Table of contents

Notch and Kuzbanian

Notch and the disintegrin metalloprotease encoded by the kuzbanian (kuz) gene are both required for a lateral inhibition process during Drosophila neurogenesis. A mutant Kuz protein lacking protease activity acts as a dominant-negative form in Drosophila. Expression of such a dominant-negative Kuz protein can perturb lateral inhibition in Xenopus, leading to the overproduction of primary neurons. This suggests an evolutionarily conserved role for Kuz (Pan, 1997).

Genetic and biochemical evidence is provided that Notch is an in vivo substrate for the Kuz protease, and that this cleavage may be part of the normal biosynthesis of functional Notch proteins. However, additional evidence, provided below, indicates that Delta and not Notch is the target of Kuz (Qi, 1999). Flies were generated that express, under heatshock control, dominant negative Kuzbanian (KUZDN). Heat pulses applied during third instar larval stage result in supernumerary macrochaetes only, while heat pulses applied during early pupal stages result in supernumerary microchaetes only. These time points match the periods when SOPs for each bristle type are selected from pools of equivalent cells, suggesting that KUZDN interferes with lateral inhibition during the selection of SOPs. KUZDN was expressed in the morphogenetic furrow of the developing eye under the control of the rough promoter. Flies carrying the rough/KUZDN transgene have supernumerary photoreceptor cells in each ommatidium. kuz is also required for axonal extension at later stages of neural development. Embryos expressing KUZDN in developing neurons show major defects in axonal pathways, such as disruption of longitudinal axonal tracts (Pan, 1997).

kuzbanian acts genetically upstream of Notch. If kuz should act genetically downstream of N then the combination of an activated form of Notch (Nact) and kuz should display the kuz phenotype of extra microchaetes. Conversely, if kuz acts genetically upstream of N then the combination of Nact and kuz should display the Nact phenotype of missing microchaetes. The combination of Nact and kuz display the Nact phenotype, suggesting the kuz acts genetically upstream of N (Pan, 1997).

Expression of KUZDN in cultured cells expressing Notch abolishes a 100 kDA Notch species while a 300 kDa Notch species is not greatly affected. KUZDN also affects the 100kDa species in third instar imaginal discs. After the induction of KUZDN by heat shock, the 100 kDa species disappears; by 4 hours after induction the 100 kDa species is almost undetectable, while the 300 kDa species has accumulated to a higher level. Only the 300 kDA species of Notch is detected in kuz null embryos (Pan, 1997).

Loss of Kuzbanian, a member of the ADAM family of metalloproteases, produces neurogenic phenotypes in Drosophila. It has been suggested that this results from a requirement for kuzbanian-mediated cleavage of the Notch ligand Delta. Using transgenic Drosophila expressing transmembrane Notch proteins, it has been shown that kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. Kuzbanian can physically associate with Notch and removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. These data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch (Lieber, 2002).

Pan and Rubin (1997) originally proposed that Kuz cleaves Notch. This proposal is in accord with the cell-autonomous requirement for kuz both in Drosophila and in C. elegans. More recently it has been suggested that the phenotypes resulting from loss of kuz are attributable to its role in the processing of Dl (Qi, 1999), although in that case the requirement for kuz would be non-cell-autonomous. This study shows that Kuz can associate with N, and that removal of kuz activity from S2 cells, which do not express Dl, blocks the processing of ligand-independent gain-of-function N molecules. In vivo, a gain-of-function N molecule that is completely Dl-independent displays an absolute requirement for kuz. These results show that kuz can mediate the cleavage of N, and are therefore in agreement with Pan and Rubin's original proposal. However, whereas Pan and Rubin proposed that kuz mediates S1 cleavage, the data suggest that kuz is responsible for S2 cleavage (Lieber, 2002).

The role of kuz/ADAM10 in the N pathway in vertebrates is uncertain. It has been shown that expression of a dominant-negative form of mouse Kuz causes the overproduction of neurons in Xenopus (Pan, 1997) and inhibits Delta-1-like-induced transactivation of a HES-1 reporter in HeLa cells expressing Notch-1. Yet both S2 and S3 cleavages of a ligand-independent N protein occur in cells derived from kuz mutant mice. Whereas mammals have a single kuz/ADAM10 gene, Drosophila has two: kuz, the focus of this work, and another ADAM10 homolog. Perhaps Drosophila kuz has evolved a function distinct from that of ADAM10 (Lieber, 2002).

Although both LNLexA and LNRLexA (mutant LexA tagged N proteins consisting solely of the the soluble cytoplasmic domain of N) function in the absence of the ligand Dl, the activity of LNRLexA, deleted for a fewer number of amino acids than is LNLexA, is greater than that of LNLexA. This difference is not caused by an enhanced affinity of Kuz for LNRLexA, as both associate equally with Kuz in S2 cells. This suggests that the difference in activity of these gain-of-function N molecules results from an enhanced ability of LNRLexA to be cleaved by Kuz. In fact, the interaction of LN and LNR with Kuz is no greater than that of wild-type N, and the association of Kuz with any of the N molecules occurs in the absence of Dl. In this regard, the association of Kuz with N is like that of Kuz with ephrin-A2, which forms a stable complex with Kuz prior to Eph receptor binding. The binding of clustered Eph receptors to the Kuz-ephrin-A2 complex activates Kuz and triggers ephrin-A2 cleavage. Likewise, the binding of Dl to the Kuz-N complex could activate Kuz and trigger N cleavage (Lieber, 2002).

A curious result is the difference in Kuz dependence of LNRLexA and DeltaEGF1-18 LNRLexA (for NDeltaEGF1-18 LNRLexA) in vivo. Two explanations are offered for this observation. The first takes into account the difference in Dl responsiveness between Delta1-18 LNRLexA and LNRLexA. LNRLexA, which has reduced activity in the absence of Dl, still retains some function in the absence of Kuz, whereas Delta1-18 LNRLexA, which is completely Dl-independent, cannot function in the absence of kuz. One possible explanation for the discrepancy is that there are two pathways that mediate N cleavage and function in embryos. One pathway requires Dl but is independent of Kuz, and the other pathway requires Kuz but is independent of Dl. LNRLexA can operate in both pathways, so that upon removal of either Dl or Kuz, LNRLexA still functions via the alternative remaining pathway. Delta1-18 LNRLexA can only operate in the kuz pathway, so that upon removal of kuz it is nonfunctional. There is, however, no strong evidence pointing to a Dl-independent N pathway involving cleavage in embryos, and given the requirement for Dl in the germ line for the differentiation of follicle cells, the generation of embryos that are maternally Dl null is not straightforward (Lieber, 2002).

Therefore the hypothesis is favored that the difference in kuz-dependence of LNRLexA and Delta1-18 LNRLexA in vivo is owing to their differing abilities to be cleaved by TACE. TACE, another member of the ADAM family of metalloproteases, mediates S2 cleavage of mammalian N in vitro. Drosophila S2 cells do not contain any detectable TACE RNA, and exogenous TACE only poorly complements the RNAi-mediated loss of kuz activity. The restoration of some S3 product upon expression of TACE is in accord with the residual activity of LNRLexA in kuz embryos, and in vitro TACE and N do interact, albeit less well than do Kuz and N. In the absence of a TACE mutant, it is not possible to say for certain whether kuz and TACE have redundant functions, if another member of the ADAM family is responsible for the residual S3 cleavage, or if the residual in vivo activity is caused by the expression of LNRLexA from a heterologous promoter. Hardly any S3 product was generated from Delta1-18 LNRLexA by exogenous TACE in S2 cells that had been treated with kuz double-stranded RNA, accounting for the in vivo Kuz-dependence. It is not clear why the ability of exogenous TACE to produce S3-cleaved N differs between LNRLexA and Delta1-18 LNRLexA; however, in S2 cells, even in the presence of endogenous Kuz, Delta1-18 LNRLexA is not cleaved as well as is LNRLexA, suggesting that perhaps differences in the secondary structure of the molecules account for their differing responses to TACE (Lieber, 2002).

The pattern of cleavage products generated by expression of TACE in kuz- S2 cells also provides an explanation for the in vivo biochemical data, which had seemed to suggest that kuz is responsible for S3 cleavage. It is intriguing that both in kuz embryos and in TACE-complemented kuz- S2 cells there is an accumulation of a protein the size of S2-cleaved N, which is not efficiently cleaved further to produce S3-cleaved N. It is proposed that although TACE can cleave N at juxtamembrane sites, a large fraction of this cleavage is occurring at a site close to but distinct from the S2 site that allows for efficient S3 cleavage. This suggests that cleavage of N at any juxtamembrane site is not immediately followed by efficient S3 cleavage (Lieber, 2002).

A surprising result is the suppression of the kuz neurogenic phenotype by expression of NLexA. In fact, the size of the nervous system in NLexA kuz embryos is smaller than in LNRLexA kuz embryos. Since kuz embryos are neurogenic, the suppression must result from the overexpression of N. This, along with the fact that the suppression does not involve the association of the cytoplasmic domain of N with Su(H), suggests that in the absence of kuz, overexpressed N is competitively interacting with a protein required for neurogenesis. Wild-type N accumulates to a higher steady-state level on the cell surface than does LN or LNR. It has been shown that expression of the extracellular domain of N can disrupt the establishment of proneural clusters in the developing wing disc (Lieber, 2002).

In summary, it has been shown that in flies S2 cleavage can be mediated by kuz. This contrasts with mammalian data that suggest S2 cleavage occurs via TACE. The discrepancy might be owing to mechanistic differences between flies and mammals as has also been shown for S1 cleavage (Lieber, 2002).

Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. Given the similar phenotypes produced by loss of Notch signaling and loss-of-function mutations for kuz, it has been suggested that Kuz may be involved in the cleavage of N (Pan, 1996). This hypothesis is not corroborated by recent biochemical studies, indicating that the functionally crucial cleavage of N in the trans-Golgi network is catalyzed by a furinlike convertase (Logeat, 1997). These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands (Qi, 1999).

A genetic screen to identify modifiers of the phenotpes associated with the constitutive expression of a dominant negative transgene of kuz (kuzDN) in developing imaginal discs has identified Delta as an interacting gene (X. Wu, W. Wang, and T. Xu, unpublished observation reported by Qi, 1999). Flies expressing this dominant negative kuz construct, despite carrying a wild-type complement of kuz, become semi-lethal when heterozygous for a loss-of-function Delta mutation (X. Wu, W. Wang, and T, Xu, unpublished observation reported by Qi, 1999). In contrast, Delta duplications rescue the phenotypes associated with kuzDN. The kuzDN flies display extra vein material (especially deltas at the ends of the longitudinal veins); wing notching (observed with a low penetrance); extra bristles on the notum, and they also have small rough eyes. When kuzDN flies carry three, as opposed to the normal two, copies of wild-type Notch the bristle and eye phenotypes are not affected, nor are the vein deltas altered. However, the kuzDN phenotypes are effectively suppressed by Delta duplications, indicating that a higher copy number of Dl molecules is capable of overriding the effects of the kuzDN construct (Qi, 1999).

Delta has been shown to be cleaved by Kuz in transfected cultured cells, with the release of the extracellular domain of Delta. Sequencing reveals a putative propeptide processing site that is conserved in all Delta homologs. There is a distinct absence of cleaved Delta in kuz minus embryos but no difference in the processing of Notch. The biological activity of Delta extracellular domain can be detected in culture. Ligand-dependent Notch activation has been demonstrated in cortical neurons, which express endogenous Notch receptors, causing morphological changes as well as retractions of neurites. The same effects are observed when neurons are cultured in the presence of the extracellular domain of Delta. The importance of additional cleavages in Dl, the mode of activity of full-length Dl, and whether the second ligand Serate is also processed are critical questions to resolve. It is now apparent that future analysis of Delta in Notch signaling events must consider its potential as a diffusable ligand (Qi, 1999).

Notch interaction with Wingless

The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Frizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression, and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during Drosophila development is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch (Wesley, 1999).

Notch, a cell surface receptor, is required for the production of different types of cells during Drosophila development. Notch activates expression of one set of genes in response to the ligand Delta and another set of genes in response to the ligand Wingless. Just how Notch initiates these different intracellular activities has been the focus of this study. Cultured cells expressing Notch were treated with either Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta results in accumulation of ~120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch does not accumulate in cells treated with Wingless; rather, the ~350-kDa full-length Notch molecules accumulate. These results indicate that N responds differently to binding by Delta and by Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane. It is proposed that the N receptor is a 'switch' for activation of different signaling pathways during development. Dl binds the EGF-like repeats 11-12 region to shunt the N120-Su(H) complex into the nucleus, turning on the expression of Dl-related genes. Wg binds the EGF-like repeats 19-36 region to send a transcriptional activator to the nucleus, turning on the expression of Wg-related genes (Wesley, 2000a).

Schneider cells expressing Notch (S2-N cells) treated with Dl for 1h accumulate ~120-kDa N molecules (N120). Dl binds N in the extracellular region, including EGF-like repeats 11 and 12). S2 cells expressing N molecules lacking this region, NDeltaEGF1-18, do not accumulate N120 molecules in response to treatment with Dl. This indicates that N120 accumulates in response to Dl binding N. N120 is the complete intracellular domain and is similar to the ~120-kDa N intracellular domain molecule shown to accumulate in vivo in response to D (Wesley, 2000a).

N120 molecules do not accumulate in S2-N cells treated with Wg for 1, 2, or 5 hours. However, S2-N cells treated with Wg for 5h accumulates ~350-kDa N molecules (N350) but not S2-N cells treated with Dl. N350 is the full-length co-linear N molecule containing both the intracellular and extracellular domains. Wg binds N in the EGF-like repeats 19-36 region. S2 cells expressing N molecules lacking this region, NDeltaEGF19-36, do not accumulate co-linear molecules when treated with Wg for 5h. In contrast, truncated, co-linear NDeltaEGF19-36 molecules containing the Wg binding sites accumulate upon treatment with Wg. These results indicate that accumulation of N350 in S2-N cells is in response to Wg binding N. Accumulation of N350 molecules is also discernible in cells treated with Wg for 2h when the blots are exposed to film for shorter periods. In contrast to Wg-treated cells, Dl-treated cells in the same blots always have lower levels of N350 compared with the levels in untreated cells (Wesley, 2000a).

Accumulation of N350 molecules in Wg-treated cells is not due to activity of the endogenous Notch gene, which is rearranged in S2 cells. It is not due to a general increase or stabilization of all proteins in the cells: all N molecules do not accumulate, and the total protein levels in the three lanes are comparable. It is also not due to a Wg effect that is unrelated to N binding but retards N processing for cell surface presentation. Otherwise, co-linear NDeltaEGF19-36 would have also accumulated, but it did not. Thus, whereas Dl binding full-length N results in accumulation of N120, Wg binding results in accumulation of the co-linear N350 (Wesley, 2000a).

Treatment of S2-N cells with Dl or Wg for 2 h also results in accumulation of ~55-kDa N molecules (N55). N55 contains only the amino terminus half of the intracellular domain, requires about 2 h to accumulate, and is variably recovered after about 3 h of treatment (Wesley, 2000a).

To determine whether the responses observed in S2 cells are general N responses to treatments with Dl and Wg, the experiments were repeated with clone-8 cells that express N endogenously. The results show that N in clone-8 cells responds similarly to N in S2 cells. Treatment with Dl results in accumulation of N120 and not N350, whereas treatment with Wg results in accumulation of N350 and not N120; both Dl and Wg treatments result in accumulation of N55 molecules. The difference in levels of N350 between Dl-treated and Wg-treated cells is obvious after just 2 h of treatment. Clone-8 cells express a higher level of N55 molecules in the absence of any treatment, presumably because they also express Dl endogenously (Wesley, 2000a).

When Dl binds N in vivo, the ~120-kDa N intracellular domain is released into the cytoplasm. To determine whether the N120 in these in vitro experiments with Dl also accumulates in the cytoplasm, S2-N cells were fractionated and analyzed following treatments with Dl and Wg. Following treatment with Dl, N120 molecules accumulate in the cytoplasmic fraction. In contrast, N350 molecules accumulate in the membrane fraction following treatment with Wg. N55 molecules are not consistently detected in these experiments as they are very unstable in this fractionation and extraction procedure (Wesley, 2000a).

It is not known whether the N120 molecules that accumulate in the cytoplasm in response to Dl are the same as those present in the membranes or whether they are different molecules migrating in the same region of the gel. Membrane-tethered N intracellular domain (Nintra), untethered Nintra, and N120 migrate alongside each other in these gels. N120 molecules associated with the membranes or with the cytoplasm are probably the membrane-tethered or released N intracellular domain, respectively. Accumulation of N350 molecules in response to Wg is likely to be in the intracellular membranes associated with production of the heterodimeric cell surface receptor. N55 is derived from N350 upon activation of Notch signaling by a ligand (Wesley, 2000a).

Notch modulates Wnt signalling by associating with Armadillo/ß-catenin and regulating its transcriptional activity

The establishment and stability of cell fates during development depend on the integration of multiple signals, which ultimately modulate specific patterns of gene expression. While there is ample evidence for this integration at the level of gene regulatory sequences, little is known about its operation at other levels of cellular activity. Wnt and Notch signalling are important elements of the circuitry that regulates gene expression in development and disease. Genetic analysis has suggested that in addition to convergence on the transcription of specific genes, there are modulatory cross-regulatory interactions between these signalling pathways. The nodal point of these interactions is an activity of Notch that regulates the activity and the amount of the active/oncogenic form of Armadillo/ß-catenin. This activity of Notch is independent of that induced upon cleavage of the Notch intracellular domain, which mediates transcription through Su(H)/CBF1. The modulatory function of Notch described in this study, contributes to the establishment of a robust threshold for Wnt signalling which is likely to play important roles in both normal and pathological situations (Hayward, 2005).

A soluble form of the intracellular domain of Notch, NICD, acts as an activated Notch receptor and provides constitutive Su(H)-dependent Notch signalling. Whereas a chimera between the extracellular and transmembrane domains of the receptor tyrosine kinase (RTK) Torso and the intracellular domain of Notch (TNotch) prevents the cleavage of Notch and the translocation of its intracellular domain to the nucleus. However, this chimeric molecule is still capable of signalling, as reflected by the loss of neural precursors during neurogenesis. This signalling event is likely to be independent of Su(H) because while NICD and full length Notch are able to activate transcription of either a Su(H) reporter in vivo or the Notch target gene wingless, TNotch is unable to do so. Thus TNotch behaves as a gain-of-function allele but one specific for a particular function of Notch which might not involve Suppressor of Hairless. In agreement with this, TNotch is unable to rescue a complete loss of function of Notch (Hayward, 2005).

The inputs of Notch and Wingless signalling on the development of the wing are well characterised. Notch and Wingless signalling cooperate in the development of the wing and in the case of Notch the effects are mediated by NICD. To test if the cleavage-independent function of Notch modulates Wingless signalling, NICD and TNotch were expressed at the same time that Wingless signalling was activated either with ectopic expression of Wingless or of a constitutively active form of Armadillo, Armadillos10. This form of Armadillo lacks the Shaggy/GSK3ß phosphorylation sites and provides Wingless-independent signalling by escaping degradation by the Axin-based destruction complex. Expression of either Wingless or Armadillos10 along the AP boundary results in an expansion of the hinge region and the occasional appearance of extra wing tissue off the notum. However, the effects of the intracellular domain of Notch depend on its molecular disposition. Expression of NICD along the AP boundary induces the appearance of an ectopic wing margin and promotes the growth of the wing, while expression of TNotch leads to a slight reduction in the overall size of the wing pouch region of the disc. In the developing wing, co-expression of NICD with either Wingless or Armadillos10 leads to a synergistic effect of extra growth of the wing tissue. In contrast to NICD, TNotch is very effective in suppressing the effects of ectopic expression of Wingless and, surprisingly, also of Armadillos10 (Hayward, 2005).

Since Armadillos10 provides Wingless signalling constitutively and expression of TNotch does not affect the expression of Wingless in the third instar discs, these results argue that a Su(H)-independent Notch activity modulates Wingless signalling by targeting the activity of Armadillo. To test this further, the effects of TNotch were analyzed on the ability of Armadillos10 to induce expression of Wingless target genes, Distalless (Dll) a low threshold target of Wingless, and the proneural gene senseless (sens), which like other proneural genes, provides a high threshold target. Both are elevated and ectopic in the presence of Armadillos10, and in both cases TNotch markedly suppresses this effect (Hayward, 2005).

To test whether the effects observed are restricted to the developing wing, the effects of TNotch on the cuticle pattern of the first instar larva were monitored. In the wild-type each segment contains an anterior region decorated with denticles and a 'naked' posterior region, devoid of denticles. The extent of the 'naked' region depends on the level of Wingless signalling, and ubiquitous Wingless signalling associated with strong expression of Armadillos10 results in cuticles all devoid of denticles. By modulating the levels of expression of Armadillos10 it is possible to modulate the extent of denticle loss: weak expression leads to a patchy loss of denticles in contrast, strong expression results in ventral cuticles completely devoid of denticles. Expression of TNotch modulates the effects that Armadillos10 has on the pattern of the cuticle: while strong effects of Armadillos10 are often suppressed. This observation confirms that Notch exerts a negative modulation on Wnt signalling and suggests that this might be a general phenomenon. Altogether these observations suggest that there is an activity of Notch, independent of Su(H), which modulates the Wingless signalling pathway at or below the level of Armadillo (Hayward, 2005).

Armadillo and Notch show a high degree of co-localisation at the adherens junction of the epidermal cells of the wing disc. To test whether Notch and Armadillo are associated in the cell, Notch from developing embryos was immunoprecipitated and Armadillo was sought among the co-immunoprecipitated proteins. Two different anti-Notch antibodies were used and in both cases Armadillo protein was detected in the same protein complex as the immunoprecipitated Notch protein. Interestingly, the predominant form of Notch protein detected in these assays is unprocessed and uncleaved, suggesting that this complex is membrane associated. The reverse experiment, in which Armadillo protein is immunoprecipitated, was also undertaken; here an unprocessed and uncleaved form of Notch was found to be associated with Armadillo. Previous experiments have indicated that Dishevelled, another element of Wnt signalling, can associate with Notch in a yeast two-hybrid assay. This was confirmed and this association was shown in the same immunoprecipitates from embryos in which the complex between Notch and Armadillo was found. These results indicate that the intracellular domain of Notch and a proportion of the Armadillo protein of the cell are associated in the same protein complex. Preliminary data suggests that this association is preferentially mediated by the region C-terminal to the cdc10/ANK repeats and such an interaction might be an element in the functional interactions described in this study (Hayward, 2005).

The precise mechanism whereby Wnt proteins elicit the activity of ß-catenin is still under scrutiny but it is generally agreed that the stability and amount of cytoplasmic Armadillo/ß-catenin are rate-limiting steps in the signalling event. This pool of Armadillo/ß-catenin is under very tight control by a destruction complex assembled on Axin, which together with Shaggy/GSK3ß are the main targets of Wnt signalling. However, there is increasing evidence that high levels of cytoplasmic Armadillo/ß-catenin are not sufficient to promote Wnt signalling. Recently emphasis has been placed on the observation that Axin can regulate the activity of Armadillo/ß-catenin in a Shaggy/GSK3ß-independent manner. This has led to the conclusion that Wnt regulates the activities of Shaggy/GSK3ß and Axin co-ordinately and that there might be other factors contributing to the control of Armadillo/ß-catenin activity. Consistent with this possibility it has been reported that Wnt signalling can regulate the activity of stable oncogenic forms of ß-catenin (Hayward, 2005).

This study shows that Notch signalling provides an important input into Wnt signalling in Drosophila by associating with Armadillo and regulating its levels and activity during Wingless signalling. This activity of Notch, which is different and probably independent of that which mediates CBF1/Su(H)-dependent signalling, lies functionally downstream of Shaggy/GSK3ß and targets the concentration and activity of the hypophosphorylated form of Armadillo. It can also modulate the activity of an oncogenic form of vertebrate ß-catenin and this functional interaction between Notch and Armadillo has been shown to extend to the vertebrate system, with mNotch1 regulating the activity of ß-catenin in tissue culture cells (Hayward, 2005).

A role for Notch in the modulation of Wnt signalling has been inferred from genetic analysis. However, although these results indicate that Notch antagonises Wnt signalling, alone they do not provide insights into the mechanism of the interaction. The current study does, and it is likely that the molecular interactions that are reported in this study underpin the observed modulation of Wnt signalling by Notch. Wingless signalling can be activated in vivo in the absence of Notch and this activation does not require Dishevelled. The observations that removal of Notch in cl8 cells leads to activation of a synthetic Wnt reporter confirm this and suggest a direct regulatory effect of Notch on the mechanism of Wnt signalling. Furthermore, the effects of Notch on the activated form of Armadillo offer an explanation for why removal of Notch can bypass a requirement for Dishevelled. It may well be that even under steady state conditions there is a small amount of hypophosphorylated, active Armadillo/ß-catenin which escapes the Axin/GSK3ß mediated degradation. Given the high specific activity of this molecule, it is not surprising that there might be further mechanisms that control it. Notch appears to be an essential part of these mechanisms and in its absence this active form of Armadillo would operate even in the absence of Dishevelled. Axin is also likely to be involved in the regulation of the active form and Axin can also suppress the effects of an activated form of Armadillo. It will be of interest to explore the relationships between Notch and Axin (Hayward, 2005).

Previous studies have implicated Deltex and Dishevelled as important elements of the interaction between Notch and Wingless signalling. Both proteins bind Notch, but they do so in different places. Deltex binds to the cdc10/ANK repeats and promotes Su(H)-independent Notch signalling. Whereas, Dishevelled binds within a broad region C-terminal to this domain and reduces the Su(H)-independent activity of Notch. This study has shown that Armadillo also physically interacts with Notch, probably through the same broad region that binds Dishevelled. Mutations in Notch that impair this domain result in Notch receptors that interfere with Wnt signalling. The deletion of this region reduces the efficiency with which the intracellular domain of Notch affects the levels and activity of Armadillo. Together these observations underscore the role of this region of Notch in mediating interactions between Notch and Wnt signalling by targeting the active form of Armadillo/ß-catenin (Hayward, 2005).

The relationship between Notch and Armadillo in Drosophila extends to their vertebrate homologues, Notch1 and ß-catenin. This interaction, rather than an interaction of Dishevelled with Notch/CBF signalling, might reflect the functional relationships between the two signalling systems that have been reported during the development of the skin the immune system and in somitogenesis. In these instances Wnt and Notch drive alternative fates (skin and immune system) or act antagonistically (somites) perhaps by a combination of their individual pathways and the modulatory interaction described in this study. One consequence of this modulatory interaction might also be the observed tumour suppressor function of Notch1 in the mouse skin where removal of Notch1 results in the generation of tumours associated with an increase in the levels of active ß-catenin and Wnt signalling. While some of the elevation of ß-catenin in these cells might be a secondary consequence of activation of Wnt signalling, the current observations suggests that the loss of Notch1 can also contribute to this increase by allowing the activation of cß-catenin. In a different study carboxyl-terminal deletions in Notch1, which include the region that binds Dishevelled and Armadillo, enhanced the oncogenic effects of a chimeric E2A-PBX1 protein. It is possible that some of this effect is due to misregulation of ß-catenin in the tumours (Hayward, 2005).

In summary, Notch provides a modulatory input in the activity of Armadillo/ß-catenin. This modulation provides two functions: it establishes a threshold for Wnt signalling that is likely to play an important role in the patterning of tissues and the assignation of cell fates during development and, in addition it provides a stringent regulation of the activated form of Armadillo/ß-catenin. The second function might be crucial in pathological situations and might contribute to the understanding of Notch as a tumour suppressor (Hayward, 2005).

Notch interaction with Fringe

Fringe (Fng), an extracellular protein, determines the boundary of two different cell populations during the development of diverse structures not only in Drosophila but also in vertebrates. The identification of the proteins that physically interact with Fng is important to understand the molecular mechanisms of Fng function. As most known Fng-mediated developmental processes require Notch signaling, Notch is a strong candidate for Fng-interacting proteins. To test whether Fng binds Notch, a series of biochemical experiments were performed focusing on the EGF-like repeats 22 to 36 and Lin-Notch repeats (LNRs) of Notch, to which most Ax mutations and antineurogenic mutations map. Expressed either separately or together in transgenic Drosophila were a chimaeric Notch, named NSG, which includes the region from EGF repeat 22 to the transmembrane domain of Notch fused to an S tag and green fluorescent protein (GFP), and Fng, tagged with the Myc epitope (FngM). The extracts of the transgenic larvae were immunoprecipitated with anti-GFP or anti-Myc antibody and then purified by S-protein affinity column chromatography. This ensured that all of the positive bands in Western blots with anti-GFP antibody contained an S tag as well as a GFP tag. Because Notch is processed into two polypeptides linked by disulphide bonds in the trans-Golgi, the NSG protein could be expected to yield two polypeptides by SDS-polyacrylamide gel electrophoresis. The first product is an amino-terminal peptide of relative molecular mass 89,000 (Mr 89K), which is untagged. The second polypeptide is a carboxy-terminal peptide (NSG-C) of Mr 43K that contains an S tag and GFP. The eluents of the S-protein affinity column from extracts of animals expressing 2xNSG or 2xNSG plus 2xFngM all yield a positive 43K band as expected. When the immunoprecipitations were carried out with anti-Myc antibody, which specifically binds to FngM, the extract from the animal co-expressing NSG and FngM yields the co-precipitated 43K NSG-C peptide (Ju, 2000).

To map the domains of Notch that interact with Fng, various forms of Notch derivatives were expressed with an S tag and GFP in stably transfected S2 cell lines with or without Fng-GST (Fng fused to glutathione S-transferase). AxM1 is a missense mutation of Cys 999 to tyrosine. The extracts from these cell lines were loaded onto glutathione-Sepharose column. If NSG, or its derivatives, form a complex with Fng, it should be retained on the column with Fng-GST. NSG, LNRSG, N22-36SG and AxSG were retained with the Fng-GST protein on gutathione-Sepharose, but N1-21SG and Ax22-36SG were not. These data suggest that Fng specifically binds to Notch through the LNRs as well as the EGF22-36 and that AxM1 mutation abolishes the Fng interaction with EGF22-36 of Notch but does not affect the Fng interaction with the LNRs (Ju, 2000).

The subcellular colocalization of Fng-GST with the Notch derivatives also supports the specific Fng-Notch interaction. Examined were the subcellular localization of Fng-GST and the Notch derivatives, which were co-expressed in S2 cells. The subcellular distribution of Fng mostly overlaps with that of the Notch derivatives, which interacted with Fng in biochemical experiments (NSG, N22-36SG and LNRSG). In contrast, N1-21SG and Ax22-36SG are mainly not colocalized with Fng and do not interact. These data indicate that Fng may be present as a complex with Notch even before its secretion (Ju, 2000).

Consistent with these observations, when Fng-GST and the Notch derivatives are expressed separately and mixed in vitro, none of the Notch derivatives co-purified with Fng-GST on a glutathione-Sepharose column. In addition, when FngM and NSG are expressed separately and mixed in vitro and then analysed by glycerol-gradient centrifugation, FngM is mainly found in fractions 16-19, whereas NSG is found in fractions 2-7. This implies that Fng and Notch do not form a complex if they are not expressed in the same cells simultaneously. These data are consistent with the finding that Fng is not detected on the surface of cells expressing Notch when Fng protein is added exogenously. Therefore, Fng-Notch complex formation may occur before secretion, probably within the secretory pathway. This explains why the secretary Fng protein acts cell-autonomously in Notch-expressing cells. It is unlikely to modify Notch found on the surface of neighboring cells or cells that may be reached by secreted Fng. Fng has been proposed as a glycosyltransferase on the basis of sequence similarity. Fng may bind Notch and may modify the glycosylation of Notch during its secretion in the endoplasmic reticulum (ER) or Golgi. A comparison of the glycosylation status between the Fng-Notch complex and Notch alone will be of considerable interest (Ju, 2000).

Genetic evidence from Ax mutants suggests that the Notch EGF 22-36 are involved in the Notch-Fng interaction in vivo. In wild-type mid-third Drosophila wing imaginal discs, the expression of Serrate (Ser), the vestigial (vg) boundary enhancer-lacZ (vgBE) and wingless (wg) are upregulated by Fng and Notch near or at the dorsoventral (D-V) boundary but not in dorsal interior cells. In contrast, in AxM1 mutants Ser and vgBE expression are expanded into all dorsal cells, including interior cells. This expansion may be due to disturbance of the Fng-Notch interaction through the EGF22-36 of Notch by the Ax mutation (Ju, 2000).

To test this, Fng was ectopically expressed in AxM1 mutant wing discs using UAS-fng and a patched (ptc)-Gal4 driver. In wild-type imaginal discs, ectopic expression of Fng in the ptc pattern induces vgBE, Wg and Delta (Dl) only in ventral cells in a single stripe along the A/P boundary. In AxM1 mutants, however, Ser, Wg, vgBE and Dl expression are strongly induced by ectopic Fng even in dorsal cells. The level of Ser expression in the dorsal cells expressing ectopic Fng is much higher than that in other dorsal cells. The effects of ectopic Fng expression on Ser, Wg, vgBE, and Dl expression are also similar in Ax28 homozygous and Ax16172/Ax28 heterozygous mutant wing discs, although the extent of the effect varies. Ax mutations do not affect the response of dorsal or ventral cells to Ser or Dl as significantly as their response to Fng. This implies that the induction of Ser, Dl and vgBE by Fng in Ax mutant dorsal cells may not be mediated by Ser (Ju, 2000).

To confirm the effects of Fng on Ser expression in AxM1 mutants, homozygous fng null mitotic clones were generated in the AxM1 mutant background. Notably, homozygous fng- dorsal cells of AxM1 mutant wing discs do not express Ser. Ser expression in the interior dorsal cells of AxM1 mutant therefore requires Fng function, and the AxM1 mutation disrupts the normal downstream regulatory effects of the Fng-Notch interaction (Ju, 2000).

The LNRs of Notch function as a repressor domain in Notch signaling. Since Ax mutations prevent EGF22-36 of Notch from binding to Fng but do not affect Fng binding to the LNRs of Notch, one of the possible mechanisms of Fng-dependent Notch target gene activation in Ax mutants is that the Ax mutation allows Fng to bind to LNRs alone rather than to EGF 22-36. This may dampen or silence the repressor function of the LNRs, and might, in turn, make Notch more sensitive to Delta. Alternatively, Ax mutations may mimic the conformation of Notch bound with a ligand and Fng may help this form of Notch to be converted to a fully activated form by antagonizing the repressor function of the LNRs (Ju, 2000).

Fringe has been proposed to execute its boundary determining function by inhibiting the Notch response to Ser and potentiating the Notch response to Dl. Because Fng does not bind Ser or Dl, modulation of Notch signaling by Fng is directly mediated by the complex formation of Fng and Notch during their secretory transits. Notch bound to Fng may have preferential affinity or sensitivity to Delta, whereas free Notch may have a higher affinity or sensitivity to Ser. Upon Dl binding to the Fng-Notch complex, Fng may also antagonize the repressor function of the LNRs and facilitate the activation of Notch signaling (Ju, 2000).

Signaling via the Notch receptor is a key regulator of many developmental processes. The differential responsiveness of Notch-expressing cells to the ligands Delta and Serrate is controlled by Fringe, itself essential for normal patterning in Drosophila and vertebrates. The mechanism of Fringe action, however, is not known. The protein has an amino-terminal hydrophobic stretch resembling a cleaved signal peptide, which has led to the widespread assumption that it is a secreted signaling molecule. It also has distant homology to bacterial glycosyltransferases, although it is not clear if this reflects a shared enzymatic activity, or merely a related structure. A functional epitope-tagged form of Drosophila Fringe is localized in the Golgi apparatus. When the putative signal peptide is replaced by a confirmed one, Fringe no longer accumulates in the Golgi, but is instead efficiently secreted. This change in localization dramatically reduces its biological activity, implying that the wild-type protein normally acts inside the cell. Fringe specifically binds the nucleoside diphosphate UDP, a feature of many glycosyltransferases. Furthermore, specific mutation of a DxD motif (in the single-letter amino acid code where x is any amino acid), a hallmark of most glycosyltransferases that use nucleoside diphosphate sugars, does not affect the Golgi localisation of the protein but completely eliminates in vivo activity. These results indicate that Fringe does not exert its effects outside of the cell, but rather acts in the Golgi apparatus, apparently as a glycosyltransferase. They suggest that alteration in receptor glycosylation can regulate the relative efficiency of different ligands (Munro, 2000).

The suggestion that Fringe is secreted came from the amino-terminal hydrophobic stretch, which was predicted to be a leader peptide. Golgi glycosyltransferases are, almost without exception, Type II membrane proteins with a single transmembrane domain within the first 5-50 residues of the amino terminus. Because the transmembrane domains of Golgi proteins are usually shorter than those of plasma membrane proteins, their amino-terminal regions can appear similar to signal peptides. Indeed, the best current signal-peptide prediction programs incorrectly predict known mammalian glycosyltransferases to have cleaved amino termini. The fact that, when attached to a confirmed signal peptide, Fringe is secreted and has a smaller apparent size, strongly suggests that the amino-terminal hydrophobic stretch is not normally cleaved, but rather is a transmembrane domain typical of Golgi glycosyltransferases. A previous examination of Fringe expressed in transgenic flies concluded that the protein was being secreted on the basis that it could be detected outside of the expression domain of the GAL4 expression driver being used. This 'extracellular' Fringe appeared in a punctate pattern strikingly similar to the Golgi localization described here, raising the possibility that it was in fact weak ectopic expression of the protein, caused by leakiness of the Gal4 driver. Furthermore, even if a small amount of Fringe is secreted, forcing its secretion dramatically reduces its activity, indicating that it is the intracellular form, rather than the secreted form that regulates Notch signaling. An intracellular site of action for Fringe is also easier to reconcile with the protein's observed cell autonomy in regulating Notch (Munro, 2000 and references therein).

Specific modification by Fringe of one or more of the O-linked structures on the Notch EGF repeats could alter the binding affinity of the ligands Delta and Serrate. Specifically, genetic evidence implies that Serrate binding and/or activation of Notch would be inhibited by Fringe-mediated glycosylation, whereas Delta binding and/or activation would be enhanced. Delta and Serrate both bind EGF repeats 11 and 12 of Notch, the latter of which contains a well conserved site for O-linked glucosylation. However, the situation may be more complex, as Abruptex mutations in Notch that map to EGF repeats 24, 25, 27 or 29 have interestingly been found to be insensitive to modulation by Fringe. It may be that recognition or modification of many of the Notch repeats is critical for affecting ligand binding (Munro, 2000 and references therein).

This study establishes the biochemical mechanism of Fringe action. Fringe is a secreted protein that both positively and negatively modulates the ability of Notch ligands to activate Notch signaling. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. Biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signaling has been obtained by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signaling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signaling pathways (Moloney, 2000).

A site-specific mutation was generated in Drosophila Fng, D236-D237-D238 to D236-E237-E238 [D-Fng(DEE)], to show that the GlcNAc-transferase activity of Fringe is essential for it to modulate Notch signaling in vivo. This aspartic acid patch is highly conserved among galactosyltransferases and several GlcNAc-transferases. Conservative changes within the DDD motif disrupt the catalytic activity of these enzymes without destabilizing their overall structure. Indeed, affinity-purified D-Fng(DEE) is enzymatically inactive (Moloney, 2000).

The biological activity of fngDEE was examined by using an ectopic expression assay. The influence of fng on Notch signaling results in establishment of a stripe of Notch activation along the borders of fng expression. Ectopic expression of wild-type Fringe proteins under the control of patched regulatory sequences eliminates the normal Fringe expression border in the middle of the wing and induces an ectopic Fringe expression border in ventral cells. This results in corresponding changes in the pattern of Notch activation, as judged by Wingless expression in developing wing imaginal discs and by formation of margin bristles in adult wings. Strikingly, ectopic expression of fngDEE fails to cause any discernible effect on Notch activation in the wing, or on any other aspects of Drosophila development. Thus, the GlcNAc-transferase activity of Fringe is essential for it to modulate Notch signaling (Moloney, 2000).

Glycosylation modifies protein activities in various biological processes. This study reports the functions of a novel UDP-sugar transporter (UST74C, an alternative name for Fringe connection (Frc), which is localized to the Golgi apparatus in cellular signalling of Drosophila. Mutants in the frc gene exhibit phenotypes resembling wingless and Notch mutants. Both Fringe-dependent and Fringe-independent Notch pathways are affected, and both glycosylation and proteolytic maturation of Notch are defective in mutant larvae. The results suggest that changes in nucleotide-sugar levels can differently affect Wingless and two distinct aspects of Notch signalling (Goto, 2001).

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. A new gene, fringe connection (frc), is described that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue (Selva, 2001).

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).

Notch is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. Endocytic trafficking of Notch plays important roles in the activation and downregulation of this receptor. A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. It was recently proposed that O-fut1 acts as a chaperon for Notch in the endoplasmic reticulum and is required for Notch to exit the endoplasmic reticulum. O-fut1 has additional functions in the endocytic transportation of Notch. O-fut1 is indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which is independent of the O-fucosyltransferase activity of O-fut1. O-fut1 promotes the turnover of Notch, which consequently downregulates Notch signaling. O-fut1 formed a stable complex with the extracellular domain of Notch. In addition, O-fut1 protein added to conditioned medium and endocytosed is sufficient to rescue normal Notch transportation to the early endosome in O-fut1 knockdown cells. Thus, an extracellular interaction between Notch and O-fut1 is essential for the normal endocytic transportation of Notch. It is proposed that O-fut1 is the first example, except for ligands, of a molecule that is required extracellularly for receptor transportation by endocytosis (Sasamura, 2007).

Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila

Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, the genetic effects were studied of mutating Gmd and Gmer on fucose modifications in Drosophila. These mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin 2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. These results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances (Ayukawa, 2012).

Dual roles of O-glucose glycans redundant with monosaccharide O-fucose on Notch in Notch trafficking

The extracellular domain of Notch contains multiple EGF-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The potential functional interactions between these glycans are just beginning to be understood. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. Loss of the terminal dixylose of O-glucose-linked saccharides has little effect on Notch signaling. Analyses of double mutants of shams and other genes required for glycan modifications reveals that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose are required in distinct Notch-trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, these results suggest that these potential redundancies may lead to a serious underestimation of glycan functions (Matsumoto, 2016).

Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling

Notch signaling is broadly used to regulate cell fate decisions. A novel gene, rumi, has been identified with a temperature-sensitive Notch phenotype. At 28°-30°C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18°C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells, but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. These studies show that Rumi is a protein O-glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2 sequence. These data indicate that by O-glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane (Acar, 2008).

In all contexts examined, rumi is essential for Notch signaling in a ts manner, i.e. lateral inhibition, asymmetric division and inductive signaling. Homozygous rumi animals are viable and fertile when kept at 18°C and exhibit a mild lateral inhibition defect and a modest Delta wing vein phenotype. rumi animals raised at 25°C show a very significant decrease in viability and fertility. At this temperature, there is a failure in the cell fate specification process. At 28°-30°C a full-blown Notch phenotype is observed in all tissues examined, and homozygous mutants can only reach the third instar stage because of the wild-type maternal component. The difference between the requirements for Rumi at 25°C and 28-30°C is also reflected in genetic interaction studies; an extra copy of Notch is able to improve Notch signaling in rumi mutants raised at 25°C (partial requirement), but not at 28°-30°C (full requirement). Altogether these observations indicate that loss of rumi phenocopies loss of Notch in a temperature-dependent fashion (Acar, 2008).

Multiple lines of evidence suggest that Rumi functions in the signal-receiving cell. MARCM experiments indicate that overexpression of Notch ligands in rumi mutant cells is able to induce signaling, suggesting that Rumi function is not required in the signal-sending cell. However, cells that are mutant for rumi are not able to receive the signal, even when ligands are overexpressed in adjacent cells. Of note, the only component of the Notch signaling pathway in flies with multiple O-glucosylation sites is the Notch protein itself, besides Delta which contains a single predicted site (Acar, 2008).

Since an upregulation of Notch protein was observed in rumi mutant clones, it was hypothesized that Notch might be trapped in the ER and fail to reach the membrane at the restrictive temperature. However, an accumulation of Notch was observed at the surface of rumi mutant cells. In addition, a lack of an unfolded protein response was observed, and a lack of expansion of the ER in rumi clones raised at the restrictive temperature. These data raised the possibility that Notch present at the cell surface may not interact with its ligands at the restrictive temperature. However, the data suggest that the Notch-Delta interaction is not decreased at 28°C, but rather that the cleavage of Notch at the membrane is impaired. Hence the data indicate that the S2 cleavage of Notch is impaired in rumi mutant signal-receiving cells (Acar, 2008).

Most proteins with a CAP10 domain contain a signal peptide and an ER retention signal. The CAP10 gene was first discovered in the fungus Cryptococcus neoformans. The CAP proteins (CAP10, 59, 60 and 64) are referred to as putative polysaccharide modifiers as they affect extracellular polysaccharide capsule formation. The data indicate that knockdown of Rumi in S2 cells results in loss of O-glucosylation at Serines in C1-X-S-X-P-C2 sites on numerous EGF repeats. No effects were seen on levels of O-fucosylation. In vitro assays with purified Rumi demonstrate that it can catalyze the transfer of glucose from UDP-glucose to an EGF repeat with the consensus sequence. Hence, Rumi encodes the first identified protein O-glucosyltransferase. Rumi shares several common features with enzymes responsible for addition of O-fucose to EGF repeats and thrombospondin type 1 repeats (TSRs), Pofut1 and Pofut2, respectively. These proteins are soluble, ER localized and only modify properly folded structures (EGF repeats for Pofut1, thrombospondin type 1 repeat for Pofut2). Preliminary studies using crude lysates suggest that the mammalian form of the protein O-glucosyltransferase (presumably a Rumi homologue) can also distinguish folded from unfolded structures. The ER localization and ability to distinguish folded from unfolded structures suggests that all of these enzymes may function in folding and/or quality control (Acar, 2008).

Unlike Ofut1, which is reported to have important non-enzymatic functions, the results indicate that the function of Rumi resides in the O-glucosyltransferase activity. It is therefore proposed that preventing the addition of O-glucose to Notch causes a ts phenotype. It is propose that the O-glucose glycans may function to hold the NECD in a stable conformation needed for proper function, especially at higher temperatures. For example, O-glucosylation of Notch might be a prerequisite for conformational changes in the NECD that are proposed to promote the S2 cleavage. Alternatively, addition of O-glucose might be required for another posttranslational modification. The importance of O-glucosylation of Notch is also supported by studies showing that elimination of individual O-glucosylation sites in mouse Notch1 impairs activation in cell-based Notch signaling assays (Acar, 2008).

Lack of O-glucosylation at the restrictive temperature does not block the ER-to-membrane transport and ligand interactions but disrupts Notch cleavage. These data, together with accumulation of Notch intracellularly and at the cell membrane in rumi cells suggest that lack of O-glucose modification causes a folding problem which impairs Notch function. Trafficking problems upstream of S3 cleavage have been documented to cause accumulation of Notch and ectopic activation of Notch signaling. For example, loss of Lethal giant discs (Lgd), a protein required for proper trafficking of Notch, causes ectopic activation of Notch in a ligand-independent manner. The data show that the loss of rumi suppresses the ectopic activation of Notch in lgd mutant cells, suggesting that the lack of O-glucosylation prevents the ligand-independent activation of Notch in the absence of Lgd (Acar, 2008).

In summary, these data uncover a novel mechanism for enzymatic regulation of Notch signaling in Drosophila by a protein O-glucosyltransferase, and provide an in vivo model to study the role of O-glucosylation in developmental signaling. Given the evolutionary conservation of Notch signaling and the presence of conserved O-glucosylation motifs in other Notch proteins, addition of glucose may be required for proper folding and cleavage in many species (Acar, 2008).

Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling

Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, a mutational analysis of these glucosylation sites was carried out and their effects on Notch activity were determined in vivo. The results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. These data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling (Leonardi, 2011).

These studies indicate that the Notch receptor is the key target of the protein O-glucosyltransferase Rumi in the Notch signaling pathway, as the temperature-sensitive loss of Notch signaling observed in rumi mutants can be recapitulated by mutations in the O-glucosylation motifs of Notch. In the mouse, a single knock-in mutation that abolishes the O-fucosylation of EGF12 of Notch1 results in decreased ligand binding of Notch1 and behaves as a hypomorphic allele (Ge, 2008). Furthermore, overexpression studies in Drosophila indicate that a single O-fucose mutation in EGF12 significantly increases the activation of Notch by Serrate, most likely owing to an accompanying increase observed in the binding of Notch to Serrate. However, the data indicate that no single O-glucosylation motif, including that in EGF12, is essential for Drosophila Notch signaling. Even though O-glucose sites in EGF10-15 make a significant contribution to Notch signaling at high temperature, the N-/Y; Ngt-10_15/+ males (bearing a genomic transgene lacking 6 EGF repeats) only show Notch loss-of-function phenotypes at 25°C or higher and still reach the pharate adult stage at 30°C. These observations suggest a role for other O-glucose residues, in agreement with the mild decrease in the activity of the Notch Ngt-16_35 transgene. The activity of Ngt-10_20 is considerably less than that of Ngt-10_15, but mutating only the O-glucose sites on EGF16-20 does not affect the ability of Ngt-16_20 to rescue the lethality and the bristle and leg phenotypes of a Notch null allele. These examples, together with similar comparisons between the various other mutant transgenes, indicate that all O-glucose residues contribute in additive and redundant fashions to ensure robust Notch signaling, especially at high temperatures (Leonardi, 2011).

It is proposed that rather than a local contribution to facilitate specific lectin-type interactions, the O-glucose residues on Notch EGF repeats function globally to maintain the Notch extracellular domain in a conformation that is permissive for signaling. Based on this model, in wild-type flies the O-glucose residues on Notch act as a buffer to ensure robust Notch signaling, especially at high temperature. Several lines of evidence support this idea. First, Notch proteins with a smaller number of O-glucose mutations signal better and are more resistant to increased temperatures than Notch proteins with a greater number of mutations. Second, at low temperature, the function of Notch is less dependent on the number of O-glucose residues, as evidenced by the similarity of the N-/Y; Ngt-10_20/+, N-/Y; Ngt-10_35/+ and N-/Y; Ngt-4_35/+ phenotypes at 18°C. Third, increasing the dosage of Ngt-10_15 can rescue the bristle and leg phenotypes of the N55e11 allele at 30°C, indicating that even though O-glucose residues on EGF10-15 play a prominent role in preventing the temperature-dependent loss of Notch signaling, a lack of O-glucose in this region can be compensated by O-glucose on other EGF repeats when the level of Notchgt-10_15 is increased (Leonardi, 2011).

Biochemical, X-ray crystallography and genetic experiments have established that deletion of the LIN-12/Notch (LNR) motif from Drosophila and mammalian Notch proteins results in ligand-independent S2 cleavage and activation of Notch. It has been proposed that endocytosis of the Notch-bound ligand into the signal-sending cell applies a pulling force to the Notch extracellular domain and thereby leads, in a stepwise fashion, to LNR dissociation and heterodimer relaxation, which will ultimately expose the S2 cleavage site. The complete suppression of the ligand-independent NotchγLNR-LexA overexpression phenotypes in rumi MARCM clones suggests that the cross-talk between the EGF repeats and the heterodimerization region of Notch is not solely mediated by the LNR motif. The data further suggest that O-glucosylation of Notch by Rumi is required at a step that is common between ligand-dependent and ligand-independent forms of Notch activation. Mutations in the heterodimerization region of human NOTCH1 result in ligand-independent activation of NOTCH1 and thereby promote the development of T-cell acute lymphoblastic leukemia. Accordingly, decreasing NOTCH1 O-glucosylation using a Rumi inhibitor might offer a potential therapeutic avenue for this disease (Leonardi, 2011).

Based on these observations and the gradual increase in the severity of phenotypes caused by the loss of rumi or loss of O-glucose sites upon temperature increase, it is proposed that the ability of the Notch protein to undergo S2 cleavage gradually declines as the temperature increases. However, the broad distribution of O-glucose residues across the extracellular domain of Notch ensures that at the tissue and organismal levels, no significant decline in Notch signaling occurs at high temperatures and therefore wild-type flies raised at 30-32°C do not show Notch loss-of-function phenotypes. Of note, qRT-PCR data on the control cells show a modest, yet statistically significant, decrease in E(spl)m3 expression at higher temperatures, suggesting that the buffering role of O-glucose residues is not 100% efficient at the molecular level (Leonardi, 2011).

A close homolog of fly Rumi is the primary, if not the only, protein O-glucosyltransferase in the mouse (Poglut1) (Fernandez-Valdivia, 2011). shRNA-mediated Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes characteristic of loss of Notch signaling, including a severe decrease in the S3 cleavage of Notch1, without affecting the binding of Notch to the jagged 1 and delta-like 1 ligands. The number and distribution of the EGF repeats with a C1-X-S-X-P-C2 O-glucosylation motif are similar in vertebrate and fly Notch proteins, and mammalian Notch1 and Notch2 have been shown to harbor O-linked glucos. Altogether, these observations suggest that the biologically relevant O-glucose residues on mammalian Notch proteins are likely to be broadly distributed in their extracellular domains (Leonardi, 2011).

O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, this study generated a knock-in mutant of O-fut1 (O-fut1R245A knock-in), which expresses a mutant protein that lacks O-fucosyltransferase activity, but maintains the N-specific chaperon activity. Using O-fut1R245A knock-in and other gene mutations that abolish the O-fucosylation of N, it was found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. It was also showm that the redundant function of these two modifications is responsible for the presence of N at the cell surface. These findings elucidate how different forms of glycosylation on a protein can influence the protein's functions (Ushio, 2014: PubMed).

Gfr is involved in the fucosylation of Notch-linked glycans

Congenital disorder of glycosylation IIc (CDG IIc), also termed leukocyte adhesion deficiency II, is a recessive syndrome characterized by slowed growth, mental retardation, and severe immunodeficiency. Recently, the gene responsible for CDG IIc was found to encode a GDP-fucose transporter. This study investigated the possible cause of the developmental defects in CDG IIc patients by using a Drosophila model. Biochemically, it was demonstrated that a Drosophila homolog of the GDP-fucose transporter, the Golgi GDP-fucose transporter (Gfr), specifically transports GDP-fucose in vitro. To understand the function of the Gfr gene, null mutants of Gfr were generated in Drosophila. The phenotypes of the Drosophila Gfr mutants were rescued by the human GDP-fucose transporter transgene. The phenotype analyses revealed that Notch (N) signaling was deficient in these Gfr mutants. GDP-fucose is known to be essential for the fucosylation of N-linked glycans and for O-fucosylation, and both fucose modifications are present on N. These results suggest that Gfr is involved in the fucosylation of N-linked glycans on N and its O-fucosylation, as well as those of bulk proteins. However, despite the essential role of N O-fucosylation during development, the Gfr homozygote is viable. Thus, these results also indicate that the Drosophila genome encodes at least another GDP-fucose transporter that is involved in the O-fucosylation of N. Finally, it was found that mammalian Gfr is required for N signaling in mammalian cultured cells. Therefore, the results implicate reduced N signaling in the pathology of CDG IIc (Ishikawa, 2005).

The GDP-fucose transporter is a multitransmembrane protein that transports GDP-fucose into the Golgi lumen from the cytoplasm, in which it is synthesized. Its activity is absent in CDG IIc individuals. Notch signaling was reduced in flies with mutant Gfr, a Drosophila ortholog of the human GDP-fucose transporter gene. Furthermore, mammalian Gfr was also required for ligand-dependent activation of N signaling in mammalian cultured cells. Therefore, these findings imply that a defect in N signaling is responsible for the pathogenesis of CDG IIc, at least in part. Interestingly, diseases caused by defects in N signaling components, such as Alagille syndrome and CADASIL, are occasionally associated with mental retardation. Alagille syndrome is an autosomal-dominant disorder characterized by growth and mental retardation. Positional cloning studies have revealed that Alagille syndrome is caused by mutations in the Jagged1 gene, the human homolog of Drosophila Serrate, one of the N ligands. CADASIL is an autosomal-dominant vascular disorder associated with migraine with aura, mood disorders, recurrent subcortical ischemic strokes, progressive cognitive decline, dementia, and premature death. CADASIL is caused by mutations in the Notch3 gene. Therefore, these results imply that a reduction of N signaling may be responsible for the mental retardation observed in CDG IIc patients. Moreover, in view of the fact that N signaling regulates lymphocyte development and function in mammals, it is highly probable that a deficiency of N signaling is responsible for the immunodeficiency associated with CDG IIc (Ishikawa, 2005 and references therein).

GDP-fucose is essential for the terminal fucosylation of N-linked glycans and O-fucose, and both fucose modifications are reported to occur on mammalian Notch1; these modifications are thought to be conserved in Drosophila. In CDG IIc fibroblasts, the bulk addition of fucose as a terminal modification of N-linked glycans is severely diminished, whereas bulk protein O-fucosylation is not affected. However, the reduction of N O-fucosylation accounts for at least a portion of the defects seen in the Gfr mutants, although it is difficult to evaluate the contribution of N N-glycan fucosylation to these defects at this time. These results suggest that, in the Gfr1 mutant, the fucosylation of N-glycans drastically declines but the effect on O-fucosylation is subtle. These distinct sensitivities to the reduction of GDP-fucose may be accounted for by previously published results that the fucosylation of N-glycans requires a higher concentration of GDP-fucose than O-fucosylation. These results also suggest that at least one other GDP-fucose transporter, which is sufficient for N O-fucosylation, is present in Drosophila. It was reported that the majority of GDP-fucose transport activity is found in the Golgi rather than in the ER, in which the O-fucosylation of N occurs. However, given that a low concentration of GDP-fucose is sufficient for O-fucosylation, a putative GDP-fucose transporter that has low GDP-fucose transport activity might be present in the ER. It is speculated that the cold-sensitive activity of this GDP-fucose transporter is rate limiting in the Gfr1 mutant. In this mutant, the wing nicking occurs in different regions of the wing than in the N heterozygote. The wing nick in the Gfr1 mutant is often in the anterior and posterior regions of the wing margin, whereas nicking occurs mostly in the wing tip in the N mutant. These observations may suggest that another GDP-fucose transporter is more active in the center of the wing pouch. Notably, the symptoms of CDG IIc are partially suppressed by the oral administration of fucose, which is made into GDP-fucose through the salvage pathway, indicating the presence of a GDP-fucose transporter activity in these patients. Therefore, a GDP-fucose transporter, which is required for O-fucosylation, may be conserved between Drosophila and mammals (Ishikawa, 2005).

Staining for lectin is not enhanced by the overexpression of Gfr in the wild-type wing imaginal disc and Wg expression is not altered in these discs, suggesting that the overexpression of Gfr does not affect N signaling. It was also found that Gfr overexpression driven by various Gal4 drivers does not cause any detectable adult phenotype. This result raises the possibility that gene therapy involving the introduction of Gfr transgenes into somatic cells might be used to cure CDG IIc, because the likelihood of side effects is probably low (Ishikawa, 2005).

Distinct functional units of the Golgi complex in Drosophila cells as revealed by the distribution of Fringe connection - Notch and the Golgi complex

A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. This study shows that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disc cells are functionally diverse. The UDP-sugar transporter Fringe-Connection (Frc) is localized to a subset of the Golgi units distinct from those harboring Sulfateless (Sfl), which modifies glucosaminoglycans (GAGs), and from those harboring the protease Rhomboid (Rho), which processes the glycoprotein Spitz (Spi). Whereas the glycosylation and function of Notch are affected in imaginal discs of frc mutants, those of Spi and of GAG core proteins are not, even though Frc transports a broad range of glycosylation substrates, suggesting that Golgi units containing Frc and those containing Sfl or Rho are functionally separable. Distinct Golgi units containing Frc and Rho in embryos can also be separated biochemically by immunoisolation techniques. Tn-antigen glycan is shown to be localized only in a subset of the Golgi units distributed basally in a polarized cell. It is proposed that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification (Yano, 2005).

The pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are >300 glycosylenzymes in humans and >100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization/compartmentalization of glycosylenzymes (Yano, 2005).

The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae. Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly. In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase, although there has been no evidence to date to indicate functional differences among the Golgi fragments (Yano, 2005).

A Drosophila UDP-sugar transporter, Fringe connection (Frc) transports a broad range of UDP-sugars that can be used for the synthesis of various glycans, including N-linked types, GAGs, and mucin types. Interestingly, despite its broad specificity, loss-of-function studies have revealed that Frc is selectively required for Notch glycosylation, but not for GAG synthesis. This observation prompted a study at Frc localization; in this study, it was found that Frc is localized only to a subset of Golgi fragments in Drosophila discs and embryos (Yano, 2005).

Frc, Sfl, a glycosylenzyme of GAGs, and Rho, a processing enzyme of Spi glycoprotein, are localized to distinct Golgi fragments, which are referred to as 'Golgi units,' in Drosophila cells. frc mutants do not exhibit defects in the glycosylation and function of Spi nor do they exhibit defects in glycosylation or function of GAG core proteins. Moreover, biochemically separated distinct Golgi units containing Frc and Rho were isolated by immunoisolation technique. This study clearly shows that there are functionally distinct Golgi units in a Drosophila cell (Yano, 2005).

The Golgi complex is a stack of cis-, medial-, and transcisternae in mammalian cells. In contrast, Golgi markers often do not overlap with each other in Saccharomyces cerevisiae, in which the Golgi cisternae are not stacked but disassembled. The Golgi cisternae of Drosophila are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase. To determine whether Drosophila imaginal disc cells have assembled or disassembled Golgi cisternae, the localizations were compared of the cis-cisternal marker dGM130, the transcisternal marker Syntaxin16 (Syx16), and the Golgi-tethered 120-kDa protein, which is commonly used to detect the Golgi complex in Drosophila. The 120-kDa protein was identified by immunoaffinity purification and protein sequencing as a Drosophila homolog of the vertebrate 160-kDa medial Golgi sialoglycoprotein (MG160), which resides uniformly in the medial-cisternae of the Golgi apparatus in vertebrate cells. An antibody specific for the 120-kDa protein also stained numerous Golgi fragments in imaginal disc cells. More than 80% of immunoreactivity for the 120-kDa protein was colocalized with both dGM130 and Syx16, suggesting that 120-kDa protein-positive fragments of the Golgi complex indeed comprise assembled cisternae; these fragments are referred to as 'Golgi units.' The distributions of the 120-kDa protein, dGM130, and peanut agglutinin (PNA), another transcisternal marker, also show that the markers are closely apposed but not identical, suggesting that the Golgi units are polarized. Interestingly, most of the PNA-positive transcisternae are oriented toward the basal side of the cell, within the Golgi complex, whereas most of the GM130-positive cis-cisternae are oriented toward the apical side of the cell. The cis-to-trans polarity of each Golgi unit thus appears to be correlated with the apico-basal polarity of the disc cells (Yano, 2005).

Drosophila mutant larvae defective in the UDP-sugar transporter Frc manifest a highly selective phenotype: the lack of Notch glycosylation in the presence of normal GAG synthesis (Goto, 2001). This limited phenotype was unexpected, given that Frc exhibits a broad specificity for UDP sugars used in the synthesis of various glycans including N-linked types, GAGs, and mucin types. However, given that the frcR29 allele studied previously (Goto, 2001) is hypomorphic, whether the selective glycosylation defect might be a consequence of partial loss of Frc activity was examined. With the use of imprecise excision, a new allele, frcRY34, was generated the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frcR29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frcRY34 or frcR29 mutants was 4.2% and 24.4% of that in the wild type, respectively. About 1 kb of the gene, including the transcription initiation site, was deleted in the frcRY34 allele. Together, these observations suggest that frcRY34 is essentially a null allele (Yano, 2005).

Clonal cells of the frcRY34 mutant exhibit normal levels of GAGs, as detected by immunostaining with the 3G10 antibody, whereas the amount of GAGs was reduced in clones of tout-velu (ttv) mutant cells. Given that GAGs are required for signaling by Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp),the expression was examined of corresponding target genes [patched (ptc) for Hh signaling and Dll for Wg and Dpp signaling] in the wing discs of the frcRY34 mutant. Expression of ptc and that of Dll in the ventral compartment of the wing discs were unaffected in the mutant clones, suggestive of normal GAG function (Yano, 2005).

Given that Notch glycosylation by Fringe (Fng), a fucose-specific ß1,3-N-acetylglucosaminyltransferase, requires Frc activity, Notch glycosylation was examined in the frcRY34 mutant. The frcRY34 mutant clones in the dorsal compartment, but not those in the ventral compartment, of the wing discs induce wg expression at their borders, as has been observed with fng mutant clones, suggesting that Notch glycosylation is impaired in the frcRY34 mutant. The ectopic expression of Wg induced by the frcRY34 mutant clones is likely responsible for the observed induction of Dll expression in the dorsal compartment (Yano, 2005).

To determine why the loss of a UDP-sugar transporter with a broad specificity selectively affects Notch glycosylation, the subcellular localization of Frc was examined. Frc tagged with the Myc epitope was expressed in imaginal discs under the control of the arm-Gal4 driver. The Gal4-induced expression of Frc-Myc rescues the frc mutant phenotype, suggesting that Frc-Myc is functional and properly localized. Immunostaining of imaginal discs of wild-type larvae expressing Frc-Myc with antibodies to Myc and to the 120-kDa protein revealed that Frc localizes to only a small subset of Golgi units. Thus, it is hypothesized that the Golgi units might be functionally heterogeneous, and that those containing Frc might modify some proteins, including Notch, but not others (Yano, 2005).

To test this hypothesis, the localizations of various molecules involved in protein modification in the Golgi complex were compared with that of Frc. It was found that Sfl is also restricted to a subset of Golgi units, but that its distribution does not overlap with that of Frc. This differential localization of Sfl and Frc might thus explain the observation that frc mutant clones in wing discs do not show any defect in GAG synthesis by Sfl (Yano, 2005).

The Spi-processing enzyme Rho was also localized to a subset of Golgi units distinct from those containing Frc, in addition to its presence in other compartments. This result indicates the existence of at least two types of Golgi units, those containing Rho and those containing Frc. To determine whether these two types of Golgi units differ functionally, the glycosylation state and function of Spi were examined in frc mutants (Yano, 2005).

Given that the extent of Notch glycosylation, as detected by wheat germ agglutinin (WGA), is markedly reduced in frc mutants compared with that in the wild-type background (Goto, 2001), whether the WGA-reactive glycan of Spi is also affected by frc mutation was examined. Myc epitope-tagged Spi was expressed in the wild type or the frcRY34 mutant. Spi-Myc was then precipitated from larval homogenates with antibodies to Myc and was examined for its glycosylation by SDS/PAGE and subsequent blot analysis with WGA. The reactivity of the Spi glycan with WGA was similar in the frc mutant and in the wild type. Whether the frcRY34 mutation affects the Spi glycan was examined by mobility shift analysis. The electrophoretic mobility of glycosylated Spi from the wild type was also similar to that from the frc mutant. Deglycosylation of Spi by neuraminidase, peptide-N-glycosidase (PNGase) F, and O-glycanases also increased its mobility to the same extent in wild-type and frc mutant larvae, suggesting that the core protein is not affected by the frc mutation. Together, these results indicate that the function of Frc is not necessary for formation of the Spi glycan (Yano, 2005).

Spi function was evaluated by examining developmental processes such as photoreceptor recruitment and bract formation, both of which require Spi activation. During eye development, although Spi is not necessary for the primary induction of the photoreceptor R8, it is required for the subsequent recruitment of R1 to R7. Given that photoreceptors R1 to R8 express ELAV and that R1 and R6 express Bar, the expression of these proteins was examined in frc mutants. In mutants harboring the hypomorphic allele frcR29, all photoreceptors are normally induced, although their direction is irregular as seen in fringe or Notch mutants. Similar results were obtained by clonal analysis of frcRY34 mutants. Spi function in photoreceptor recruitment thus did not appear to be impaired in the frc mutants. The frcR29 mutant also formed normal bracts on malformed legs. Tests were performed for genetic interaction between rho and frc mutations in wing vein formation. The rhove1 mutant is viable but shows partial loss of L3-5 veins. This phenotype is also apparent in rhove1, frcRY34/rhove1, frc+ flies, suggesting that Frc does not affect Rho function. From these results, it is concluded that the function of the Rho-Spi pathway is not affected by frc mutation (Yano, 2005).

To confirm that the Golgi units containing Frc and those containing Rho are distinct, whether these Golgi units could be selectively isolated was tested by using antibodies to Myc (for Myc-tagged Frc) or HA (for HA-tagged Rho). Because it is difficult to collect enough of the imaginal discs, the starting material was switched to embryos, and whether Frc and Rho are also localized to distinct Golgi units in embryos was examined. Frc-Myc and Rho-HA were coexpressed in the embryos by the arm-Gal4 driver; immunostaining with antibodies to Myc and to HA revealed that the Golgi units containing Frc-Myc (45.4% of total Golgi units) and those containing Rho-HA (43.0% of total Golgi units) are largely distinct: only 11.6% of total Golgi units were positive for both Frc-Myc and Rho-HA. Immunoisolation was attempted from embryonic lysates by using either antibody to Myc or HA and how much Frc-Myc and Rho-HA were coisolated in each immunoisolate was examined. When Frc-Myc was immunoisolated with an antibody to Myc, the recovery of Frc-Myc was 5.7 times greater than that of Rho-HA. Moreover, when Rho-HA was immunoisolated with an antibody to HA, the recovery of Rho-HA was 18.3 times greater than that of Frc-Myc. The immunoblot analysis of these immunoisolates with the anti-120-kDa antibody confirmed that the Golgi units were concentrated in these immunoisolates. These results support the notion that Frc-Myc-containing fraction is distinct and can be separated from Rho-HA-containing fraction (Yano, 2005).

Whether these distinct Golgi units contain different constituents was examined. Fringe (Fng) is one of the candidate molecules that may be colocalized with Frc. Therefore, expression of ectopically expressed Fng was examined in Rho- and Frc-containing immunoisolates. It was found that expression of Fng in Frc-containing immunoisolates was 26 times greater than in Rho-containing immunoisolates, supporting the idea that Fng is localized in the Frc-positive Golgi units rather than the Rho-positive Golgi units. It was also confirmed by immunostaining analysis that Fng colocalizes mostly with Frc (88.1% of the FNG-positive Golgi units), but not with Rho (16.6% of the Fng-positive Golgi units), by immunostaining analysis (Yano, 2005).

The data suggest that different Golgi units perform different functions, a notion that is also supported by the observation that Tn antigen (O-linked N-acetylgalactosamine) was detected in only a subset of Golgi units in imaginal eye disc cells. In addition, it was found that most of these Tn antigen-positive Golgi units are distributed in the basal region of the disc cells, suggesting that the differential distribution of Golgi units might contribute to the apicobasal polarity of glycan distribution (Yano, 2005).

In contrast to the larval stage, Frc is required for GAG synthesis at the early embryonic stage (Goto, 2001; Selva, 2001). To determine why the Frc requirement for GAG synthesis differs between the embryonic and larval stages, embryos expressing Frc-Myc were stained with antibodies to Sfl and to Myc. Sfl was found to be colocalized with Frc, likely explaining the importance of Frc for GAG synthesis at the embryonic stage. In addition, this embryonic requirement of Frc for GAG synthesis excludes the possibility that the selective defects in Notch and not in GAG synthesis observed in frc mutant larvae are caused by the selective Frc-dependent transport of a subset of UDP-sugars used only for glycosylation of Notch but not for GAGs synthesis (Yano, 2005).

It summary, these results provide evidence for the existence of functionally distinct Golgi units in Drosophila cells. Such functional heterogeneity of Golgi units is likely responsible for the diversity of protein glycosylation. At least two types of Golgi units containing either Frc or Sfl are present in larval disc cells. Two distinct sets of proteins, exemplified by Notch and GAG core proteins, might thus be selectively transported to Frc- or Sfl-containing Golgi units, respectively, where they undergo glycosylation by different sets of molecules (Yano, 2005).

The variety of Golgi units might be established by separate transport of secretory proteins and glycosylenzymes from the endoplasmic reticulum (ER) to the distinct Golgi units. In yeast, glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in vesicles distinct from those containing other secretory protein. Given that the GAG core protein Dally in Drosophila is anchored to the membrane by GPI, it is possible that Dally and Notch are loaded into distinct vesicles as they exit the ER (Yano, 2005).

Combinations of glycosylenzymes and transporters, such as Sfl and Frc, contained in Golgi units of Drosophila differ not only between embryos and larval disc cells but also among cell types. For example, Frc is localized to all Golgi units in salivary gland cells at the larval stage. It has also been shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the Gurken precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region. The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes (Yano, 2005).

The functional diversity of Golgi units also might contribute to the polarized distribution of glycans along the apicobasal axis of cells. It was found that Tn antigen is synthesized in the basal Golgi units of larval disc cells. Furthermore, certain types of glycans are distributed along the apicobasal axis of pupal ommatidia. These glycans might thus be synthesized differentially in the Golgi units that are asymmetrically distributed along the apicobasal axis and then be secreted at either the apical or basal cell surface (Yano, 2005).

Whereas Golgi units are dispersed throughout Drosophila cells, the Golgi complex in mammalian cells is thought to be a single entity that is located in the pericentriolar region through its association with the microtubule-organizing center in interphase and which is fragmented at the onset of mitosis. The Golgi fragments apparent in mammalian cells during mitosis are highly similar to the Golgi units of Drosophila cells in both electron and confocal microscopic images. The mammalian Golgi complex during interphase may therefore be comprised of functionally distinct units that are associated with the microtubule-organizing center and connected with each other (Yano, 2005).

The Big brain aquaporin is required for endosome maturation and Notch receptor trafficking

Activity of the big brain (bib) gene influences Notch signaling during Drosophila nervous system development. This study demonstrates that Bib, which belongs to the aquaporin family of channel proteins, is required for endosome maturation in Drosophila epithelial cells. In Drosophila mutants for hrs, lethal(2) giant discs (lgd), and genes encoding ESCRT complex proteins, mislocalization of Notch in endosomes is associated with the accumulation of other receptors and ligands In the absence of Bib, early endosomes arrest and form abnormal clusters, and cells exhibit reduced acidification of endocytic trafficking organelles. Bib acts downstream of Hrs in early endosome morphogenesis and regulates biogenesis of endocytic compartments prior to the formation of Rab7-containing late endosomes. Abnormal endosome morphology caused by loss of Bib is accompanied by overaccumulation of Notch, Delta, and other signaling molecules as well as reduced intracellular trafficking of Notch to nuclei. Analysis of several endosomal trafficking mutants reveals a correlation between endosomal acidification and levels of Notch signaling. These findings reveal an unprecedented role for an aquaporin in endosome maturation, trafficking, and acidification (Kanwar, 2008).

Mutants in the big brain gene were among the first Drosophila mutants isolated with specific patterning defects in embryonic neurogenesis. Together with five other founding members of the 'neurogenic' gene family, Notch, Delta, mastermind, Enhancer of split, and neuralized, the bib locus prevents ectopic neuroblast specification and lethal hypertrophy of the embryonic nervous system. Subsequent molecular analyses have demonstrated that the five other genes encode core components of the Notch signaling pathway, but the relevance of Bib function to Notch activation has long remained unclear. Based on the current findings, it is proposed that the primary defect caused by loss of Bib function is a failure in endosome maturation, which indirectly impairs transmission of the activated Notch signal during its endosomal trafficking (Kanwar, 2008).

The results support a model in which the initial steps of Notch activation, including ligand binding, ectodomain removal, endocytosis, and biochemical cleavage of the Notch receptor by the γ-secretase complex, do not require Bib. However, these events are associated with the entry of ligand-activated Notch into endosome trafficking compartments (Vaccari, 2008), and efficient movement of Notch through these compartments depends upon Bib-promoted maturation of the endosomes. The morphological features of the bib endosome arrest phenotype could potentially account for the specificity of the bib mutant effects on Notch signaling as well as the partial preservation of Notch signaling in the absence of Bib function. Unlike other major developmental signaling pathways that involve signal amplification through an effector cascade, Notch signaling relies upon direct cleavage of Notch to produce stoichiometric amounts of NICD needed for transcriptional regulation of target genes. The tightly packed early endosomes in bib mutant cells might prevent proper intracellular trafficking of most but not all NICD, thus leading to reduced but not completely blocked Notch signaling. Arguing against this idea, however, is the finding that bib lgd double mutant cells exhibit a rather different phenotype of enlarged, possibly arrested MVB (multivesicular body)-like structures accompanied by suppression of the ectopic Notch activation seen in lgd single mutants. Hence, the specific early endosome arrest seen in bib mutants is not required per se for the observed negative effects on Notch signaling, and instead Bib seems capable of facilitating Notch activity at different steps along the endosome-lysosome pathway (Kanwar, 2008).

A clustering phenotype of early endosomes similar to that in bib mutant cells has been observed upon loss of KIF16B, a kinesin-3 microtubule motor that regulates plus-end motility of early endosomes, their intracellular distribution, and their ability to convert into late endosomes, maintaining the crucial balance between receptor recycling and degradation. Bib may regulate the assembly or activity of these key determinants, such that loss of Bib disrupts dynamic interactions of endosomes with the cytoskeleton and causes clustering through an arrest in motility (Kanwar, 2008).

How does the Bib aquaporin promote the endosome maturation? Mammalian aquaporins act as tetramers, each subunit of which forms a channel through which water, ions, or other small solutes are transported. The channel is formed by two NPA motif-containing loops that project into the lipid bilayer from opposite sides, and residues located near the narrowest channel point influence permeability characteristics through size restriction and electrostatic repulsion. Bib lacks a conserved histidine that is present in all water-transporting aquaporins, and Drosophila Bib expressed in Xenopus oocytes is permeable to cations but not water molecules (Yanochko, 2002; Kanwar, 2008 and references therein).

There are two primary models for Bib function that are suggested by these findings. In one model, Bib directly alters a biochemical property of endosomes by transporting ions across the limiting membrane. In the second model, the ion channel domain of Bib acts as a sensor for ion flux across the membrane, perhaps linking endosomes to different cytoplasmic factors in response to changes in endosome ion concentrations. With respect to the first model, endosomes undergo increasing acidification as they mature from early endosomes to lysosomes, and acidification plays a causal role in the formation of multivesicular/late-endosome-like membrane structures in vivo and in biochemical liposome reconstitution studies. The progression from early to late endosomes is also accompanied by a conversion from Rab5 to Rab7 GTPases. The absence of Rab7 in bib mutant endosomes along with their reduced acidification suggests that endosomal pH regulation by Bib may modulate aspects of intralumenal vesicle generation and endosome biogenesis. These processes could also influence interactions of endosomes with the cytoskeletal network, contributing to the bib endosome clustering phenotype as noted above (Kanwar, 2008).

The pH gradient across invaginating endosomal membranes is tightly regulated and could potentiate Notch signaling efficiency by additional mechanisms. First, the pH gradient might be needed for proper invagination or other membrane curvature events, involving dynamic changes in membrane composition that facilitate receptor/ligand dissociation or influence interactions of Notch with γ-secretase. Indeed, subtle changes in membrane lipid composition affect Notch and Egfr endosome sorting and signaling in Drosophila imaginal tissues. In addition, the catalytic activity of γ-secretase itself is optimal in a low pH environment. Although biochemical data indicate that Bib is unlikely to be an essential cofactor for γ-secretase, a caveat is that the cell lysis procedure might itself cause pH changes or other effects that bypass a normal γ-secretase requirement for Bib in intact cells (Kanwar, 2008).

Endosome-associated ion channels have been implicated in organelle acidification and biogenesis. Loss of endosome/lysosome-associated chloride ion channels leads to a bone resorption disorder in humans and defective receptor endocytosis and recycling in knockout mice. Cells lacking Bib exhibit a pronounced reduction in the acidification of endosomal organelles, even in combinations with other trafficking mutants that produce different abnormal endosome morphologies. Thus, the idea is favored that the ion channel activity of Bib is directly involved in the progressive acidification of the endosome compartments. Moreover, inhibition of vacuolar ATPase impairs endosomal acidification and protein trafficking to late endosomes, leading to the proposal that vacuolar ATPase is a component of an endosomal pH-sensing machinery that recruits cytosolic factors to endosomes. Bib might function in a similar manner, mediating pH-dependent interactions between endosomes and the cytoskeleton or recruiting cytoplasmic factors needed for endosome maturation and/or transport. Further studies will be required to elucidate the relationship of Bib to other ion channels and vacuolar ATPases implicated in endosome acidification, as well as to the cytoskeletal and trafficking machinery (Kanwar, 2008).

The involvement of Bib in endosome biogenesis illustrates the complexity of endocytosis-dependent membrane trafficking and its relationship to signal transduction. The striking defects in endosome maturation in cells lacking Bib reveal an unprecedented role for an aquaporin in organelle biogenesis. The findings also demonstrate that impairment of a specific step of endosome maturation can have profound yet relatively specific effects on a single developmental signaling pathway. Further analysis will be needed to understand the biophysical role of Bib in endosome maturation and acidification and to determine if mammalian aquaporins might also regulate endosomal processes and the membrane trafficking of internalized signaling molecules (Kanwar, 2008).

The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for subtype-specific glial gene expression

The development, organization and function of central nervous systems depend on interactions between neurons and glial cells. However, the molecular signals that regulate neuron-glial communication remain elusive. In the ventral nerve cord of Drosophila, the close association of the longitudinal glia (LG) with the neuropil provides an excellent opportunity to identify and characterize neuron-glial signals in vivo. This study found that the activity and restricted expression of the glycosyltransferase Fringe (Fng) renders a subset of LG sensitive to activation of signaling through the Notch (N) receptor. This is the first report showing that modulation of N signaling by Fng is important for CNS development in any organism. In each hemisegment of the nerve cord the transcription factor Prospero (Pros) is selectively expressed in the six most anterior LG. Pros expression is specifically reduced in fng mutants, and is blocked by antagonism of the N pathway. The N ligand Delta (Dl), which is expressed by a subset of neurons, cooperates with Fng for N signaling in the anterior LG, leading to subtype-specific expression of Pros. Furthermore, ectopic Pros expression in posterior LG can be triggered by Fng, and by Dl derived from neurons but not glia. This effect can be mimicked by direct activation of the N pathway within glia. These genetic studies suggest that Fng sensitizes N on glia to axon-derived Dl and that enhanced neuron-glial communication through this ligand-receptor pair is required for the proper molecular diversity of glial cell subtypes in the developing nervous system (Thomas, 2007).

This study identified Fng as a means by which a specific subtype of glia, the anterior LG, are made sensitive to N activation, evidence was provided that Dl, expressed on axons, activates N signaling in these glia leading to subtype-specific gene expression. Fng is required for maintenance of Pros expression in the anterior LG, which can also be blocked by antagonism of the N pathway with no effect on their survival or positioning. This is in contrast with studies of pros mutants, which found a role for Pros earlier in CNS development in establishing glial cell number. The role of Pros in mature LG is poorly understood, but it has been proposed to retain mitotic potential in these cells for use in repair or remodeling of the nervous system in subsequent larval or adult stages. It will be important to determine the consequences of lost Pros expression from mature anterior LG, and whether additional features and functions of the anterior LG are controlled by N signaling from axons (Thomas, 2007).

The importance of glycosylation for N function has been demonstrated in vivo. The addition of O-linked fucose to EGF repeats in the N extracellular domain is essential for all N activities and is mediated by O-fucosyltransferase-1 (O-fut1). By contrast, Fng is selectively used in specific developmental contexts, and has been best studied in the formation of borders among cells in developing imaginal tissues. Fng catalyzes the addition of GlcNac to O-linked fucose, to which galactose is then added. The resulting trisaccharide is the minimal O-fucose glycan to support Fng modulation of Notch signaling. Fng activity reduces the sensitivity of N for the ligand Ser but increases its sensitivity for Dl. By contrast with imaginal discs, in which modulation of N sensitivity to both ligands appears to be important, loss of Fng in LG resulted in reduced N activation only, consistent with reduced response to Dl. Expression of Pros in LG can be triggered by Dl derived from neurons but not glia, and this effect can be mimicked by direct activation of the N pathway within glia. Genetic experiments implicate neuron-derived Dl as the relevant N ligand for Pros expression in anterior LG, consistent with the ability of Fng to sensitize N to signaling by Dl. Enriched Fng expression in the anterior LG probably renders them differentially sensitive to sustained N signaling from Dl-expressing axons (Thomas, 2007).

The final divisions of the six LG precursors that give rise to 12 LG are thought to be symmetric, with low levels of Pros first distributed evenly between sibling cells after division. However, Pros is maintained and in fact upregulated in the anterior LG, and downregulated in sibling LG that migrate posteriorly. fng transcripts first appear to be expressed in all LG, then become enriched in the anterior LG and reduced in the posterior LG. It is speculated that refinement of fng expression may involve a positive feedback mechanism to consolidate and enhance N signaling in the anterior LG, since preliminary evidence suggests that N signaling can positively influence fng expression in the LG (Thomas, 2007).

Like Pros, Glutamine synthetase 2 (Gs2) is specifically expressed in the anterior LG but not posterior LG, indicating that these are functionally distinct glial subtypes with respect to their ability to recycle the neurotransmitter glutamate. The specificity of N signaling for Pros but not Gs2 indicates that N signaling is unlikely to influence cell fate decisions in the LG lineage and that Fng is unlikely to be the primary determinant of anterior versus posterior LG identity. Rather, Fng probably serves to consolidate this distinction through sustained N signaling (Thomas, 2007).

NICD is a potent activator of Pros expression in the posterior LG. This leads to a consideration of what factors limit Pros expression to the anterior LG in wild-type animals, since posterior LG are indeed capable of expressing Pros in response to constitutive N activity. (1) Based on analysis of fng mutants and Fng misexpression, it is proposed that Fng is a major determinant. The finding that misexpression of Fng causes ectopic Pros in posterior LG supports the argument that Dl-expressing axons do not contact the anterior LG only. It is likely that they make contact with at least some of the posterior LG. Therefore, in wild-type animals, in which Fng is reduced on posterior LG, contact from the subset of Dl axons is alone not sufficient to drive Pros expression. (2) Misexpression of Dl in all postmitotic neurons led to ectopic expression of Pros in posterior LG, indicating that the restricted expression of Dl on a subset of neurons also limits N activation. (3) N appears to be expressed in most or all LG, though it was also found that overexpression of full-length N caused ectopic expression of Pros. From these data a threshold model is proposed for N activation in LG that invokes a combination of factors, including Fng-regulated N sensitivity, exposure of N to ligand, N expression levels, and perhaps others. Increasing any of these factors can provide sufficient signaling for ectopic Pros induction in posterior LG. In wild-type embryos, these factors are also likely to combine with one another in the anterior LG to achieve supra-threshold N signaling and sustained Pros expression during normal development (Thomas, 2007).

Signaling through N is important for glial cell development in Drosophila, although it is context-dependent. Both an embryonic sensory lineage and the subperineurial CNS glial lineage utilize N activation to promote Gcm expression and glial fate. By contrast, in the sensory organ of adult flies, antagonism of N leads to Gcm expression in the glial precursor cell. In vertebrates, signaling through Notch receptors promotes the differentiation of peripheral glia, Müller glia, radial glia and mature oligodendrocytes. A Fng ortholog, lunatic fringe, is expressed in the developing mouse brain in a pattern consistent with glial progenitors. It will be interesting to determine whether Fng-related proteins in vertebrates have a role in glial cell differentiation, and whether they too can modulate N sensitivity and the context of N signaling between neurons and glia (Thomas, 2007).

Notch Interaction with Scabrous

The mechanisms that establish and sharpen pattern across epithelia are poorly understood. In the developing nervous system, the first pattern elements appear as 'proneural clusters'. In the morphogenetic furrow of the immature Drosophila retina, proneural clusters emerge in a wave as a patterned array of 6 to 10 cell groups, which are recognizable by expression of Atonal, a basic helix-loop-helix transcription factor that is required to establish and pattern the first cell fate. The establishment and subsequent patterning of Atonal expression requires activity of the signaling transmembrane receptor Notch. In vivo and biochemical evidence is presented that the secreted protein Scabrous associates with Notch, and can stabilize Notch protein at the surface. The result is a regulation of Notch activity that sharpens proneural cluster boundaries and ensures establishment of single pioneer neurons (Powell, 2001).

In the morphogenetic furrow, Atonal's expression can be divided into four steps:(1) it is expressed as a broad, unpatterned stripe; (2) expression is then upregulated into evenly spaced proneural clusters;(3) in these proneural clusters, a 2 to 3 cell 'R8 equivalence group' emerges, and (4) expression narrows to identify a single cell in this group as the R8 photoreceptor neuron, the first cell type of the developing retina. In each step, as cells lose Atonal expression they concurrently gain expression of negative regulators, such as members of the E(spl) (Enhancer of Split) complex. Expression of E(spl) initially requires the presence of Atonal, and is subsequently amplified by the Notch signaling pathway to downregulate proneural bHLH expression and function. E(spl), therefore, represents one reporter of Notch activity (Powell, 2001).

Patterning of Atonal and E(spl) expression requires the normal activity of Scabrous, a secreted fibrinogen-related protein with a potential for association with components of the extracellular matrix. In the retina, Scabrous protein first appears in the proneural clusters, mirroring Atonal expression by narrowing to the R8 equivalence group, and eventually R8 alone. This expression is dependent on Atonal activity, which indicates that the scabrous locus may be a direct target of Atonal (Powell, 2001).

Genotypically null scaBP2 proneural clusters are poorly spaced with poorly defined borders. Broadened E(spl) expression throughout much of the proneural cluster region is one potential cause of this imprecision, suggesting that Notch activity is altered in sca BP2 mutants. These observations suggest that initial broad, low-level Atonal expression activates broad, low-level E(spl) expression, and that Scabrous is required to refine the complementary Atonal and E(spl) expression in the proneural cluster region -- events also associated with Notch activity (Powell, 2001).

A 1 hour pulse of ectopic Scabrous results in rapid loss of Atonal within 2 h; E(spl) shows low, diffuse expression that is lost within 4 h. This ectopic expression of Scabrous leads to aberrant patterning of R8s in a manner similar to that of the phenotypes observed in scabrous loss-of-function alleles. The loss of the initial broad stripe of Atonal, a Notch-dependent step, suggests that ectopic Scabrous can lead to a disruption of Notch function, a result consistent with overexpression studies in the Drosophila wing (Powell, 2001).

Subsequent resolution of Atonal expression in the equivalence group, also a Notch-dependent step, is delayed in scaBP2 mutants. Delay of Atonal resolution can result in ectopic R8s; indeed, 40% of scaBP2 ommatidia contain two or three R8s derived from the R8 equivalence group. R8s were observed to be both adjacent and non-adjacent, indicating that selection may be stochastic (Powell, 2001).

These results suggest that Scabrous influences the establishment or maintenance of boundaries of Notch activity during both proneural-cluster and R8 equivalence-group maturation. To investigate the nature of this influence, transient expression of full-length Notch protein (N350) in Drosophila S2 tissue-culture cells was examined by Western analysis. Transiently induced N350 is depleted gradually over 7 h; this loss is accelerated in the presence of Delta. In contrast, addition of Scabrous leads to an accumulation of N350 and smaller Notch fragments, even in the presence of Delta. Accumulation of Notch is not due to induction of the endogenous Notch gene, which is rearranged in S2 cells. Cell-surface biotinylation analysis indicates that the presence of Scabrous significantly stabilizes N350 at the cell surface; however, the functional nature of this stabilization remains unknown (Powell, 2001).

Genetic and histological studies have suggested a function for Scabrous as a secreted Notch ligand. However, attempts to show this interaction biochemically have been unsuccessful. This issue was reexamined using two independent assays. Notch is expressed throughout embryogenesis, whereas Scabrous protein is detected in young (2.5-6.5 h) but not more mature (12.5-16.5 h) embryos. Immunoprecipitation with a mouse polyclonal anti-Scabrous antibody leads to a complex with a relative molecular mass of 300,000 (Mr 300K) that contains both Scabrous and Notch protein. The size and composition of the 300K complex indicates that it may include a truncated form of Notch previously characterized in vivo. As controls, crosslinked complexes recovered by an anti-Delta antibody included Notch, but complexes recovered by an anti-Fasciclin III antibody did not (Powell, 2001).

To investigate binding in vivo, the fact that diffusible ligands can be stabilized specifically in cells expressing their natural receptor was used. In the midpupal eye, Notch is expressed exclusively in a set of interommatidial cells, the secondary and tertiary pigment cells. Scabrous is not expressed in the retina at this developmental stage. Transient heat-shock induction of hs-scabrous pupae results in a ubiquitous pulse of Scabrous protein that is processed, secreted and rapidly lost from most cells in as little as 5 min after the end of heat shock. However, Scabrous is retained in secondary/tertiary cells, the same cells that express the Notch receptor. Retention of Scabrous occurs in apically localized vesicles commonly observed with secreted ligands and their receptors, including Notch. This cell-type-specific retention suggests that a receptor for Scabrous is found specifically in the secondary/tertiary cells at this stage of development (Powell, 2001).

The expression pattern of Notch was altered through an inducible heat-shock promoter. Co-expression of Scabrous and Notch in all retinal cells results in the stabilization of Scabrous in all cells. Similarly, ubiquitous expression of an inactive form of Notch that lacks its intracellular domain (NdeltaICN) is also sufficient to stabilize exogenous Scabrous in all cells, indicating that Scabrous may associate with the extracellular domain of Notch. This also indicates that stabilization of Scabrous does not require the signaling activity of Notch. As predicted, a membrane-tethered form of the intracellular domain (NdeltaECN) does not retain Scabrous (Powell, 2001).

The extracellular domain of Notch includes 36 epidermal growth factor (EGF)-like repeats. Two demonstrated ligands of Notch, Delta and Serrate, require EGF-like repeats 11 and 12 for binding. Ubiquitous expression of forms of Notch that delete repeats 1-18 or 9-17 (Ndelta1-18 and Ndelta9-17) is still sufficient to stabilize Scabrous protein in all retinal cells; however, Ndelta19-36 and N delta17-26 fail to retain Scabrous protein. The inability of Ndelta19-36 or Ndelta17-26 to retain Scabrous suggests that the site of Notch/Scabrous association lies within repeats 19-26. Consistent with this view, Scabrous protein is able to promote stabilization of Ndelta1-18 that was expressed on the surface of S2 cells, but failed to stabilize Ndelta19-36 (Powell, 2001).

These data support the view that Scabrous and Notch form a receptor-ligand pair; however, the possibility that binding requires other cofactors or adapters cannot be ruled out. These results indicating stabilization suggest that Scabrous may act to scaffold Notch protein to the extracellular matrix and downregulate Notch activity in at least some experimental situations in order to sharpen local boundaries. Consistent with this view, the sharp boundaries between proneural clusters are lost in scaBP2 clonal patches, indicating that Scabrous may act over a short distance. Notably, when a scaBP2 clonal patch crosses the morphogenetic furrow, high E(spl) expression typically coincides with the boundary of the clone (Powell, 2001).

The observed complexity of their in vivo interactions leaves unresolved the precise mechanism by which Scabrous regulates Notch at boundaries. One model suggests that similar to Fringe, Scabrous provides a means by which Notch activity can be altered in the absence of changes in ligand expression, although the tissues and possibly the mechanisms, are different (Powell, 2001).

Table of contents

Notch continued: Biological Overview | Evolutionary Homologs | Regulation | Post-transcriptional regulation of Notch mRNA | Developmental Biology | Effects of Mutation | References

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