Protein Interactions

Serrate functions as an alternative ligand capable of activating Notch (Gu, 1995). The Notch protein has 36 EGF-like repeats, two of which (numbers 11 and 12) are required for the interaction with the Delta and Serrate ligands (de Celis, 1993). In addition, a specific mutation of the Notch locus, called Abruptex, alters EGF repeats 24-29, reducing the ability of Notch to bind to Notch ligands (de Celis, 1994).

During wing development in Drosophila, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of specialized cells that express Wingless (Wg) and organize wing growth and patterning. Three genes, fringe (fng), Serrate (Ser) and Delta (Dl), are involved in the cellular interactions leading to Notch activation. The relationship between these genes has been investigated by a combination of expression and coexpression studies in the Drosophila wing. Ser is normally expressed in dorsal cells while Dl is initially expressed by all wing cells. However, their expression soon becomes restricted to the dorso-ventral boundary. In order to study Ser and Dl signaling between dorsal and ventral cells, Dl and Ser were expressed ectopically along the anterior side of the anterior-posterior compartment boundary. These experiments confirm that Ser and Dl induce and maintain each other's expression by a positive feedback loop. Importantly, their ability to induce each others expression is dorsal-ventrally asymmetric, because Ser induces Dl strongly in ventral cells, but only very weakly in dorsal cells, whereas Dl induces Ser expression in dorsal cells, but not in ventral cells. fng is expressed specifically by dorsal cells and functions to position and restrict this feedback loop to the developing dorsal-ventral boundary. This is achieved by fng through a cell-autonomous mechanism that inhibits a cell's ability to respond to Serrate protein and potentiates its ability to respond to Delta protein.

To determine the effects of Fringe protein on Ser and Dl activity, margin gene expression and margin bristle formation were asayed while coexpressing these proteins in ventral cells. Ectopic expression of Ser leads to ectopic wing-margin gene expression and adult wing-margin formation in ventral cells along the edges of the ectopic Ser stripe. Misexpression of Fng in ventral cells inhibits these effects of Ser activity, demonstrating that Fng can inhibit Ser signaling. Misexpression of Dl induces ectopic wing-margin formation and Ser expression in dorsal cells but not in ventral cells. However, when Fng is misexpressed in ventral cells, Dl induces Ser expression and margin formation in both dorsal and ventral cells. Thus Fng potentiates Dl signaling, allowing ventral cells to respond to Dl just as dorsal cells normally do. Experiments show that Fng inhibits Ser activity only when it is expressd in receiving cells, and not when expression is restricted to Ser-signaling cells. Activated Notch can induce both Ser and Dl, and activated Notch has similar effects on dorsal and ventral cells, implying that Fng exerts its effects upstream of Notch activation. Because Fng is extracellular, this implies that activity of cell-associated Fng protein differentially modulates the binding and/or activation of Notch by its two ligands. (Panin, 1997).

In the developing imaginal wing disc of Drosophila, cells at the dorsoventral boundary require localized Notch activity for specification of the wing margin. The Notch ligands Serrate and Delta are required on opposite sides of the presumptive wing margin and, even though activated forms of Notch generate responses on both sides of the dorsoventral boundary, each ligand generates a compartment-specific response. Serrate, which is expressed in the dorsal compartment, does not signal in the dorsal regions due to the action of the fringe gene product. Using ectopic expression, it is shown that regulation of Serrate by Fringe occurs at the level of protein and not Serrate transcription. Furthermore, replacement of the N-terminal region of Serrate with the corresponding region of Delta abolishes the ability of Fringe to regulate Serrate without altering Serrate-specific signaling (Fleming, 1997).

The Notch signaling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signaling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signaling to the dorsoventral boundary of the developing wing blade. The results are reported of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development (Klein, 1998).

Serrate, known to be regulated by Apterous, is not required for the initiation of wing development but rather for the expansion and early patterning of the wing primordium. apterous is expressed in dorsal cells of the wing disc; in the absence of ap, the wing blade does not develop, an effect thought to be due to the loss of Ser expression. Consistent with this, ectopic expression of Ser, or of fringe (fng), which leads to the expression of Ser, rescues the loss of the wing in ap mutants. However, the Ser and ap mutant phenotypes are not identical: while in the absence of ap there is no trace of the wing blade, Ser mutants do bear a small marginless wing blade. To explain this difference, the expression of wingless (wg) and vestigial (vg) were compared during wing development in these mutants. The expression of vg decays at the beginning of the third instar in Ser mutants, but is never activated in the wing region of ap mutants. In Ser mutants, the expression of wg never spreads along the wing disc DV interface and resolves into two rings, which fate map the small wing characteristic of Ser mutants. However, in ap mutants, wg expression comes to outline a single circle of expression, which defines proximal hinge structures and the absence of a wing blade, a deficit that is characteristic of these mutants. This is a phenotype very similar to that of vg null alleles. These results suggest that the phenotype of ap mutants cannot be accounted for simply by the absence of Ser. In ap mutants, the development of the wing blade is never initiated; in the absence of Ser, this process is initiated normally, but is aborted early on. Furthermore, since the expansion of wg expression in the AP direction is required for the establishment of the proper size of the primordium, its failure to occur in Ser mutants indicates that, in addition to its role in the establishment of the wing margin, Ser is required to define the proper size of the wing primordium (Klein, 1998).

apterous is also also required for the expression of a second Notch ligand, Delta. The initial stages of the development of the wing blade require Notch signaling and lead to the activation of the vestigial boundary enhancer (vgBE) at the interface between dorsal and ventral cells. Ser is not involved in this event and therefore there ought to be another Notch ligand, under the control of ap, that is responsible for the activation of the vgBE. The product of the Delta gene is a good candidate for this function. During the second instar, Dl is expressed throughout the wing disc, but it is slightly upregulated over the ventral region and shortly afterwards, its pattern of expression is identical to that of vgBE, i.e. a 2- to 3- cell-wide stripe that straddles the DV interface. Furthermore, the expression of Delta is similar to that of the vgBE in Ser and ap mutant discs: in ap mutant wing discs, expression of Dl is lost at the time when the wing primordium is induced, whereas in Ser mutants expression is detected until early third instar. This suggests that Delta might be the activating ligand for the Notch-dependent expression of the vgBE, which operates in the absence of Ser. Consistent with this possibility, ectopic expression of Dl can rescue the loss of wing blade tissue and of wing margin characteristic of ap and Ser mutants (Klein, 1998).

Serrate, in turn, along with Delta, refines Notch function at the DV interface. After the establishment and expansion of the wing primordium, there is a new requirement for Notch signalling in the growth and patterning of the wing blade. In this process, both Serrate and Delta act as ligands for Notch and, as in earlier stages, have different patterns of gene expression, which suggests that they might have different functions. However, ectopic expression of either Dl or Ser will rescue the loss of wing tissue and of wing margin characteristic of ap mutants, and this raises the question of why are there two different ligands to achieve the activation of Notch in these early stages of wing development. It is believed that coexpression of both ligands might result in a different degree of Notch activation than would be achieved individually -- this might allow for a finer degree of regulation for Notch activity. In the presence of Serrate, Delta is able to signal although with a reduced activity level, which perhaps reflects a competition between Serrate and Delta for Notch (Klein, 1998).

A feedback mechanism exists in which Fringe functions to inhibit Serrate by targeting Notch. In contrast to Delta, the effects of ectopic expression of Ser on wild-type discs are restricted to ventral cells. This has led to the suggestion that there is an inhibitor of Serrate activity in dorsal cells and that this inhibitor is under the control of ap. Consistent with this proposal, ectopic expression of Ser in ap mutants is found to be able to induce the expression of downstream targets of Notch in 'dorsal' cells. A variety of arguments have led to the proposal that the dorsal inhibitor of Serrate function is encoded by the fng gene. For example, ectopic expression of Ser with ptcGAL4 results in the activation of Notch targets in two parallel stripes in ventral cells of the developing wing blade, and this can be observed as early as the beginning of the third instar. When Ser is coexpressed with fng, the anterior stripe, but not the posterior one, is lost completely in late third instar discs. Correspondingly the ectopically induced margin structures are reduced to a posterior stripe with characteristics of the posterior compartment. While Fringe suppresses the function of Serrate cell autonomously, it enhances its signaling ability in a nonautonomous manner. Fringe is thought to dampen Serrate signaling by affecting its interaction with Notch, but no evidence has been presented to support this suggestion. Increasing the concentration of Notch appears to be able to titrate the effects of fng. Furthermore, the effects of ectopic expression of fng are partially suppressed by the expression of Notch with fng and are exaggerated by expressing dominant negative Notch molecules with fng. Altogether, these results strongly suggest that a target of Fringe activity is the Notch molecule itself (Klein, 1998).

Fringe functions to inhibit Serrate signaling via Notch. The activity of Fringe can inhibit Serrate signaling by enhancing the intrinsic dominant negative activity of Serrate over Notch. Expression of Ser throughout the late wing disc leads to a strong broadening of the wing veins and a moderate increase in the number of bristles in the notum. Both of these neurogenic phenotypes can be suppressed by coexpressing Notch with Ser, indicating that they are due to a dominant negative effect of Serrate. Ectopic expression of fng alone in the same pattern results in nicked wings with normal veins and a reduction of bristles in the notum, which is associated with the loss of sensory organ precursors. Coexpression of fng with Ser suppresses the extra vein phenotype caused by misexpression of Ser and, therefore, supports the notion that Fringe reduces the ability of Serrate to bind Notch. Fringe is shown to impinge on Notch signaling by the observation that the action of Fringe requires the activity of Su(H). Fringe is not able to rescue the defects caused by Su(H) mutants (Klein, 1998).

Molecular evidence has established that direct heterotypic interactions occur between the Drosophila receptor Notch and the ligands Delta and Serrate, and that homotypic interactions occur between Delta molecules on opposing cell surfaces. Using an aggregation assay developed for Drosophila cultured cells, the affinities of these interactions have been compared. The heterotypic interactions between Notch and the ligands Delta and Serrate have higher affinities than homotypic interactions between Delta molecules. Contrary to previous suggestions, the evidence implies that the interactions between Serrate and Notch are similar in affinity to those between Delta and Notch. Fringe does not detectably affect the ligand-receptor interactions of the Notch pathway in cultured cells. Furthermore, Serrate, like Delta, is a transmembrane ligand that can participate in reciprocal trans-endocytosis of ligand and receptor between expressing cells. These findings imply that qualitative differences between Delta- and Serrate-mediated Notch signaling depend on characteristics other than intrinsic ligand-receptor affinities or the ability to participate in reciprocal ligand and receptor trans-endocytosis (Klueg, 1999).

The Delta extracellular domain and intracellular domain are taken up by adjacent Notch+ Drosophila cultured cells. Using antibodies against the Serrate extracellular domain or a MYC epitope tag in the intracellular domain, it was asked whether the entire Serrate molecule is transferred into neighboring Notch+ cells. In Drosophila Serrate-Notch cell aggregates, the Serrate extracellular domain and intracellular domain are found to be taken up by neighboring Notch+ cells. The fraction of Notch+ cells adjacent to one or more SerrateWTMYC+ cells that contain vesicles positive for the Serrate extracellular domain is 17%. This is similar to the fraction of Notch+ cells that contain vesicles positive for the Serrate intracellular domain (15%). The fraction of Notch+ cells that contain vesicles positive for the Delta extracellular domain (27%) was recorded in parallel as a control (Klueg, 1999).

Delta+ cells adjacent to Notch+ cells contain vesicles positive for the Notch intracellular domain, demonstrating that the entire Notch molecule can be taken up by adjacent Delta+ cells. To determine whether similar trans-endocytosis occurs during Serrate-Notch interactions, trans-endocytosis of Notch into SerrateWTMYC+ cultured cells was examined. Notch is found to be trans-endocytosed, and the percentage of SerrateWTMYC+ cells that contain vesicles positive for the Notch intracellular domain is 38%, similar to the percentage of Delta+ cells that contain vesicles positive for the Notch intracellular domain. Heterotypic aggregates formed after 15 minutes of aggregation were examined, and it was found that trans-endocytosis of Delta and Notch occurs shortly after their interaction is initiated. Similar observations have been repeated for Serrate+-Notch+ heterotypic cell aggregates. Both Serrate and Notch are trans-endocytosed shortly after interaction is initiated. Delta, however, is not taken up by adjacent Delta+ cells in homotypic aggregates (Klueg, 1999).

The activation of Notch is regulated both by the temporal and spatial distribution of the ligands and by the expression of proteins such as Fringe (Fng) that are able to modulate ligand-receptor interactions. This was first evident in the developing wing, where Notch activity results in the expression of genes such as wingless and cut in a narrow 2- to 4-cell-wide domain at the dorsoventral boundary. In this process, Fng influences the effectiveness of the interactions between Notch and its ligands by preventing Ser-mediated activation and potentiating Notch activation by Dl. The localized activation of Notch initially occurs because Apterous promotes the expression of both Ser and Fng in dorsal cells, while the inhibitory effect of Fng on Ser/Notch restricts Ser signaling primarily to ventral cells. At the same time, the effect of Fng on Dl has the consequence that ventral Dl-expressing cells signal primarily to dorsal cells. A similar process occurs in the eye, where again the compartment-specific expression of fng allows localized activation of Notch at the eye dorsoventral boundary (de Celis, 2000 and references therein).

Conventionally Dl and Ser are considered activating ligands of Notch and, in many instances, their elimination has non-autonomous effects on development that are characteristic of a membrane-associated ligand. However, in the Drosophila wing and eye, both Notch ligands have also been shown to have cell-autonomous inhibitory effects on the activity of the receptor. Thus, the elimination of both ligands in clones of cells in the wing can result in Notch activation within the clone, detected as ectopic ct expression, indicating that a normal function of Dl and Ser is to prevent Notch activation within the cells in which they are expressed. In addition, ectopic expression of Dl or Ser in groups of cells causes Notch activation only in the adjacent cells. Consistent with the suggestion that the inhibitory activity of the ligands relies on interactions occurring between molecules within the same cell, the negative effects of ectopically expressed Ser can be alleviated by co-expression of full-length Notch. The negative effect of the ligands could be instrumental in determining the polarity of Notch signaling: cells expressing higher levels of ligand would have reduced Notch responsiveness compared to adjacent cells with lower ligand levels and hence Notch would be more readily activated in the cells with relatively less ligand. The concept that the relative levels of Notch and Dl are important for signaling is also evident from the phenotypes caused by varying the dosage of these genes. Finally, Dl and Notch have been seen to co-localize on the surface of cultured cells, suggesting that they could interact in the plasma membrane. However, the antagonistic interactions could be occurring anywhere within the cell and the functional domain of Notch involved in this process has not been characterised (de Celis, 2000 and references therein).

The extracellular domain of Notch contains an array of 36 EGF repeats, two of which, repeats 11 and 12, are necessary for direct interactions between Notch with Delta and Serrate. An investigation has been carried out of the function of a region of the Notch extracellular domain where several missense mutations, called Abruptex, are localized. These Notch alleles are characterized by complex complementation patterns and phenotypes that are the opposite of those observed with a loss of Notch function. In Abruptex mutant wing discs, only the negative effects of the ligands and Fringe are affected, resulting in the failure to restrict the expression of cut and wingless to the dorsoventral boundary. It is suggested that Abruptex alleles identify a domain in the Notch protein that mediates the interactions between Notch, its ligands and Fringe that result in suppression of Notch activity (de Celis, 2000).

In wild-type discs, the response of Notch to Dl and Ser is affected by the presence of Fng, which is expressed in dorsal cells. Since the domain of Fng expression corresponds to the region where Dl loses its capacity to antagonize Notch in NAx mutants, an analysis was carried out to see whether NAx mutations have an altered sensitivity to Fng by comparing the consequences of ectopic fng expression in wild-type and NAx discs. As with the ectopic ligand expression, clones of cells expressing fng that cross the dorsoventral boundary inhibit expression of ct except at the clone borders. When the Fng-expressing clones lie in the ventral compartment, ct is induced in the cells at the boundary of the clone, with the result that ct is detected in neighboring fng+ and fng- cells. The ability of Fng to prevent ct expression is reduced when Fng-expressing clones are induced in NAx mutant backgrounds. In a weak NAx allelic combination, the expression of ct is still highest at clone boudaries, but significant expression is detected within the clone. In the more severe mutants, the Fng-expressing cells have little or no inhibitory effect on ct, and there are high levels of Ct throughout the clone. Similar effects are seen when fng misexpression is driven by Gal4-sal. Normally this causes an inhibition of ct expression at the dorsoventral boundary; in NAx mutant discs, however, Ct is detected throughout most of the domain of ectopic Fng-expression. If the NAx domain is significant in the interactions between Notch and Fng, the NAx mutations should modify phenotypes caused by alterations in fng expression. In the allele fngD4, fng is expressed throughout the wing pouch, causing severe scalloping of the wing margin. This correlates with the loss of ct and wg expression at the dorsoventral boundary and the expansion of vvl/drifter expression. In NAx heterozygous flies, the phenotype of fngD4 is reduced both at the level of wing scalloping and the expression of dorsoventral boundary markers. In hemizygous NAx males, both the expression of ct and vvl and the adult phenotype are similar to the expression and phenotype typical of NAx. Taken together, these results suggest that NAx proteins are also deficient in some activity related to the capability of Fng to restrict Notch activity (de Celis, 2000).

The amino-acid sequence of Fng indicates that it could be a glycosyltransferase. Since NAx mutations affect the extracellular domain of Notch, the fact that the NAx alleles have altered behavior with respect to Fng suggests that the mutated domain could be a target for Fng-mediated glycosylation. If the NAx mutations perturb glycosylation of Notch by Fng, this might explain why they only affect the activity of Notch in the imaginal discs and not in the early embryo, since fng is only required at later stages of development. NAx alleles also affect several processes, such as sensory organ development and vein cell differentiation, that do not seem to require fng activity. This indicates that the NAx domain also affects negative interactions between Notch with Dl and Ser independent of fng function (de Celis, 2000).

The results shown here indicate that the NAx domain of Notch is only necessary to mediate the functions of Fng and the ligands that result in the suppression of Notch activity. A comparison between the effects on Fng, Dl and Ser indicates that the interactions between these molecules and Notch are affected to different extents by NAx mutations. For example, although the dominant negative effects of Dl and Fng are dramatically reduced in NAx alleles, these mutations do not appear to compromise the potentiating effect of Fng on Dl activation, since there is still a strong bias towards Dl activity in the dorsal domain where Fng is present. Similarly high levels of ectopic Ser can efficiently suppress Notch activity in NAx backgrounds, even though the phenotype of NAx mutant discs indicates that NAx mutations perturb the dominant negative effects of Ser when it is expressed at normal levels. Each NAx allele has a characteristic strength that is reflected in its phenotype and in the extent of ectopic ct activation. Furthermore, heteroallelic combinations between NAx alleles often result in synergistic phenotypes, a phenomenon called negative complementation. This suggests that the correct conformation of the NAx domain in Notch multimers is critical for the efficiency of the interactions between Notch, its ligands and Fng that determine suppression of Notch activity (de Celis, 2000).

The Delta and Serrate proteins interact with the extracellular domain of the Notch receptor and initiate signaling through the receptor. The two ligands are very similar in structure and have been shown to be interchangeable experimentally; however, loss of function analysis indicates that they have different functions during development and analysis of their signaling during wing development indicates that the Fringe protein can discriminate between the two ligands. This raises the possibility that the signaling of Delta and Serrate through Notch requires different domains of the Notch protein. This possibility has been tested by examining the ability of Delta and Serrate to interact and signal with Notch molecules in which different domains have been deleted. This analysis has shown that EGF-like repeats 11 and 12, the RAM-23 and cdc10/ankyrin repeats and the region C-terminal to the cdc10/ankyrin repeats of Notch are necessary for both Delta and Serrate to signal via Notch. They also indicate, however, that Delta and Serrate utilize EGF-like repeats 24-26 of Notch for signaling, but there are significant differences in the way they utilize these repeats (Lawrence, 2000).

Expression of Delta and Serrate with molecules that lack EGF-like repeats 24-26 produce differing results. Delta can signal through a Notch protein that lacks these repeats, but Serrate cannot. This requirement for EGF-like repeats 24-26 is surprising and indicates that the influence of these repeats on the interactions between Notch and its ligands in cell culture and in vivo might reflect a requirement for these repeats in signaling. Interestingly, there are differences in the way Delta and Serrate require these repeats. In the case of Serrate, the requirement for signaling is absolute, whereas in the case of Delta it is conditional on the presence of EGF-like repeats 17-19, which have been shownto be structurally related to 24-26. These differences are made more clear when comparing the interactions of Delta and Serrate with the dominant negative extracellular Notch constructs (ECNs). Delta cannot signal with full length Notch molecules that lack repeats 10-12 or 17-19 and 24-26, nor interact with ECN molecules that lack these repeats. Although Serrate cannot signal with FLN molecules lacking these repeats, it can, however, interact with the corresponding ECN molecules. This suggests that whereas in the case of Delta a direct interaction with Notch amounts to signaling, this might not be sufficient for Serrate. In the case of Serrate, further modifications of Notch or interactions with other proteins might be required to trigger a signal (Lawrence, 2000).

Notch modulation by O-fucosyltransferase 1 is essential for Notch interaction with its ligands and for Fringe function

Notch and its ligands are modified by a protein O-fucosyltransferase (O-fut1, also known as Neurotic or Ofut1) that attaches fucose to a serine or threonine within EGF domains. By using RNAi to decrease O-fut1 expression in Drosophila, it has been demonstrated that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for O-fut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. Therefore, O-fut1 activity is required for the cell's ability to receive ligand signals, and would thus be consistent with the hypothesis that the key substrate of O-fut1 is Notch. The transcription of O-fut1 is developmentally regulated, and surprisingly, overexpression of O-fut1 inhibits Notch signaling. Together, these results indicate that O-fut1 is a core component of the Notch pathway, one that is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development (Okajima, 2002).

A mutation has been isolated in the gene encoding O-fucosyltransferase, and analysis of the mutant phenotype confirms the RNAi studies and reveals an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. A novel maternal neurogenic gene, neurotic, is essential for Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. neurotic has been shown to be required for Fringe activity. fringe encodes a fucose-specific ß1, 3 N-acetylglucosaminyltransferase that modulates Notch receptor activity. Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. These results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions (Sasamura, 2003).

Since O-fucose on Notch has been shown to act as a molecular scaffold for GlcNAc that is elongated by Fng, one would expect that the phenotypes of O-fucosyltransferase mutant might be the same as those of fng. Unexpectedly, however, the nti and fng mutant phenotypes are quite different. Strikingly, the embryonic neurogenic phenotype that is evident in nti mutant, and is an indication of its essential role in Notch signalling, is not evident in fng mutants. Furthermore, it is thought that Fng does not have a significant role in lateral inhibition, while it is involved in the generation of the cell boundary between cells expressing Fng and cells not expressing Fng. Additionally, an in vitro binding assay revealed that Nti is essential for binding between Notch and Delta. Based on the previous findings and the present results, it is proposed that O-glycosylation of Notch EGF repeats has two distinct roles for binding to Delta. (1) O-fucosylation catalysed by Nti is an absolute requirement for binding between Notch and the ligand, and this binding is sufficient to accomplish lateral inhibition. For this function, no additional glycosylation to O-fucose residue is required. This idea is also supported by the observation that in the tissues and organisms that do not express fng, Delta is competent to activate the Notch receptor. In this respect, it is worth noting that in the C. elegans genome there is a highly conserved nti, while a fng homolog is not found. (2) Addition of GlcNAc to the O-fucose residue by Fng enhances the interactions between Notch and Delta, modulating the receptor-ligand interactions. In fact, the expression of fng shows a high degree of regional specificity, and the boundary of the cells expressing and not expressing Fng often defines the border of distinct tissue structures. Thus, the region-specific expression of fng allows modulation of Notch signalling, resulting in generation of complex structure of organs. As expected from the second function of Nti, its function is essential for Fng-dependent modulation of Notch signalling as well as Fng-independent function. In the wing disc, nti is epistatic to fng, and fng requires nti to induce Wg at the dorsal and ventral compartment boundary. Additionally, in the in vitro binding assay, Fng depends on Nti to enhance the binding between Notch and Delta. These lines of evidence indicate that Nti is involved in Fng-dependent modulation of Notch signalling, which is consistent with an O-glycan structure of the Notch EGF repeats (Sasamura, 2003).

To investigate the molecular basis for the requirement for O-linked fucose on Notch, an assay was carried out of the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Downregulation of O-fut1 by RNAi in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. Conversely, over-expression of O-fut1 in cultured cells increases Serrate-Notch binding but inhibits DeltaNotch binding. These effects of O-fut1 are consistent with the consequences of O-fut1 overexpression on Notch signaling in vivo. Intriguingly, they are also the opposite of, and are suppressed by, expression of the glycosyltransferase Fringe, which specifically modifies O-linked fucose. Thus, Notch-ligand interactions are dependent upon both the presence and the type of O-fucose glycans (Okajima, 2003).

The requirement for O-Fut1 in Notch signaling has been demonstrated by RNAi in Drosophila (Okajima, 2002), and by a targeted mutation in the murine Pofut1 gene. One line from a large scale screen for lethal transposable element insertions in Drosophila has an insertion in the 3' end of O-fut1, and is predicted to result in replacement of the seven C-terminal amino acids of O-fut1 with four different amino acids followed by a stop codon. To confirm that this insertion creates an O-fut1 mutation, animals in which patches of cells were made homozygous mutant for this allele were examined by mitotic recombination. These animals exhibit classic Notch mutant phenotypes, such as wing notching, thickened wing veins, and loss of sensory bristles on the notum, consistent with the phenotypes generated by RNAi of O-fut1 (Okajima, 2002). In developing wing imaginal discs, the expression of targets of Notch signaling, such as Wingless, is lost in cells mutant for O-fut1. This mutation (referred to hereafter as O-fut1SH) thus provides an independent demonstration of the requirement for O-fut1 for Notch signaling in Drosophila, and indicates that the seven C-terminal amino acids of O-fut1 are essential for function in vivo. The last four amino acids of O-fut1 conform to a consensus signal for retention in the endoplasmic reticulum, and experiments are in progress to determine whether the loss of function in O-fut1SH is due to loss of enzymatic activity or to mislocalization (Okajima, 2003).

The studies presented here indicate that O-fucosylation is required for the physical binding of Notch to its ligands Dl and Ser. These binding studies are consistent with prior genetic studies, which positioned a requirement for O-fucosylation in signal receiving cells, upstream of the cleavages associated with Notch activation (Okajima, 2002). Although the current results do not exclude the possibility that O-fucose glycans could also act at other steps, and indeed some influence of O-fut1 RNAi on secretion of Notch extracellular domain fusion proteins is detected, the requirement for O-fucose for Notch-ligand binding can in principle account for the requirement for O-fut1 in Notch signaling (Okajima, 2003).

Notably, O-fut1 is required for efficient binding of Notch to both Ser and Dl. This is consistent with the severe Notch phenotypes observed in vivo when O-fut1 is impaired by mutation or RNAi. By contrast, elongation of O-fucose by the GlcNAc transferase Fringe exerts opposing influences on the ability of Notch to bind to Ser and Dl. Fringe has clear and reproducible effects on both Dl-Notch and Ser-Notch binding. Importantly, these effects of Fringe on Notch-ligand binding recapitulate its effects on signaling by these two ligands in Drosophila. The ability of both the O-fucose monosaccharide and elongated forms of O-fucose to influence Notch-ligand binding, the influence of O-fucosylation on binding by both ligands, and the consistent correlations between the effects of O-fucosylation on binding in vitro and its effects on signaling in vivo all argue that O-fucose glycans act at the ligand binding step of Notch signaling (Okajima, 2003).

Beyond their importance to understanding regulation of Notch signaling, these observations thus provide a striking example of glycosylation as a mechanism for modulating protein-protein interactions. With the determination that O-fucosylation affects Notch-ligand binding, attention must now be turned to elucidating the mechanistic basis for this effect (Okajima, 2003). O-fut1 and Fringe always act in Notch-expressing cells to influence Notch signaling and Notch-ligand binding: this implicates Notch itself as the relevant substrate. However, the actual sites of glycosylation on Notch that mediate the effects of these glycosyltransferases remain to be identified. It is also not yet clear whether the importance of O-fucosylation reflects a role for lectin-like recognition of Notch by its ligands or other co-factors, or whether instead O-fucose glycans influence Notch-ligand binding indirectly, by altering the conformation or oligomerization of Notch (Okajima, 2003).

By contrast to the positive requirement for O-fut1 demonstrated by RNAi, over-expression of O-fut1 enhances Ser-Notch binding but inhibits Dl-Notch binding. It is intriguing that elevated O-fut1 expression provides a mechanism for differentially modulating the ability of different Notch ligands to interact with the Notch receptor. Previously, Fringe was the only factor known that could discriminate between the ability of Delta to activate Notch and that of Serrate to activate Notch. Indeed, elevated O-fut1 expression might be a mechanism for increasing the sensitivity of cells to the presence or absence of Fringe. In vivo, Fringe only affects a subset of Notch signaling events, and it remains unclear why certain processes are sensitive to Fringe whilst others are insensitive. Although O-fut1 action is the opposite of Fringe, its effects can be blocked by Fringe; therefore, the relative impact of Fringe on Dl-Notch or Ser-Notch interactions is expected to be greater in tissues where O-fut1 is expressed at higher levels. Indeed, even though expression of Fringe alone has no obvious effect on the patterning of notal bristles, it has a strong effect when O-fut1 is also overexpressed. Overexpression of O-fut1 inhibits Dl-Notch signaling, resulting in the formation of excess sensory bristles, but this effect is partially inhibited by co-expression with Fringe (Okajima, 2002). In addition to increasing the sensitivity of Notch signaling events to the presence or absence of Fringe, elevated O-fut1 expression presents a potential mechanism for modulating Notch signaling independently of Fringe. Although the in vivo relevance of Notch-ligand modulation by increased expression of O-fut1 at endogenous levels of expression remains uncertain, it is noted that certain tissues, such as the lymph gland, express much higher levels of O-fut1 than surrounding cells (Okajima, 2002). Intriguingly then, in most Drosophila tissues Dl is the sole or major Notch ligand. However, in the larval lymph gland, a role for Notch signaling in regulating cell fate decisions during hematopoeisis has recently been described, and Ser, rather than Dl, is the ligand that regulates Notch in this tissue. These observations provide some support for the possibility that transcriptional regulation of O-fut1 might provide a mechanism for Notch pathway regulation (Okajima, 2002), and suggest developmental contexts in which this issue may be investigated further (Okajima, 2003).

An O-fucose site in the ligand binding domain inhibits Notch activation by Serrate

Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. This study describes the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. The results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicates that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling (Lei, 2003).

To begin to identify EGF domains whose O-fucosylation influences Notch activation, S was substituted at the O-fucose attachment site for A, and T for V. A or V can be found at this position in other EGF repeats of Notch or its ligands, and hence are unlikely to cause disruptions of EGF structure. Focus was placed on four EGF repeats of Notch: 12, 24, 26 and 31. EGF24, EGF26 and EGF31 were chosen because they lie in or near the region of Notch to which the NAx alleles map, and because they contain highly conserved O-fucose sites that conform to the original consensus sequence. EGF12 was chosen because it corresponds to one of two EGF repeats identified as necessary and sufficient for Notch-ligand binding in a cell aggregation assay, and because it contains a potential O-fucose site in all cloned Notch receptors with 36 EGF repeats. Although this site does not conform to the original consensus for O-fucosylation, EGF12 of Notch1 has been shown to be glycosylated by O-FucT-1 and Fringe in CHO cells. O-fucosylation of Drosophila Notch EGF12 in Drosophila cells was confirmed by assessing the ability of a fragment of Notch isolated from S2 cells to serve as an in vitro substrate for Fringe (Lei, 2003).

Therefore, EGF12 is a biologically relevant site of O-fucosylation. O-fucose is attached to an S or T. Consequently, when that amino acid is changed to one that lacks a terminal hydroxyl group, O-fucosylation of the EGF domain cannot occur. Consistent with this, the S to A mutation eliminates the ability of a Notch fragment including EGF12 to serve as a substrate for Fringe. For several reasons, the observed differences between N-EGF12f and wild-type Notch can be attributed to this absence of glycosylation, rather than to the amino acid change per se. Substitution of an S with an A is a conservative change, and the two amino acids differ only by an oxygen atom. A is found at this location in other EGF repeats (e.g., EGF36 of Drosophila Notch, and EGF7 and EGF19 of mammalian Notch1), and hence is unlikely to disrupt the EGF structure. Indeed, this same mutation in EGF26 does not result in a detectable phenotype. A distinct amino acid change in EGF12, the E491V mutation in NM1, results in a strong loss-of-function phenotype, as would be predicted for a gross structural change in the ligand-binding domain (Lei, 2003).

By contrast, the phenotype of N-EGF12f is consistent with that which would be expected of a Notch receptor that had lost a functional site of glycosylation by Fringe. Expression of N-EGF12f results in an ectopic activation of Notch in dorsal wing cells that is insensitive to Fringe, yet dependent upon endogenous ligand expression. Binding studies further show that Serrate is able to bind to this mutant form of Notch even in the presence of Fringe, which contrasts with the lack of detectable Serrate binding to wild-type Notch expressed in the presence of Fringe. Based on these observations, it is concluded that EGF12 is an essential site for inhibition of Serrate-to-Notch signaling by the Fringe glycosyltransferase (Lei, 2003).

Although the O-fucose site in EGF12 is essential for Fringe inhibition of Serrate signaling in the wing, Fringe still reduces N-EGF12f:AP-Serrate binding. The decrease in binding is not sufficient to prevent N-EGF12f activation, but there must nonetheless be multiple sites that can contribute to the inhibition of Serrate signaling by Fringe. There must also be distinct sites that mediate the potentiation of Delta-Notch signaling by Fringe, because N-EGF12f:AP-Delta binding is potentiated almost as effectively as N:AP-Delta binding. Importantly then, the effects of Fringe on Delta versus Serrate signaling appear to be mediated, at least to some extent, through distinct sites of O-fucosylation (Lei, 2003).

Notch ligands activate Notch receptors expressed by neighboring cells, but inhibit Notch receptors expressed by the same cell. Elevated expression of the Notch extracellular domain can also inhibit the ability of ligands to signal to neighboring cells. Thus, one apparent consequence of the transmembrane nature of Notch ligands is that Notch activation depends not simply on the ability of ligand to bind receptor, but also on a competition between intracellular and intercellular interactions. Previously, most attention has focused on the impact of different levels of expression on this competition. But the balance in this competition can also be shifted by adjusting the affinity between Notch and its ligands. Indeed, even though most studies have focused on the ability of Fringe to inhibit the response of a cell to Serrate, the ability of cells to send a Serrate signal appears to be enhanced by co-expression with Fringe, which is consistent with the idea that decreasing intracellular Serrate-Notch interactions increases the amount of Serrate available to signal to neighboring cells (Lei, 2003).

Cell-based binding assays indicate that the O-fucose site in EGF12 is not just important for Fringe-dependent inhibition: even the presence of the O-fucose monosaccharide at this site inhibits Serrate binding. The presence of an inhibitory site of O-fucosylation in EGF12 was unexpected given the general positive requirement for O-fucose in Notch signaling. However, the presence of an inhibitory site can be rationalized in terms of a competition between intracellular and intercellular Notch-ligand interactions. The competition model implies that it is important, at least in certain contexts, for Notch not to bind too strongly to its ligands. One such context is probably the DV boundary of the Drosophila wing, because Notch ligands are expressed on both sides of the compartment boundary, and Notch is activated on both sides of the compartment boundary. Thus, it is suggested that N-EGF12f is unable to rescue normal Notch activation at the DV boundary because its increased affinity for ligands enhances intracellular binding to a degree that interferes with the ability of a cell to send and receive Notch signals. Notably, EGF12 is apparently essential for both intercellular and intracellular Notch-ligand interactions (Lei, 2003).

The highly conserved presence of an O-fucose site in EGF12 suggests that inhibition of ligand binding by the O-fucosylation of EGF12 might be of widespread importance. However, if O-fucosylation of EGF12 was constitutive, it would simply counteract the positive influence of O-fucosylation at other sites. If, by contrast, O-fucosylation of EGF12 was regulated, then differential O-fucosylation of EGF12 could occur, and could serve as a mechanism of Notch regulation. Intriguingly then, EGF12 is distinguished from other potential O-fucose sites by the presence of an acidic amino acid (E or D) at the -2 position relative to the O-fucose attachment site. None of the other EGF repeats in Notch contain an acidic amino acid at this position, yet 13/15 Notch receptor proteins contain an acidic amino acid at this position in EGF12. It is not yet known what fraction of Notch receptors in a cell are modified at any of the potential sites of O-fucosylation, but the presence of this conserved sequence difference suggests that EGF12 might be O-fucosylated under different conditions, or with a different efficiency, than other EGF domains, and hence that differential fucosylation of this site might serve as a regulatory mechanism (Lei, 2003).

Mind-bomb and Neuralized are two distinct E3 ubiquitin ligases that have complementary functions in the regulation of Delta and Serrate signaling in Drosophila

Signaling by the Notch ligands Delta (Dl) and Serrate (Ser) regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind-bomb (Mib), have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. Drosophila mind bomb (D-mib) regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. It is concluded that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila (Le Borgne, 2005).

Cell-to-cell signaling mediated by receptors of the Notch (N) family has been implicated in various developmental decisions in organisms ranging from nematodes to mammals. N is well-known for its role in lateral inhibition, a key patterning process that organizes the regular spacing of distinct cell types within groups of equipotent cells. Additionally, N mediates inductive signaling between cells with distinct identities. In both signaling events, N signals via a conserved mechanism that involves the cleavage and release from the membrane of the N intracellular domain that acts as a transcriptional co-activator for DNA-binding proteins of the CBF1/Suppressor of Hairless/Lag-2 (CSL) family (Le Borgne, 2005).

Two transmembrane ligands of N are known in Drosophila, Delta (Dl) and Serrate (Ser). Dl and Ser have distinct functions. For instance, Dl (but not Ser) is essential for lateral inhibition during early neurogenesis in the embryo. Conversely, Ser (but not Dl) is specifically required for segmental patterning. Some developmental decisions, however, require the activity of both genes: Dl and Ser are both required for the specification of wing margin cells during imaginal development. These different requirements for Dl and Ser appear to primarily result from their non-overlapping expression patterns rather than from distinct signaling properties. Consistent with this interpretation, Dl and Ser have been proposed to act redundantly in the sensory bristle lineage where they are co-expressed. Furthermore, Dl and Ser appear to be partially interchangeable because the forced expression of Ser can partially rescue the Dl neurogenic phenotype. Additionally, the ectopic expression of Dl can partially rescue the Ser wing phenotype. The notion that Dl and Ser have similar signaling properties has, however, recently been challenged by the observation that human homologs of Dl and Ser have distinct instructive signaling activity (Le Borgne, 2005).

Endocytosis has recently emerged as a key mechanism regulating the signaling activity of Dl. (1) Clonal analysis in Drosophila has suggested that dynamin-dependent endocytosis is required not only in signal-receiving cells but also in signal-sending cells to promote N activation. (2) Mutant Dl proteins that are endocytosis defective exhibit reduced signaling activity (Parks, 2000). (3) Two distinct E3 ubiquitin ligases, Neuralized (Neur) and Mind-bomb (Mib), have recently been shown to regulate Dl endocytosis and N activation in Drosophila and Danio rerio, respectively. Ubiquitin is a 76-amino-acid polypeptide that is covalently linked to substrates in a multi-step process that involves a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3). E3s recognize specific substrates and catalyze the transfer of ubiquitin to the protein substrate. Ubiquitin was first identified as a tag for proteins destined for degradation. More recently, ubiquitin has also been shown to serve as a signal for endocytosis. Mib in D. rerio and Neur in Drosophila and Xenopus have been shown to associate with Dl, regulate Dl ubiquitination, and promote its endocytosis. Moreover, genetic and transplantation studies have indicated that both Neur and Mib act in a non-autonomous manner, indicating that endocytosis of Dl is associated with increased Dl signaling activity. Finally, epsin, a regulator of endocytosis that contains a ubiquitin-interacting motif and that is known in Drosophila as Liquid facet, is essential for Dl signaling. In one study, Liquid facets was proposed to target Dl to an endocytic recycling compartment, suggesting that recycling of Dl may be required for signaling. Accordingly, signaling would not be linked directly to endocytosis, but endocytosis would be prerequisite for signaling. How endocytosis of Dl leads to the activation of N remains to be elucidated. Also, whether the signaling activity of Ser is similarly regulated by endocytosis is not known (Le Borgne, 2005 and references therein).

While genetic analysis has revealed that neur in Drosophila and mib in D. rerio are strictly required for N signaling, knockout studies of mouse Neur1 have indicated that NEUR1 is not strictly required for N signaling. One possible explanation is functional redundancy with the mouse Neur2 gene. Conversely, the function of Drosophila mib (D-mib), the homolog of D. rerio mib gene has not previously been characterized (Le Borgne, 2005).

To establish the respective roles of these two distinct E3 ligases in the context of a single model organism, the function of the Drosophila D-mib gene was studied. D-mib, like D. rerio Mib, appears to regulate Dl signaling during leg segmentation, wing vein formation, and lateral inhibition in the adult notum. D-mib is specifically required for Ser endocytosis and signaling during wing development, indicating for the first time that endocytosis regulates Ser signaling. Interestingly, the D-mib activity was found necessary for a subset of N signaling events that are distinct from those requiring the activity of the neur gene. Nevertheless, the ectopic expression of Neur compensates for the loss of D-mib activity in the wing, indicating that Neur and D-mib have overlapping functions. It is concluded that D-mib and Neur are two structurally distinct proteins with similar molecular activities but distinct and complementary functions in Drosophila (Le Borgne, 2005).

This analysis first establishes that D-mib regulates Ser signaling during wing development. (1) Clonal analysis revealed that the activity of the D-mib gene is specifically required in dorsal cells for the expression of Cut at the wing margin. (2) Expression of D-mib in the dorsal Ser-signaling cells is sufficient to rescue the D-mib mutant wing phenotype. (3) Results from an in vivo antibody uptake assay indicate that the endocytosis of Ser (but not of Dl) was strongly inhibited in D-mib mutant cells. This inhibition correlates with the strong accumulation of Ser (but not Dl) at the apical cortex of D-mib mutant cells. Thus, an essential function of D-mib in the wing is to regulate the endocytosis of Ser in dorsal cells to non-autonomously promote the activation of N along the D-V boundary. By analogy, the defective growth of the eye tissue may similarly result from the lack of Ser signaling and of N activation along the D-V boundary. Because (1) D-mib co-localizes with Ser at the apical cortex of wing disc cells, (2) acts in a RING-finger-dependent manner to regulate Ser endocytosis in S2 cells, and (3) physically associates with Ser in co-immunoprecipitation experiments, D-mib may ubiquitinate Ser and directly regulate its endocytosis (Le Borgne, 2005).

This analysis further suggests that endocytosis of Ser is required for Ser signaling. This conclusion is consistent with observations made earlier showing that secreted versions of Ser cannot activate N but instead antagonize Ser signaling. Thus, endocytosis of both N ligands appears to be strictly required for N activation in Drosophila. Different models have been proposed to explain how endocytosis of the ligand, which removes the ligand from the cell surface, results in N receptor activation. Interestingly, the strong requirement for Dl and Ser endocytosis seen in Drosophila is not conserved in Caenorhabditis elegans, in which secreted ligands have been shown to be functional. Noticeably, there is no C. elegans Mib homolog, and the function of C. elegans neur (F10D7.5) is not known. It is speculated that endocytosis of the ligands may have evolved as a means to ensure tight spatial regulation of the activation of Notch (Le Borgne, 2005).

This analysis also establishes that the activity of the D-mib gene is required for a subset of N signaling events that are distinct from those that require the activity of the neur gene. The D-mib gene regulates wing margin formation, leg segmentation, and vein formation, whereas none of these three processes depend on neur gene activity. Conversely, the activity of the neur gene is essential for binary cell-fate decisions in the bristle lineage that do not require the activity of the D-mib gene (no bristle defects were seen in D-mib mutant flies). The activity of the neur gene is also required for lateral inhibition during neurogenesis in embryos and pupae. This process is largely independent of D-mib gene activity since the complete loss of D-mib function resulted only in a mild neurogenic phenotype in the notum. These data thus indicate that the neur and D-mib genes have largely distinct and complementary functions in Drosophila. Whether a similar functional relationship between Neur and D-mib exists in vertebrates awaits the study of the D. rerio neur genes and/or of the murine Mib and Neur genes (Le Borgne, 2005).

The functional differences observed between D-mib and neur cannot be simply explained by obvious differences in molecular activity and/or substrate specificity. Both Neur and D-mib physically interact with Dl and promote the down-regulation of Dl from the apical membrane when overexpressed. Furthermore, Dl signaling appears to require the activity of either Neur or D-mib, depending on the developmental contexts. Specific aspects of the D-mib phenotype in legs and in the notum cannot simply result from loss of Ser signaling and are consistent with reduced Dl signaling, suggesting that D-mib regulates Dl signaling. Consistent with this interpretation, overexpression studies indicate that D-mib up-regulates the signaling activity of Dl, whereas a dominant-negative form of D-mib inhibits it. It is noted, however, that no clear defects in Dl subcellular localization and/or trafficking were observed in D-mib mutant cells. It is conceivable that the contribution of D-mib to the endocytosis of Dl is masked by the activity of D-mib-independent processes that may, or may not, be linked to Dl signaling. It has also been shown that, reciprocally, Neur and D-mib may similarly regulate Ser. Neur and D-mib similarly promote down-regulation of Ser from the cell surface when overexpressed. Moreover, D-mib binds Ser and regulates Ser signaling. Whether endogenous Neur binds and activates Ser remains to be tested. However, the ability of Neur to rescue the D-mib mutant wing phenotype when expressed in dorsal cells strongly indicates that Neur can promote Ser signaling. Together, these data indicate that Neur and D-mib have similar molecular activities (Le Borgne, 2005).

D-mib and Neur may have identical molecular activities but distinct expression patterns, hence distinct functions at the level of the organism. Consistent with this possibility, D-mib is uniformly distributed in imaginal discs, whereas Neur is specifically detected in sensory cells. Importantly, the rescue of the D-mib mutant phenotype by ectopic expression of Neur strongly supports this interpretation. This result further suggests that Neur can regulate Ser signaling. Consistent with this idea, overexpression of Neur in imaginal discs results in a strong reduction of Ser accumulation at the apical cortex. Thus, despite their obvious structural differences, Neur and D-mib appear to act similarly to promote the endocytosis of Dl and Ser. Nevertheless, the observation that D-mib can not compensate for the loss of neur activity in the embryo indicates that D-mib and Neur have overlapping rather than identical molecular activities (Le Borgne, 2005).

In conclusion, Neur and D-mib appear to have similar molecular activities in the regulation of Dl and Ser endocytosis but distinct developmental functions in Drosophila. The conservation from Drosophila to mammals of these two structurally distinct but functionally similar E3 ubiquitin ligases is likely to reflect a combination of evolutionary advantages associated with: (1) specialized expression pattern, as evidenced by the cell-specific expression of the neur gene in sensory organ precursor cells, (2) specialized function, as suggested by the role of murine MIB in TNFα signaling and (3) regulation of protein stability, localization, and/or activity. For instance, Neur, but not D-mib, localizes asymmetrically during asymmetric sensory organ precursor cell divisions (Le Borgne, 2005).

The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta

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).

Loss- and gain-of-function analyses indicate that the major function of D-mib is to regulate Notch signal transduction. Since Delta is a bona fide substrate of zebrafish Mib, tests were performed for a physical association of D-mib and Delta by co-immunoprecipitation. Cultured cells were co-transfected with Delta and various D-mib expression vectors, and co-immunoprecipitation was performed in both directions. Although Delta did not successfully co-immunoprecipitate full-length D-mib, it did associate with all isoforms that contain the D-mib N terminus and lack the C-terminal RING finger (namely D-mib-N, D-mibDelta3RF and D-mibDeltaRF. Conversely, these same D-mib isoforms efficiently co-immunoprecipitate Delta; full-length D-mib also shows modest association with Delta in this direction. It was consistently observed that the presence of full-length D-mib reduces Delta levels, which might account for why this interaction is poorly detected. Notably, D-mib-N shows the strongest interaction with Delta. In fact, immunoprecipitated D-mib-N brings down both full-length Delta and cleaved DeltaIC, consistent with a direct interaction between the N terminus of D-mib and the intracellular domain of Delta. A truncated D-mib protein lacking the N-terminal domain (D-mib-C) shows no binding to Delta, demonstrating that this region is crucial for association with Delta (Lai, 2005).

Physical association between D-mib proteins and Serrate was tested. D-mib:Serrate interactions appear to be somewhat weaker than D-mib:Delta interactions; however, the overall profile of the different D-mib truncations in association with Serrate and Delta is identical. These findings lead to the conclusion that the N terminus of D-mib mediates physical association with both Drosophila DSL ligands. In addition, full-length D-mib similarly reduces the accumulation of Serrate, indicating that D-mib downregulates both DSL ligands (Lai, 2005).

In vitro data correlate well with in vivo studies, in that all RING-finger-deleted D-mib isoforms that retain the ability to associate with DSL ligands (D-mib-N, D-mibDeltaRF and D-mib3DeltaRF) have at least some ability to inhibit Notch signaling. However, full specificity and activity of D-mib requires inclusion of the ankyrin repeats and the two non-canonical RING fingers. Curiously, there is no significant similarity at the primary amino acid level between the intracellular domains of Delta and Serrate. In this regard, it is relevant to note that Xenopus Neur (X-Neur) robustly regulates Drosophila Delta in vivo, even though there is no significant similarity between the intracellular domains of Delta and X-Delta. D-mib and Neur may therefore recognize a more hidden, possibly structural, feature that is shared by DSL ligands (Lai, 2005).

The interplay between Delta and Serrate proteins and ubiquitin ligases in Notch signaling

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).

Role of conserved intracellular motifs in Ser signalling, cis-inhibition and endocytosis

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). This study identifies two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels (Glittenberg, 2006; full text of article).

Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. By generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, tests were performed of each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand-expressing cells. First, it was discovered that deletion or mutation of both Ubiquitin interaction motifs (UIM) destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the Epsin-N-terminal homology (ENTH domain) was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, it was found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand-expressing cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. It is proposed that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin (Xie, 2012).

Epsin is a complex multi-modular protein that functions differently in different contexts. Each Lqf isoform has two UIMs, two Clathrin binding motifs (CBMs), seven DPW motifs that bind the AP-2 endocytic adapter complex, and two NPF motifs that bind EH-domain-containing endocytic factors such as Eps15. In C. elegans, Drosophila, and mice, Epsin is needed specifically in Notch ligand cells. The structure/function analysis of Epsin performed in this study shows that modules of Epsin associate with the internalization step of endocytosis - the lipid binding function of the ENTH domain and the C-terminal modules that bind proteins present in Clathrin-coated vesicles - are required for Epsin's function in Notch ligand cells. In addition, it was shown that a UIM is necessary (Xie, 2012).

The dispensability of the Cdc42 GAP binding function of the ENTH domain suggests that in ligand cells the primary role of Drosophila Epsin, unlike yeast Ent1, is not regulation of actin dynamics. The other known function of the ENTH domain is the endocytic function, and the results suggest that the ability of the ENTH domain to interact with PIP2 explains why it is needed for maximal Epsin function in Notch ligand cells. These observations are consistent with the lack of typical Notch signaling defects in Drosophila cdc42 mutants. In contrast, flies with mutations in genes for either of two actin regulators, the Arp2/3 complex and WASp, do have notal bristle defects indicative of Notch signaling failure. The notal bristle phenotype described in this study is not due to failure of the Epsin-dependent endocytosis of ligand that activates Notch in all cell types, but instead to failure of ligand transcytosis required in only some cell types to relocalize ligand prior to signaling. The absence of the Arp2/3 complex or WASp in mutants inhibits signaling by blocking traffic of endocytosed Delta to apical microvilli of sensory organ precursors. Whether or not Delta transcytosis in sensory organ precursors also depends on Epsin is unknown. If Epsin is involved, it may be interesting to use the Epsin variant transgenes generated in this study to determine whether or not the Cdc42 GAP interaction function of the ENTH domain is required (Xie, 2012).

There are two types of UIMs: single-sided UIMs that bind one Ubiquitin, and double-sided UIMs that bind two Ubiquitins simultaneously. As the affinity between a UIM and Ubiquitin is low, successful interaction between a mono-ubiquitinated protein and a UIM-containing protein is thought to require either one double-sided UIM, or two single-sided UIMs. Epsins have single-sided UIMs, and so the observation that only one single-sided UIM is required for Drosophila Epsin function in Notch signaling is unexpected. The simplest explanation is that Notch ligands use multiple mono-Ubiquitins or Ubiquitin chains as a signal for Epsin-mediated internalization (Xie, 2012).

Two distinct Lysine residues in the intracellular domains of both Delta and Serrate have been implicated as important for the function of each ligand. In the case of Serrate, simultaneous mutation of both of these Lysines results in a Serrate ligand that can neither activate Notch nor be endocytosed in wing discs. These observations identify two particular Lysines as candidates for the critical Ub attachments, but do not distinguish whether one or both Lysines are required. In the case of Delta, single mutation of either of two specific Lysines results in accumulation of Delta at the cell surface of eye discs and failure to signal. Although Delta is thought to be mono-ubiquitinated, these results suggest the possibility that Delta is multiply mono-ubiquitinated. An alternative explanation for Epsin's ability to promote ligand endocytosis with a single UIM is that mono-ubiquitinated ligands cluster to generate an environment where multiple Ubiquitins attract Epsin to ligand at the plasma membrane (Xie, 2012).

There is compelling evidence that in somatic cells, Notch ligand endocytosis associated with signaling is Clathrin-dependent. First, there are exceedingly strong genetic interactions between the Clathrin heavy chain (Chc) gene and lqf, the gene for Epsin. Flies with only one Chc+ gene copy are wild-type, but this condition is lethal in homozygotes for a normally viable hypomorphic allele of lqf. Second, the Clathrin-coated vesicle uncoating protein Auxilin is, like Epsin, required specifically for Notch signaling in Drosophila and in ligand cells. Given the clear involvement of Clathrin and the lack of strong genetic interaction between α-Adaptin (the gene for an AP-2 subunit) and lqf, the simplest model for Epsin function in Notch signaling was as an adapter protein that links Clathrin and the plasma membrane, independent of AP-2. This model predicted that direct interaction between Epsin and Clathrin would be necessary, and thus the most surprising result of this work is that deletion of the CBMs had no detectable effect on Epsin activity. The dispensability of the CBMs rules out models where Epsin acts as a monomeric Clathrin adapter that links ligand to Clathrin cages (Xie, 2012).

In the Drosophila female germline, Notch signaling requires Epsin but neither Clathrin nor Auxilin. Although this is surprising, Epsin has been shown to function in Clathrin-independent internalization of ubiquitinated transmembrane cargos in vertebrate cell culture. Epsin must therefore function differently in Notch signaling in the female germline than in somatic cells. It is speculated that the ENTH domain and UIMs may be required in germline cells to guide the ubiquitinated proteins into Q6 an endocytic vesicle. However, it is not clear how any of the characterized modules within Epsin's C-terminus might be involved in Clathrin-independent endocytosis. It would be of interest to use the transgenes that were generated in this study to determine which motifs are required in the female germline. Additional experiments could potentially identify unknown C-terminal interaction motifs used in Clathrin-independent endocytosis (Xie, 2012).

Does Epsin function in the same way in the embryo, eye, and wing? The experiments began with the assumption that Epsin functions through the same mechanism in all signaling contexts, and thus it was expected the same Epsin modules would be required for Epsin function in all contexts. Epsin appears to be required in every Notch signaling event and thus could be regarded as a core component of Notch signaling. It therefore seems reasonable to expect that Epsin would function in the same manner in all tissues. The female germline is apparently an exception. Nevertheless, in the three assays used for Epsin activity - rescue of lethality and eye morphology defects due to lqf mutations and rescue of the ability of lqf null cells to activate Cut expression in cells at the D/V boundary in the wing disc - only subtle differences were detected between the eye and the wing in the activity of two Epsin variants, δENTH and δUIM. (The only major difference was with the highly artificial Epsin variant, 4XNPF.) Despite these differences, it is thought that Epsin likely functions the same way in the eye and wing, as well as during embryogenesis. For one, the differences in activity that were observed be explained easily without invoking different mechanisms for Epsin in the eye and wing. Importantly, no even one case was observed where modules were essential in one context (embryogenesis, eye, or wing development) and dispensable in another one. In fact, it is possible to observe all-or-none differences in requirements for Epsin modules. Epsin was found to function outside of Notch ligand cells and modules were found that were dispensable completely in this context yet absolutely essential for Epsin's function in ligand cells (Xie, 2012).

Notch ligands require ubiquitination and (usually) Clathrin-dependent endocytosis, and formation of Clathrin-coated vesicles requires adapter proteins that link the plasma membrane with Clathrin. The absolute necessity of at least one UIM and the observation that the lipid-binding function of the ENTH domain plays a role in ligand cells suggests that Epsin indeed binds ubiquitinated Notch ligands at the plasma membrane.However, as an Epsin derivative lacking CBMs functions as well as wild-type Epsin in ligand cells, the essential role of Epsin in Notch signaling cannot be as a monomeric Clathrin adapter that links Clathrin directly to ligand at the plasma membrane. As any pair of the three types of modules is sufficient for Epsin function (CBMs+DPWs, CBMs+NPFs, or DPWs+NPFs), Epsin must be able to support Notch activation by linking ligand to Clathrin in a variety of different ways. It is speculated that Eps15, the second Drosophila Epsin, is involved because of the three EH-domain proteins in Drosophila (Eps15, Dap160, Past1), none have Clathrin binding motifs, and Eps15 is the only one with motifs for a known Clathrin-binding protein (AP-2). From analysis of mutant phenotypes and genetic interaction studies, there is no evidence for the involvement of Eps15 nor AP-2 in Notch signaling (Xie, 2012). The results presented in this study suggest that Eps15 and AP-2 may play redundant roles in the presence of intact Epsin and this idea could be tested with additional genetic experiments. In light of the evidence indicating a requirement for Clathrin in ligand cells (outside of the germline), the results suggest that Epsin is required absolutely for Notch signaling not because it generates a special endocytic environment, but simply because it is the only UIM-containing endocytic protein with the appropriate complement of interaction modules to target ubiquitinated cargo to Clathrin-coated vesicles (Xie, 2012).

Functional analysis of the NHR2 domain indicates that oligomerization of Neuralized regulates ubiquitination and endocytosis of Delta during Notch signaling

The Notch pathway plays an integral role in development by regulating cell fate in a wide variety of multicellular organisms. A critical step in the activation of Notch signaling is the endocytosis of the Notch ligands Delta and Serrate. Ligand endocytosis is regulated by one of two E3 ubiquitin ligases, Neuralized (Neur) or Mind bomb. Neur is comprised of a C-terminal RING domain, which is required for Delta ubiquitination, and two Neur homology repeat (NHR) domains. Previous studies have shown that the NHR1 domain is required for Delta trafficking. This study shows that the NHR1 domain also affects the binding and internalization of Serrate. Furthermore, it was shown that the NHR2 domain is required for Neur function and that a point mutation in the NHR2 domain (Gly430) abolishes Neur ubiquitination activity and affects ligand internalization. Finally, evidence is provided that Neur can form oligomers in both cultured cells and fly tissues, which regulate Neur activity and, by extension, ligand internalization (Liu, 2012).

Neur is an E3-ubiquitin ligase that plays an essential role in Notch signaling by regulating the endocytosis of Notch ligands. It contains NHR domains, which are rare and conserved between vertebrates and invertebrates but not present in viruses, bacteria, fungi, or plants. In the Drosophila proteome, besides Neuralized, there are two other NHR-containing proteins, CG3894 and Bluestreak. In mammals, proteins containing NHR domains (also known as NEUZ) include the β-catenin regulator OzzE3 and lung-inducible Neuralized-related C3HC4 RING protein (LINCR). Recent studies reported that the human homologue of Bluestreak serves to localize to the centrosome. Although the general role of the NHR domains is unclear, these domains tend to cluster, and most proteins contain two to six NHR domains. The significance of having more than one NHR domain in one protein is to yet be determined (Liu, 2012).

This study has investigated the role of the highly conserved NHR domains in Neur function. The NHR1 domain alone mediates the interaction between Neur and both Delta and Serrate. It was also shown that the NHR2 domain is required for Neur function, and while it is not required for the interaction with Notch ligands, it is involved in Dl internalization, a critical step in Notch activation. Moreover, the NHR domains play a role in Neur oligomerization, which in turn could contribute to Neur ubiquitination activity and ligand endocytosis (Liu, 2012).

The NHR1 domain mediates the interaction between Neur and the Notch ligands Delta and Serrate. Previous studies have shown that the NHR1 domain of Neur is both necessary and sufficient for the interaction with the Notch ligand Dl. Specifically, it was found that a point mutation in a highly conserved glycine residue within the NHR1 domain (G167E) abolishes the ability of Neur to bind Delta. Whether the NHR1 domain was also required to bind to Serrate, however, was unknown. In fact, in vitro studies in vertebrates suggested that the NHR2 domain in mouse Neuralized- like 1 (Neurl1) is sufficient to bind to Jagged1, the mouse orthologue of Serrate. In contrast, the current study found that the NHR2 domain is not required for Neur to bind Serrate in Drosophila and that the interaction is mediated entirely by the NHR1 domain. Other studies reported previously that the motifs on Dl and Ser that mediate the interaction with Neur are conserved. In comparisons of protein sequences, Jagged1 and Serrate share 40.7% similarity overall, while the overall similarity between Jagged1 and Dl is 33.8%. The NHR1 and NHR2 domains from Drosophila Neuralized have the same degree of amino acid similarity with the mouse Neurl1 NHR2 domain (33%). Since there is no clear correlation between protein sequence similarity and the ability of either the NHR1 or the NHR2 domain to interact with Notch ligands, whether NHR1 or NHR2 is important for interacting with ligands is likely to be species dependent. The Neur-Ser interaction was found to be abrogated by the G167E mutation in the NHR1 domain. Given that the NeurG167E mutant still retains ubiquitination activity, it is unlikely to affect overall protein folding. A previously reported structural analysis of the Drosophila NHR1 domain suggested that Gly167 resides in a hydrophobic core and that the Gly167 mutation presumably destabilizes the surrounding microenvironment. Therefore, the Gly167 mutation may result in spatial changes in the neighboring residues of the core, thus abolishing binding to ligands (Liu, 2012).

The G430E mutation reveals a distinct role for the NHR2 domain in the regulation of Neur activity and Delta trafficking. The data demonstrate that the NHR2 domain is required for Neur function in vivo. NeurG430E fails to rescue neur mutant embryos, while NeurNHR2 has some residual activity, which suggests that they affect different aspects of Neur function. The expression of NeurG430 in a heterozygous background, which does not have a neurogenic phenotype on its own, resulted in a significant increase in the percentage of neurogenic embryos, suggesting that NeurG430E has a negative effect on Neur function. In contrast, NeurNHR2 overexpression did not have any effect on heterozygous embryos, suggesting that it behaves as a loss-of-function allele. Despite the fact that the two NHR2 mutant proteins NeurG430E and NeurNHR2 behave differently, they both localize to the plasma membrane in the presence of Delta both in vitro and in vivo, and they are both capable of binding to the Notch ligands Delta and Serrate. However, both mutant proteins affect the extent of Dl internalization to various degrees. NeurG430E exhibits severely compromised ubiquitination activity and is no longer capable of inducing Delta internalization. NeurNHR2, on the other hand, retains ubiquitination activity but is much less efficient than WT Neur at directing Dl internalization in vivo or in Kc cells. The precise mechanism by which NeurNHR2 affects Delta endocytosis is unclear (Liu, 2012).

One possibility is that the NHR2 domain is required for Neur oligomerization. Neur was shown to form NHR domain-mediated oligomers by coimmunoprecipitation experiments. Therefore, the deletion of the NHR2 domain (NeurNHR2) may simply reduce the oligomerization potential of Neur, leading to a decrease in ligand endocytosis. In contrast, the point mutation (NeurG430) might disrupt the overall structure of the NHR2 domain, preventing oligomerization and resulting in a protein that has no ubiquitination activity and therefore can no longer internalize ligands. However, this model cannot fully explain the data, which show that NHR2 does not prevent Neur oligomerization and that Neur oligomerization still occurs in the absence of any NHR domains, suggesting that while the NHR domains may play a role in oligomerization, they are not necessary for this process. The data also show that although the G430E mutant loses ubiquitination activity, the double mutant containing the NHR1 deletion and the G430E mutation retains ubiquitination activity, which argues that G430E does not affect the overall folding of the NHR2 domain (Liu, 2012).

Another possibility is that the NHR1 and NHR2 domains initially form an intramolecular structure that is inactive and must be resolved for ubiquitination to promote ligand internalization. The G430E mutation may lock Neur into an intramolecular conformation through an NHR1-NHR2 interaction, such that this inactive form can still bind to Dl and Ser but cannot form oligomers and has no ubiquitination activity as a consequence of dysfunctional oligomerization. This model, in contrast to the former one, is supported by the data that demonstrate that the G430E mutant no longer forms oligomers and has no ubiquitination activity. Furthermore, when the NHR1 domain was removed from the G430E mutant (NeurNHR1G430E), the ubiquitination activity was restored, suggesting that the intramolecular loop can no longer form, whereas intermolecular interactions between NHR1 and NHR2 domains can occur. It may also explain the negative effect of NeurG430E on NeurWT: although NeurG430E has a reduced ability to bind WT Neur, it is still be able to sequester some portion of NeurWT into a nonfunctional intermolecular oligomer that can no longer ubiquitinate targets. In contrast, the deletion of the NHR2 domain would prevent intramolecular interactions but would be expected to have a reduced ability to form productive oligomers, leading to a defect in ligand internalization. Whether the NHR2 domain has additional roles in recruiting a protein(s) that promotes Notch ligand ubiquitination and endocytosis remains to be determined. Like Neuralized, other RING domain E3 ligases often function as oligomers, and they multimerize in different ways: some form heterodimers, such as Mdm2-MdmX, and some can form homo-oligomers, such as TRAF. The functional significance of RING E3 oligomerization is poorly defined. One previously proposed model is that the oligomerization of E3 ligases may functionally resemble the dimerization of receptor tyrosine kinases in such a way that autoubiquitination yields a mark that serves as a platform to assemble a signaling complex. Consistent with this idea, ubiquitination was seen in anti-Neur IPs when Dl was not present, consistent with the idea that Neur may be autoubiquitinated. Whether this autoubiquitination can initiate a cascade of further downstream ubiquitination events remains to be determined. It is possible that oligomerization is mediated via autoubiquitination and the interaction of ubiquitinated Neur with itself through a ubiquitin-binding motif (UIM). If so, then the NHR1-NHR2 interaction could also function to keep Neur in an inactive state by occluding the putative UIM. Such a model of Ubi-UIM complex formation was previously proposed for other endocytic proteins (Liu, 2012).

In summary, this study has shown that NHR domains are protein-protein interaction modules that are required for many aspects of Neur function. The NHR1 domain mediates the interaction between Neur and its targets Dl and Ser. Both NHR domains appear to regulate Neur activity by affecting its ability to form oligomers and/or interact with proteins required for the endocytosis of Notch ligands. Interestingly, NHR domains have been identified in several other proteins that are conserved between flies and humans. Whether Neur can form heterodimers with these other NHR-containing proteins and whether these heterodimers play a role in Notch signaling and other developmental processes remain to be determined (Liu, 2012).

Serrate-Notch-Canoe complex mediates glial-neuroepithelial cell interactions essential during Drosophila optic lobe development

It is firmly established that neuron-glia interactions are fundamental across species for the correct establishment of a functional brain. This study found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of the optic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions generating neuroblasts. The proneural gene lethal of scute (l'sc) is transiently activated by the Epidermal Growth Factor Receptor (EGFR)/Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateral neuroepithelium promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium of multiple signaling pathways, including EGFR/Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex is crucial for glial-neuroepithelial cell interactions during optic lobe development. Ser is tissue-autonomously required in the glia where it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling to avoid the premature activation of the EGFR/Ras pathway and hence of L'sc. Interestingly, different Notch activity reporters showed very different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote the expression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling (Perez-Gomez, 2013).

Cno and its vertebrate homologues AF-6/Afadin localize at epithelial AJs where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and nectin family proteins. This study found that Cno colocalizes with Notch at the AJs of NE cells in the optic lobe proliferation centers. Notch also colocalizes with its ligand Ser, which was detected at the glia, highly accumulated at the interface between NE cells and the surrounding glia. Co-immunoprecipitation experiments indicate the formation of a Ser-Notch-Cno complex in vivo, and the mutant analysis shows the functional relevance of such a complex at the glia neuroepithelium interface. The data presented in this study support the hypothesis that Cno may be stabilizing Notch at the AJs of NE cells, favoring the binding of Ser present in the adjacent glial cells. Indeed, in cno lof both Notch and Ser distribution is affected; this alteration is accompanied by an abnormally advanced proneural wave, a reminiscent phenotype to that shown by Notch lof optic lobes and also a similar phenotype found in this work in Ser lof. The activation of Notch pathway is essential to maintain the integrity of the neuroepithelium and to allow the correct progression of the proneural wave. The results show that glial Ser is responsible of such activation, promoting the expression of the m7-nuclacZ reporter in NE cells. In fact, the reduction of glial Ser either by knocking down epithelial cno or by expressing DNSer in the glia led to a decrease in the expression of the m7-nuclacZ reporter in NE cells and to an ectopic activation of the Ras/PntP1 pathway and of L'sc. It is proposed that this may be ultimately the cause of the proneural wave advance observed in those genotypes. Thus, the activation of Notch in the neuroepithelium by glial Ser, in nomal conditions, would be essential to avoid a premature activation of the EGFR/Ras/PntP1 pathway and hence of L'sc. Indeed, Notch has been shown to downregulate different EGFR/Ras signaling pathway components such as Rhomboid (Rho), required for the processing of the EGFR ligand Spitz, in other developmental contexts in which both pathways are actively cross-talking. Therefore, Notch activity in NE cells could be contributing to inhibit Rho, restricting its presence to the transition zone where Rho is very locally expressed (Perez-Gomez, 2013).

It was observed that in a WT condition Ser is present in all surface glia (perineurial and subperineurial), as shown by the expression of CD8::GFP (SerGal4>>UAS-CD8::GFP), and Notch, as tested by different reporters, is active in this tissue and highly reduced in Ser lof in the glia. This makes sense with the existence of a Ser-Notch mediated intercellular communication between the glial cells that comprise both the perineurial and subperineurial glia. Intriguingly, the knockin down and overexpression of cno in NE cells also had a clear effect on Notch activity in the glia, a reduction and an increase, respectively. This is more challenging to explain. As the cno lof in the NE led to a high reduction of both neuroepithelial Notch and glial Ser, the easiest explanation is that an 'excess' of unbound glial Ser is degraded and this impinges on the general thresholds of glial Ser, therefore causing a general reduction in the Notch activity in this tissue. This is an interesting field to explore in detail and is left open for future investigation (Perez-Gomez, 2013).

The activity of Notch in the neuroepithelium and in medulla NBs seems controversial. For example, Notch has been shown to be active in the neuroepithelium at low/null levels or in a 'salt and pepper' patter. A weak/null activity of Notch has also been reported in NBs as well as a high activation. One possibility to conciliate all these results and apparently contradictory data is that different Notch target genes used as Notch activity reporters are, in fact, differentially activated in particular regions or tissues. The results support this proposal. Four different Notch reporters were used in this study. Whereas m7-nuclacZ was expressed throughout the neuroepithelium, Gbe+Su(H)lacZ was restricted to the transition zone, although both were expressed in medulla NBs along with mβ-CD2. In addition, mβ-CD2 was strongly activated in the glia, whereas the Gbe+Su(H)lacZ and the mδ-lacZ reporters were expressed at much lower levels at this location. Differential activation of Notch targets genes has been previously reported and tissue-specific factors could contribute to this differential expression. This is an intriguing scenario to analyze in the future. The in depth analysis of other Notch reporter genes in the developing optic lobe can contribute to further clarify this issue (Perez-Gomez, 2013).

At third larval instar during optic lobe development, Dl is highly restricted to 2-3 cells at the transition zone in the neuroepithelium, where Dl activates Notch. This work has found that the other ligand of Notch, Ser, is expressed in the surrounding glia at this larval stage and it is strongly accumulated at the interface with NE cells. Ser activates Notch in the neuroepithelium and this, in turn, would contribute to restrict the activation of the Ras-PntP1 pathway and L'sc to the transition zone. Intriguingly, it was observed that Ser preferentially activates the Notch target gene m7-nuclacZ in the neuroepithelium whereas Dl activates other Notch target genes, including Gbe+Su(H)lacZ, in the transition zone. For example, the overexpression of Dl in NE cells caused an ectopic activation throughout the neuroepithelium of Gbe+Su(H)lacZ, along with dpn that also behaves as a Notch target in other systems, and a repression of m7-nuclacZ . In addition, the lof of Ser in the glia caused a striking decrease in the expression of m7-nuclacZ in the neuroepithelium. One possibility to explain these observations is that the pool of Notch associated to the AJs and activated by glial Ser is subject of particular posttranslational modifications or/and is associated with other AJs proteins (including Cno) that somehow make Notch more receptive to Ser and able to activate specific target genes (i.e., m7). In this regard, it is interesting to note that Dl ectopically expressed in the glia (i.e., repoGal4>>UAS-Dl) was not detected at the interface with NE cells, where glial Ser is highly present in contact with Notch, but Dl was restricted to the outermost surface glia (perineurial glia). This result strongly indicates that Dl cannot bind or has very low affinity for this pool of Notch at the AJs, hence being actively degraded in the subperineurial glia. This low affinity of Dl by Notch at this location further suggests that this pool of Notch at the AJs must be endowed with particular characteristics that ultimately could alter the activity properties of Su(H), explaining in turn the distinct expression pattern of Notch targets genes. Another possibility, which is not necessarily exclusive, to explain the differential activation of the Notch reporters is that they respond to different Notch thresholds. For example, m7-nuclacZ would require very low levels of Notch activation whereas Gbe+Su(H)lacZ would require high amounts of Notch signaling in NE cells. All these questions remain open for further investigation (Perez-Gomez, 2013).

Structural analysis uncovers lipid-binding properties of Notch ligands

The Notch pathway is a core cell-cell signaling system in metazoan organisms with key roles in cell-fate determination, stem cell maintenance, immune system activation, and angiogenesis. Signals are initiated by extracellular interactions of the Notch receptor with Delta/Serrate/Lag-2 (DSL) ligands, whose structure is highly conserved throughout evolution. To date, no structure or activity has been associated with the extreme N termini of the ligands, even though numerous mutations in this region of Jagged-1 ligand lead to human disease. This study demonstrates that the N terminus of human Jagged-1 is a C2 phospholipid recognition domain that binds phospholipid bilayers in a calcium-dependent fashion. Furthermore, this activity is shared by a member of the other class of Notch ligands, human Delta-like-1, and the evolutionary distant Drosophila Serrate. Targeted mutagenesis of Jagged-1 C2 domain residues implicated in calcium-dependent phospholipid binding leaves Notch interactions intact but can reduce Notch activation. These results reveal an important and previously unsuspected role for phospholipid recognition in control of this key signaling system (Chillakuri, 2013).

Cis-interactions between Notch and its ligands block ligand-independent Notch activity

The Notch pathway is integrated into numerous developmental processes and therefore is fine-tuned on many levels, including receptor production, endocytosis, and degradation. Notch is further characterized by a two-fold relationship with its Delta-Serrate (DSL) ligands, as ligands from opposing cells (trans-ligands) activate Notch, whereas ligands expressed in the same cell (cis-ligands) inhibit signaling. This study, carried out in vivo during oogenesis and in cultured Drosophila cells shows that cells without both cis and trans ligands are able to mediate Notch-dependent developmental events during Drosophila oogenesis, indicating ligand-independent Notch activity occurs when the receptor is free of cis and trans ligands. Furthermore, cis-ligands can reduce Notch activity in endogenous and genetically-induced situations of elevated trans-ligand-independent Notch signaling. It is concluded that cis-expressed ligands exert their repressive effect on Notch signaling in cases of trans-ligand independent activation, and a new function of cis-inhibition is proposed which buffers cells against accidental Notch activity (Palmer, 2014: PubMed).

Serrate: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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