the extensive Drosophila genetic screens for neurogenic genes and components of the Notch pathway; and (3) it is a large locus that might be expected to have been relatively easily hit, as has proven to be the case in zebrafish (Lai, 2005).
Although D-mib mutants have in fact been isolated, they are only very weakly neurogenic (Le Borgne, 2005; Melendez, 1995). This might partially explain how it was missed in earlier screens for components of the Notch pathway. By contrast, D-mib is absolutely required for the execution of several other Notch-regulated development events, including wing margin specification, eye growth and leg joint specification. Misexpression of full-length and dominant-negative truncations of D-mib affects Notch-mediated pattern formation even more broadly, including many settings that do not normally require D-mib. Biochemical and genetic experiments demonstrate that D-mib associates with both Drosophila DSL ligands, and promotes their internalization and signaling activity. However, dominant-negative D-mibDeltaRF binds Delta and Serrate but interferes with their normal trafficking and inhibits their signaling capacity. It is inferred that D-mibDeltaRF binding to both Delta and Serrate occludes endogenous Neur and D-mib from ubiquitinating and activating DSL ligands. This binding likely underlies the broad capacity of D-mibDeltaRF to inhibit Notch activation in virtually all settings of Notch signaling (Lai, 2005).
Curiously, NeurDeltaRF potentiates Delta signaling during wing margin induction just as full-length Neur does, even though ectopic NeurDeltaRF otherwise strongly inhibits Notch signaling. An explanation is lacking for this difference between NeurDeltaRF and D-mibDeltaRF, but it might hint at a functional difference between these DSL-regulating ubiquitin ligases. In almost every other regard, however, the activities and functions of D-mib/D-mibDeltaRF are highly reminiscent of Neur/NeurDeltaRF. In fact, D-mib can functionally replace Neur in a series of developmental decisions in vivo. Conversely, contemporaneous studies show that Neur can functionally replace D-mib during wing margin specification (Le Borgne, 2005). Nevertheless, the essential endogenous requirements for neur and D-mib are quite distinct, in that they are genetically required for different developmental processes and the respective mutants have differential effects on DSL ligands. Despite potent effects of ectopic D-mib on Delta localization and activity, Delta is mislocalized primarily only in neur mutant tissue, whereas Serrate is mislocalized primarily only in D-mib mutant tissue (Le Borgne, 2005; Lai, 2005).
This apparent specificity is unexpected, since D-mib is expressed ubiquitously, and is therefore present in all Delta-expressing cells. Does endogenous D-mib normally regulate Delta as implied by its ability to associate with Delta, induce Delta endocytosis, and potentiate Delta signaling activity? A close examination of D-mib mutants reveals certain phenotypes that are either stronger than those of Serrate mutants (i.e., leg truncation) or are more suggestive of Delta loss of function (i.e., wing vein deltas and a mildly neurogenic phenotype in the adult thorax) (Le Borgne, 2005). These observations collectively imply that another ubiquitin ligase may co-regulate Delta and thereby partially compensate for loss of D-mib. Neur is a possible, but relatively poor, candidate to supply this function. Although it has a demonstrated role in regulating Delta, neur expression in imaginal tissue is restricted mostly to neural precursors and photoreceptors. A more tantalizing candidate is D-mibl (CG17492), which may also prove to regulate DSL ligands. In support of this, systematic yeast two-hybrid screening has identified a specific interaction between D-mibl and Delta (http://pim.hybrigenics.com/pimriderext/droso/prflybase.html). Therefore, the in vivo function of D-mibl with regard to the regulation of DSL ligands deserves future investigation (Lai, 2005).
Both Drosophila DSL ligands are regulated by ubiquitin ligases that promote ligand endocytosis. Still, the mechanism by which endocytosis promotes DSL ligand activity is still unclear. An earlier proposal was that Delta endocytosis might facilitate Notch proteolytic processing by helping to unmask the S2 Notch cleavage site. Other models suggested that ligand endocytosis might promote ligand clustering or clearance of extracellular NECD. Most recently, genetic studies of the epsin Liquid facets (Lqf), an apparently DSL ligand-specific endocytic component, have led to further insight into this mechanism. In particular, a provocative model was put forth suggesting that Lqf directs Delta into an endocytic recycling compartment, and that Delta recycling back to the plasma membrane is a prerequisite for ligand activation. The finding that Serrate is similarly regulated by endocytosis via D-mib suggests further avenues for testing this model. For example, it will be informative to ask whether lqf shows defects in Serrate trafficking, or if the requirement of Serrate for D-mib can be bypassed by shunting it through an endocytic recycling pathway (Lai, 2005).
Even though Neur and D-mib promote DSL ligand activity by stimulating ligand endocytosis, they also efficiently induce ligand degradation. This might conceptually be at odds with the proposition that ligand recycling back to the plasma membrane underlies DSL ligand activation. These activities might be reconciled if ubiquitination permits a portion of the DSL ligand pool to enter the select Lqf-mediated recycling pathway, but directs the bulk of DSL ligands for degradation. Consistent with this, Lqf is strictly required for DSL ligand activation, but is not required for bulk endocytosis of DSL ligands . If ubiquitination is prerequisite for DSL ligand activation but also makes DSL ligands prone to degradation, this would prevent endless recycling of activated ligands and thereby limit the temporal extent of Notch pathway activation. The strategy of coupling activation with downregulation is seen with Notch itself. Ligand-induced Notch cleavage liberates activated Notchintra, which is a potent regulator of gene expression as a nuclear co-activator for Su(H). However, nuclear Notchintra also becomes a substrate for ubiquitination by the ubiquitin ligase Sel-10, and is rapidly degraded. Coupled activation and downregulation allows for precise temporal control of signaling by limiting the lifetime of activated signaling components (Lai, 2005).
The presence of Neur and Mib homologs in both fly and vertebrate genomes suggests that both proteins were present and regulated DSL ligands in the ancient common ancestor of these species. There is a surprising functional overlap between these structurally unrelated ubiquitin ligases in regulating DSL ligand activity. What, then, was the rationale of evolving such different proteins to perform the same function? The genetic implication that Neur and D-mib preferentially regulate Delta and Serrate, respectively, belies the ability of these enzymes to interact with and regulate the localization and signaling activity both DSL ligands. While it remains to be seen whether Neur regulates Serrate in addition to its documented substrate Delta, D-mib directly and efficiently regulates both Delta and Serrate. Therefore, these ubiquitin ligases did not obviously co-evolve with different classes of DSL ligands (Lai, 2005).
Another possible explanation lies in the curious observation that Neur is genetically required mostly in settings that involve 'lateral inhibitory' Notch signaling, wherein Notch restricts a cell fate among equipotent cells. By contrast, D-mib is required largely in settings that involve 'inductive' Notch signaling, which occurs between non-equivalent cell populations. This apparent division of labor raises the possibility that different ubiquitin ligases could help to specify the appropriate response to Notch activation in each developmental setting (Lai, 2005).
This hypothesis, however, is not particularly supported by the observations that D-mib and Neur can functionally replace each other in a variety of processes. Neither does this correlation hold up in other species, because Neur mediates lateral inhibition of neural precursors in flies, whereas Mib mediates lateral inhibition of neural precursors in fish. This latter finding highlights the plasticity in how these ubiquitin ligases have been deployed during evolution, and is consistent with a model in which fish Mib has subsumed the function of fly Neur during neurogenesis (or vice versa). This may have occurred by appropriate changes in the transcriptional regulation of these genes. Given this likely scenario, one wonders whether it might not have been more evolutionarily expedient to have diversified the function of duplicated, paralogous genes. There are indeed multiple neur and mib genes in vertebrates, and two mib genes in flies. Of course, it might be argued that a similar conundrum concerns the co-existence of the HECT domain and the RING finger/U box as unrelated protein domains that both catalyze E3 ubiquitin ligation (Lai, 2005).
How general is the requirement for DSL ligand endocytosis across evolution? The neurogenic mutant phenotypes of Drosophila neur and zebrafish mib, along with the involvement of Xenopus neur in lateral inhibition, show that DSL ligand ubiquitination is required in both invertebrates and vertebrates. However, thorough loss-of-function genetic studies are incomplete in any organism and are complicated by the duplication of neur and/or mib genes. For example, knockout of murine Neur1 does not affect Notch signaling, possibly due to functional overlap with Neur2 (Lai, 2005).
The present work clearly demonstrates that the vast majority of Notch-regulated settings during Drosophila development are strictly dependent on either Neur or D-mib. Thus, DSL ligand ubiquitination and endocytosis appears to be obligate in Drosophila. In light of this, the situation in nematodes provides an interesting possible counter-example. C. elegans lacks a recognizable Mind-bomb ortholog, but does possess a single Neur gene. However, in contrast to what has been found in Drosophila, where intracellular deletions of the DSL ligands have a dominant-negative activity, the extracellular domains of the DSL ligands LAG-2 and APX-2 can fully rescue the lag-2 mutant and can activate Notch signaling ectopically. A more recent analysis actually revealed a large family of putative secreted DSL ligands in the worm, at least one of which (DSL-1) is a bona fide DSL ligand. This suggests that nematodes may have dispensed with ubiquitination and endocytosis of DSL ligands in at least some settings of Notch signaling. Nevertheless, a nematode ortholog of epsin/Lqf (Ce-epn-1) participates in Notch signaling during germline development. The functional relationships amongst Ce-epn-1, nematode DSL ligands and any potential DSL-regulating E3s in nematodes remain to be determined (Lai, 2005).
Ligands of the Delta/Serrate/Lag2 (DSL) family must normally be endocytosed in signal-sending cells to activate Notch in signal-receiving cells. DSL internalization and signaling are promoted in zebrafish and Drosophila, respectively, by the ubiquitin ligases Mind-bomb (Mib) and Neuralized (Neur). DSL signaling activity also depends on Epsin (Liquid facets), a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Evidence is presented that the Drosophila ortholog of Mib (Dmib) is required for ubiquitination and signaling activity of DSL ligands in cells that normally do not express Neur, and can be functionally replaced by ectopically expressed Neur. Furthermore, both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and the two Drosophila DSL ligands Delta and Serrate differ in their utilization of these Dmib- and Epsin-dependent pathways: most Serrate is endocytosed via the actions of Dmib and Epsin, whereas most Delta enters by other pathways. Nevertheless, only those Serrate and Delta proteins that are internalized via the action of Dmib and Epsin can signal. These results support and extend the proposal that mono-ubiquitination of DSL ligands allows them to gain access to a select, Epsin-dependent, endocytic pathway that they must normally enter to activate Notch (Wang, 2005).
To date, two E3 ubiquitin ligases have been implicated in DSL signaling: zebrafish Mind-bomb (Mib) and Drosophila Neuralized (Neur). Both proteins have been shown to promote DSL ubiquitination, endocytosis and signaling. Moreover, loss-of-function mutations in zebrafish mib and Drosophila neur cause essentially the same hallmark phenotype exemplifying a failure of DSL-signaling in their respective organisms; namely, a dramatic hyperplasia of the embryonic nervous system at the expense of the epidermis. These observations have led to the suggestion that zebrafish Mib and Drosophila Neur are functional homologs. Yet, the two proteins show only limited sequence homology; moreover they appear to be members of distinct Mib and Neur ubiquitin ligase families, each having true orthologs in both vertebrate and invertebrate genomes. As a consequence, the relative roles of Mib and Neur are not known in any animal system and this uncertainty complicates the use of mutations in these genes to assay the role of ubiquitination in DSL endocytosis and signaling (Wang, 2005).
Using newly isolated mutations in dmib, evidence has been found that Dmib and Neur constitute functionally related ubiquitin ligases that are normally required for DSL signaling in different developmental contexts. In the developing Drosophila wing disc, Dmib is required for inductive signaling across the D-V compartment boundary, as well as for the refinement of wing vein primordia, both contexts in which Neur is normally not required or expressed. However, Dmib plays only a modest role in specifying sensory organ precursor (SOP) cells, and little or no role in the subsequent segregation of distinct cell types that form each sensory organ. Instead, Neur appears to provide the essential ubiquitin ligase activity required for DSL signaling in these latter two contexts. Similarly, in the embryo, where Neur is required for most DSL signaling events, Dmib has little or no apparent role. Indeed, embryos devoid of Dmib activity hatch as viable first instar larvae; moreover they develop into pharate adults that show only a limited subset of Notch-related mutant phenotypes, each of which appears to reflect the failure of a particular DSL signaling event that does not, normally, depend on Neur. Thus, it is inferred that Dmib and Neur share a common ubiquitin ligase activity that is essential for DSL ligands to signal (Wang, 2005).
Le Borne (2005) has published similar findings indicating a role for Dmib in DSL signaling, and the capacity of ectopic Neur to substitute for Dmib during wing development. The results differ, however, in that this analysis of clones of dmib- cells that express Ser, Dl, or the DlR+ chimera appears to indicate an absolute requirement for Dmib in sending both Drosophila DSL signals, Dl and Ser. By contrast, Le Borgne interprets his data as evidence for a regulatory rather than an obligatory role of Dmib in sending DSL signals, as well as for a lesser role of Dmib in Dl signaling compared with Ser signaling. Differences in experimental design, particularly in the means used to define or infer the identity of DSL signaling and Dmib-deficient cells, could account for the different conclusions reached (Wang, 2005).
It is noted that if Mib and Neur ligases have overlapping molecular functions in all animal systems, as they have in Drosophila, there is no compelling reason why the ligases would need to be deployed in the same way in different animal species. Instead, any given DSL-signaling process might depend on Mib in one animal system but on Neur in another, as appears to be the case for neurogenesis in zebrafish and Drosophila (Wang, 2005).
In contrast to the selective requirement for Dmib and Neur in overlapping subsets of DSL signaling contexts, Epsin is required for most or all DSL signaling events. This difference is expected if ubiquitination of DSL ligands by either Dmib or Neur is normally a prerequisite for Epsin-mediated endocytosis, and hence for signaling activity (Wang, 2005).
Do Dmib and Neur directly bind and ubiquitinate DSL ligands and thereby confer signaling activity by targeting them for Epsin-mediated endocytosis? Although ectopic Dmib and Neur activity are both associated with enhanced DSL ubiquitination, endocytosis and signaling, there is still no compelling evidence that either ligase directly binds and ubiquitinates DSL proteins, or that Dmib/Neur-dependent ubiquitination of DSL ligands confers signalng activity. However, this study shows that the obligate requirement for Dmib for Dl-signaling by wing cells can be bypassed by replacing the cytosolic domain of Dl with a random peptide, R+, that may serve as the substrate for ubiquitination by an unrelated ubiquitin ligase. This result provides in vivo evidence that Dmib/Neur-dependent ubiquitination of DSL ligands is normally essential to confer signaling activity. Moreover, the failure of the chimeric DlR+ ligand to bypass the requirment for Epsin, supports the interpretation that ubiquitination of DSL ligands confers signaling activity because it targets them for Epsin-mediated endocytosis (Wang, 2005).
During wing development, Ser and Dl both serve as unidirectional signals that specify the 'border' cell fate in cells across the D-V compartment boundary, and both are found to be equally dependent on Dmib and Epsin function for signaling activity. However, the two ligands differ in the extent to which they accumulate on the cell surface, and to which they are cleared from the surface as a consequence of Dmib and Epsin activity. Specifically, most Ser accumulates in cytosolic puncta rather than on the cell surface, whereas the reverse is the case for Dl. Furthermore, removing either Dmib or Epsin activity results in a dramatic and abnormal retention of Ser on the cell surface, whereas it has no detectable effect on the surface accumulation of Dl. Similar results for Dmib were also obtained by Le Borgne (2005). Thus, it appears that most Ser is efficiently cleared from the cell surface by the actions of Dmib and Epsin, whereas most Dl remains on the cell surface, irrespective of Dmib and Epsin activity. This unexpected difference provides two insights (Wang, 2005).
(1) In a previous analysis of the role of Epsin, focus was placed almost exclusively on Dl endocytosis and signaling and failed to obtain direct evidence that Epsin is required for normal DSL endocytosis, despite the obligate role for Epsin in sending both Dl and Ser signals. Instead, such a requirement could be detected only in experiments in which surface clearance of over-expressed Dl was abnormally enhanced by ectopically co-expressing Neur, or could only be inferred from experiments in which the requirement for Epsin was bypassed by replacing the cytosolic domain of Dl with the well-characterized endocytic recycling signal from the mammalian low density lipoprotein (LDL) receptor. By contrast, the different endocytic behavior of Ser has now allowed direct evidence to be obtained that Dmib and Epsin are both required for normal DSL endocytosis (Wang, 2005).
(2) It was found that even though bulk endocytosis of Ser depends on both Dmib and Epsin activity, neither requirement appears absolute. Instead, the accumulation of Ser can still be detected in cytosolic puncta in both Dmib- and Epsin-deficient cells. Moreover, a difference is detected in the abnormal cell surface accumulation of Ser in Dmib-deficient versus Epsin-deficient cells; significantly more Ser appears to accumulate in the absence of Dmib than in the absence of Epsin. It is inferred that both Dl and Ser are normally internalized by multiple endocytic pathways, only some of which depend on ubiquitination of the ligand, and only a subset of these that depends on Epsin. However, the two ligands normally utilize these pathways to different extents, most Ser being internalized by ubiquitin- and Epsin-dependent pathways, and most Dl being internalized by alternative pathways. It is presumed that this difference reflects the presence of different constellations of internalization signals in the two ligands, especially the presence of signals in Delta, but not Ser, that target the great majority of the protein for internalization pathways that do not depend on ubiquitination or Epsin. Nevertheless, only those molecules of Ser and Dl that are targeted by ubiquitination to enter the Epsin-dependent pathway have the capacity to activate Notch; all other routes of entry that are normally available appear to be non-productive in terms of signaling. These results reinforce previous evidence that endocytosis of DSL ligands, per se, is not sufficient to confer signaling activity; instead, DSL ligands must normally be internalized via the action of Epsin to signal (Wang, 2005).
Why must DSL ligands normally be internalized by an Epsin-dependent endocytic mechanism to activate Notch? Two general classes of explanation are considered. In the first, Epsin confers signaling activity by regulating an early event in DSL endocytosis that occurs before internalization. For example, Epsin might cluster DSL ligands in a particular way or recruit them to a select subset of coated pits or other endocytic specializations. Alternatively, Epsin-mediated invagination of these structures might control the physical tension across the ligand/receptor bridge linking the sending and receiving cell, creating a sufficiently strong or special mechanical stress necessary to induce Notch cleavage or ectodomain shedding. In the second class of models, Epsin acts by regulating a later event in DSL endocytosis that occurs after internalization. For example, Epsin might direct, or accompany, DSL proteins into a particular recycling pathway that is essential to convert or repackage them into ligands that can activate Notch upon return to the cell surface. In both cases, internalization of DSL ligands via the other endocytic routes normally available to them would not provide the necessary conditions, even in the extreme case of Dl, which appears to be internalized primarily by these other pathways (Wang, 2005).
The present results do not distinguish between these models. However, recent studies of Epsin-dependent endocytosis in mammalian tissue culture cells suggest that Epsin may direct cargo proteins to different endocytic specializations or pathways, depending on their state of ubiquitination. They also suggest that interactions between Epsin and components of the core Clathrin endocytic machinery normally regulate where and how Epsin internalizes target proteins. Both properties might govern how DSL proteins are internalized, allowing the ligands to gain access to the select endocytic pathway they need to enter to activate Notch (Wang, 2005).
Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. It is shown in this study that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. A mutation was characterized in mib1, a Drosophila homolog of zebrafish mind-bomb, another Delta-targeting ubiquitin ligase. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling (Pitsouli, 2005).
Until now, it was thought that lateral inhibition in notum SOPs was solely mediated via Dl and that Dl transcriptional upregulation in the nascent neural precursor was crucial for a Dl-N negative feedback loop to establish the neural precursor fate within a group of equivalent cells. These data have refuted both of these models, because endogenous Ser has now been shown to participate in lateral inhibition of macrochaete SOPs and either Dl or Ser uniformly expressed is able to produce a wild-type pattern of macrochaetes. Dl transcriptional upregulation in the absence of Notch signaling in proneural fields does occur, but this modulation does not appear to be a prerequisite for the specification of the wild-type neural precursor, at least in the case of macrochaetes and embryonic neuroblasts. It is possible that the genetically detected N-Dl negative feedback loop may reflect Dl and N activity rather than transcription, although a transcriptional input has been documented. An exciting possibility, given the reliance of DSL activity on ubiquitin ligases, is that this feedback loop targets transcription of neur, rather than Dl. mib1 is an unlikely target as since shows no transcriptional modulation within proneural regions (Pitsouli, 2005).
Although Neur was known to affect Dl localization and function in some instances, ubiquitin ligases were not considered as essential components of Notch signaling. The characterization of Mib1 described here and in recent papers (Lai, 2005; Le Borgne, 2005; Wang, 2005) points to a much more prominent role of these factors. mib1 appears to be required in a large number of Notch-dependent processes where neur is not expressed, e.g., the wing DV boundary. The fact that mib1 neur double mutants appear to lose all ability to perform lateral inhibition strongly supports the hypothesis that Ub ligases may always be required for Dl/Ser signaling. A comprehensive survey of Notch-dependent events with respect to neur and mib1 will test this hypothesis and may uncover additional E3 ligases with this activity; Mib2 represents a potential candidate (Pitsouli, 2005).
The intimate relation between Neur/Mib1 and DSL proteins is generally assayed in three ways: (1) physical association, (2) effects on Dl/Ser endocytosis and (3) effects on Dl/Ser signaling. All of these had been well documented for the Neur-Dl combination and, more recently, for the Mib1-Dl and Mib1-Ser combinations (Lai, 2005; Le Borgne, 2005; Wang, 2005). In the present work the final pair, Neur-Ser, has been added, using all of the above assays. The conclusion, stated simply, is that both Neur and Mib1 associate with and affect the endocytosis and function of both Dl and Ser (Pitsouli, 2005).
Ubiquitination of transmembrane proteins tags them for endocytosis, using a complex of adaptors, including epsin, which carry ubiquitin recognition domains. The simplest scenario for the role of Neur/Mib1 in Dl/Ser signaling would be that they attach ubiquitin to Dl/Ser to trigger endocytosis. Signaling would ensue, either as a consequence of recruiting/clustering ubiquitinated DSL cargo to specialized plasma membrane domains conducive to signaling, or by more elaborate routes involving DSL protein recycling through the endocytic pathway as a prerequisite for their modification/activation (Pitsouli, 2005).
Alternatively, Neur/Mib1 need not ubiquitinate the DSL proteins directly. In the ubiquitin-dependent endocytosis pathway, many of the adaptor proteins are themselves ubiquitinated, possibly favoring the formation of interconnected cargo-adaptor complexes; Neur/Mib1 could have one or more of the adaptors, including themselves, as substrates. DSL protein chimaeras become Mib1 independent if their intracellular domains are substituted with ones bearing alternative internalization motifs (Wang, 2005). Of two such artificial Mib1-independent versions of Dl, one is ubiquitination/epsin-independent (Dl-LDL-receptor fusion), whereas the other (Dl-random-peptide-R fusion) still curiously requires ubiquitination/epsin for activity (Wang, 2004). Nothing is yet known about the native Dl/Ser intracellular domains, other than the puzzling fact that they are neither similar nor evolutionarily conserved, despite apparent conservation of recognition by Neur/Mib (Pitsouli, 2005).
An even more puzzling observation in the light of this model is that some DSL proteins in C. elegans appear to be secreted. Secreted mutants of Drosophila Dl and Ser act as Notch antagonists, consistent with a requirement for endocytosis in DSL signaling. Even C. elegans LAG-2 (a transmembrane DSL) needs EPN-1 (epsin ortholog), in order to signal to GLP-1 (Notch-like) during germline differentiation, which is hard to reconcile with secreted DSL proteins. Apparently, ubiquitination/endocytosis can be bypassed in some contexts, allowing secreted DSL proteins to signal via a yet unknown process (Pitsouli, 2005).
Whatever the molecular details and variations turn out to be, it is becoming clear that ubiquination plays a prominent role in Notch signaling, in both sending and receiving cells. In the latter, Ub ligases downregulate Notch activity either at the membrane or in the nucleus. Besides downregulation, however, Notch ubiquitination is also needed for activation: ubiquitination apparently targets Notch to a compartment where it can be activated by gamma-secretase cleavage. How two ubiquitination/trafficking events, activating DSL proteins in one cell and Notch in another, might be coordinated across the extracellular space is a mystery worth investigating in the future (Pitsouli, 2005).
Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. This study reports the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Rat Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Rat Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associates with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development (Choe, 2007; full text of article).
A Drosophila model with Mib1 loss-of-function was used to examine its physiological role and interaction with p35/CDK5. A P-element insertion was identified in the fly gene of CG5841, the Drosophila ortholog of Mib1. The homozygote was affirmed to be a mib1-null allele, because it showed pupal lethality that was rescued by precise removal of the transposon in the 5' untranslated region of the gene, or by the expression of transgenic mib1. The lack of full-length mib1 expression in the allele was verified by Western blotting. In Drosophila, the synaptic structure at the larval neuromuscular junction (NMJ) is a well defined system with which to study synaptic structure and neurotransmission, and the number of synaptic boutons is an established index for synaptic growth. By counting synaptic boutons in the wild-type and the mutant larvae, it was found that the loss of mib1 causes the synaptic overgrowth, and the number of synaptic boutons increased 85%. This finding suggests that Mib1 plays a conserved negative role in the formation of synaptic structure (Choe, 2007).
The genetic interaction between p35/CDK5 and mib1 was tested by monitoring adult eye phenotype. Overexpression of p35/CDK5 causes a rough eye phenotype, whereas the heterozygotic line of the mib1 mutant had a smooth eye phenotype. Crossing the p35/CDK5 transgenic line with the mib1 mutant line showed that the partial loss of mib1 in the heterozygote enhances the rough eye phenotype induced by p35/CDK5. This result also supports the negative regulation between p35/CDK5 and Mib1 in neuronal development (Choe, 2007).
Reference names in red indicate recommended papers.
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Choe, E. A., et al. (2007). Neuronal morphogenesis is regulated by the interplay between cyclin-dependent kinase 5 and the ubiquitin ligase mind bomb 1. J. Neurosci. 27(35): 9503-12. PubMed citation; Online text
Crosnier, C., et al. (2005). Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine. Development 132: 1093-1104. 15689380
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Le Borgne, R., Remaud, S., Hamel, S. and Schweisguth, F. (2005). Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS Biol. 3(4): e96. 15760269
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Pitsouli, C. and Delidakis, C. (2005). The interplay between DSL proteins and ubiquitin ligases in Notch signaling. Development 132(18): 4041-50. 16093323
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Wang, W. and Struhl, G. (2004). Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch. Development 131: 5367-5380. 15469974
Wang, W. and Struhl, G. (2005). Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytos and signaling in Drosophila. Development 132(12): 2883-94. 15930117
Yamamoto, M., et al. (2010). Mib-Jag1-Notch signalling regulates patterning and structural roles of the notochord by controlling cell-fate decisions. Development 137(15): 2527-37. PubMed Citation: 20573700
date revised: 1 November 2010
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