InteractiveFly: GeneBrief

lethal (2) giant discs 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal (2) giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).

Imaginal disc development depends on the Drosophila tumor suppressor genes (TSGs). Fifty TSGs have been identified and the loss-of-function of many of these genes results in overproliferation of the imaginal discs. These genes can be divided into two groups based on the mutant phenotypes (Bryant, 1993; Watson, 1994). Deletion of genes belonging to the tumorous class causes cells to overproliferate and invade new regions so that eventually the epithelial and compartmental organization of the discs is lost. In contrast, the loss of genes of the hyperplastic group causes overproliferation, but does not disturb the epithelial and compartmental organization of the discs. l(2)giant discs belongs to this second group. The loss of lgd causes massive overproliferation of imaginal disc cells and extended larval life (Bryant, 1971; Klein, 2003).

It has also been observed that wingless (wg) is expressed ectopically in the pouch of lgd mutants during wing development (Buratovich, 1995). Similar phenotypes are observed, if the Notch pathway is ectopically activated during wing development, raising the possibility that the lgd mutant phenotype could stem from the ectopic activation of the Notch pathway. The Notch pathway is indeed ectopically active in lgd mutants, and hyperactivation as well as ectopic activation of the pathway accounts for the lgd phenotype during wing development. In lgd mutants, the expression of Notch target genes along the D/V boundary is expanded, indicating that Lgd is required for the restriction of Notch activity to the D/V boundary. Furthermore, the mutant phenotype of lgd is suppressed by concomitant loss of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the activation of the Notch pathway. Evidence is provided that the activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch by Lgd is not restricted to wing development and occurs during several other developmental processes, such as vein, eye, and bristle development, suggesting that Lgd suppresses the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).

Loss of lgd function leads to an overgrowth of the imaginal discs, clearly noticeable in the wing region of the wing disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a much broader domain that extends into the wing pouch (Buratovich, 1995). In addition, lgd mutant wing discs often develop a second wing pouch in the region of the anlage of the scutellum (Buratovich, 1995). Similar phenotypes are caused by gain-of-function alleles of N (for example, Abruptex) and are also observed upon expression of the activated intracellular form of Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing discs and precedes the visible morphological changes that occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The phenotype is always variable, but the overall phenotype of lgd^d7 and lgd^d10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgd^d4 and lgd^d1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in homozygotes, indicating that the observed phenotype is probably caused by the loss-of-function of the lgd gene (Klein, 2003).

The similarity between the loss of lgd function and ectopic N activation suggests that the phenotype of lgd could be caused by ectopic activation of the Notch pathway. To examine this possibility, the expression of E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the vg-boundary enhancer (vgBE) in mutant wing discs. The expression of all these markers is initiated in cells at the D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is activated in an additional stripe along the anteroposterior compartment boundary (A/P boundary), which is also dependent on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is more complex but always dependent on the activity of Notch in cells at the D/V boundary. In lgd mutant wing discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer, since a version lacking it shows no ectopic activity in the mutants. As in the case of wg, the expression of the vgBE is already expanded in early third larval wing discs. Altogether, these results show that the loss of lgd function leads to the ectopic expression of Notch target genes. This suggests that the Notch pathway is ectopically activated in lgd mutants (Klein, 2003).

All tested Notch-target genes are ectopically activated in lgd mutant wing discs or lgd mutant cell clones. The ectopic activation of Notch target genes as well as the observed overproliferation of lgd mutants is abolished in lgd;Psn double mutants. In addition, Notch target gene expression is also abolished in Psn or Su(H) mutant clones generated in lgd mutant wing imaginal discs. These data suggest that the Notch pathway becomes ectopically active in the absence of lgd function. Furthermore, the fact that Delta alone seems to provide sufficient Notch activity to sustain wing development in lgd mutants indicates that the pathway can be activated more efficiently in the mutant background. The activation of Notch is a consequence of loss of lgd function also in other developmental processes, such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).

Although most aspects of the mutant phenotype of lgd mutants can be explained by the inappropriate activation of the Notch pathway, the cell death observed during induction of lgd mutant clones has not been observed if activated forms of Notch are expressed in the wing pouch or in gain-of-function mutants of Notch, such as Ax. These facts would suggest that lgd function might also have another function for cell viability that is separable from its role in the regulation of Notch activity. However, inappropriate activation of the Notch pathway elicits apoptosis in wing pouch cells under certain circumstances. Hence, it is also possible that this aspect of the lgd mutant phenotype is a consequence of Notch activation (Klein, 2003).

The clonal analysis of lgd reveals several interesting effects. One effect is that Notch becomes activated at the boundary of Dl;Ser double mutant cell clones. At the moment, it is not clear how this activation is achieved. A likely explanation is that activation of Notch at the clone boundaries is caused by the removal of the negative effects of strong Dl and Ser expression observed during late wing development. During normal development, Dl and Ser are expressed in a dorsal and ventral band of cells adjacent to the cells at the D/V boundary in later stages of the third larval instar. Both ligands signal from there to the cells at the boundary to maintain expression of Wg and other genes. It has been shown that activation of Notch is blocked in the cells expressing the ligands because of their autonomous inhibitory effect on Notch signalling at high concentrations. Loss of Dl and Ser expression leads to the loss of the suppressive effect, and the mutant cells at the clone boundary activate expression of Notch target genes. In lgd mutants, the expression domains of Dl and Ser are expanded and the pathway can be activated more efficiently. Thus, the effect of activation of Notch at the boundary of Ser/Dl double mutant clones should also be comparably enhanced (Klein, 2003).

The analysis of the lgd mutant clones suggests that lgd acts in a cell-autonomous way. However, this autonomy is not complete, and in some cases, Notch target genes are activated in wild type cells at the boundary of lgd mutant clones. An explanation for this observation is the fact that the activation of Notch results in the expression of the ligands Dl or Ser. Clones of wing pouch cells expressing the activated form of Notch, Nintra, also activate Notch target gene expression in cells outside the clone, indicating a nonautonomous behavior of Nintra in this cases. This nonautonomous behavior is caused by the induction of the expression of the Notch ligands. The nonautonomy of Nintra is not observed in all situations. For example, if UAS Nintra is expressed with ptcGal4, activation of Notch target genes is cell-autonomous, although induction of ligand expression is observed. Hence, the nonautonomous activation of Notch target genes by Nintra is dependent on other criteria, such as the level of expression or the time span of signalling. It is likely that the observed weak nonautonomy of lgd in clones is caused by the activation of expression of Dl and Ser close to threshold levels of activity that are required to activate Notch in some cells outside the clone (Klein, 2003).

Several explanations for how the Notch pathway is activated in lgd mutants are possible. A very simple one would be that the expansion of Notch target genes in lgd mutant clones or wing discs is caused by an overproliferation of the mutant cells that cause an expansion of the expression domains of the Notch target genes. Thus, the effects on Notch signalling would be secondary. However, clones that are located in the wing pouch and do not have any contact with the normal domain of Notch activity at the D/V boundary are able to activate the expression of Notch target genes, indicating that the pathway is activated de novo. Furthermore, Notch is activated in mutant clones of wing discs of the early third instar. These discs do not show any visible overproliferation. Hence, it is very likely that the expansion of the target gene expression is not caused by a secondary effect, such as cell proliferation, but by the activation of the Notch pathway (Klein, 2003).

The expansion of Notch activity could also be caused by the loss of the suppressive effect on signalling of high concentrations of the ligands observed in the lgd mutants. Although this mode of regulation is important during the second half of the third larval instar stage, it cannot account for the ectopic activation of Notch targets in earlier wing discs observed here (Klein, 2003).

lgd could act in a parallel pathway that is required to restrict the activation of the target genes by Notch. An example of this is the Nubbin transcription factor that seems to bind to the regulatory region of at least some Notch target genes and represses their expression away from the D/V boundary. lgd could act in a similar way. However, there are important differences in the behavior of nub and lgd mutants. nub mutants do not show the overproliferation of the imaginal discs seen in lgd mutants and, in contrast to lgd, the effects of Nub on Notch target gene expression are restricted to the wing. These differences make it unlikely that both genes act in the same pathway. In agreement with these conclusions, it has been found that nub expression is not affected in lgd mutant wing imaginal discs (Klein, 2003).

A further possibility is that lgd could modulate the effectiveness of the Notch signal, e.g., by creating a threshold for Notch activity required for activation of the target genes or influencing the activity of a selector gene such as Vg for the wing. However, the activity of one target gene of Vg/sd, spalt, is not affected in lgd mutants, suggesting that the activity of the selector is not affected (Klein, 2003).

The comparison of the Ax and lgd mutant phenotype reveals a striking similarity: In Ax mutant wing discs, as in those of lgd mutants, Notch activity expands into the wing pouch. In addition, in Ax mutant wing discs, the dominant negative activity of the ligands is suppressed in a fashion similar to that observed in lgd mutants. The phenotype of both of these mutants requires the activity of the Notch ligands. Furthermore, in both mutants, the cell-autonomous suppressive effect of Fng on Notch signalling is strongly suppressed. Finally, the development of the veins and SOPs is suppressed in both mutants. The similarity of the phenotypes between lgd and Ax mutants raises the possibility that they are based by the interruption of the same process required to negatively regulate Notch activity. One argument against this conclusion is that the phenotype of the lgd^d7,Ax^MI double mutant wing discs described here is synergistic. This suggests that the genes do not act in the same regulatory mechanism. The problem with this argument is that it is not clear whether any of the known Ax mutations are abolishing the affected function completely and thus does not rule out the possibility that lgd and Ax affect the same regulatory pathway (Klein, 2003).

REGULATION

The conserved c2 domain protein Lethal (2) giant discs regulates protein trafficking in Drosophila

Drosophila sensory organ precursor (SOP) cells undergo several rounds of asymmetric cell division to generate the four different cell types that make up external sensory organs. Establishment of different fates among daughter cells of the SOP relies on differential regulation of the Notch pathway. This study identified the protein Lethal (2) giant discs (Lgd) as a critical regulator of Notch signaling in the SOP lineage. lgd encodes a conserved C2 domain protein that binds to phospholipids present on early endosomes. When Lgd function is compromised, Notch and other transmembrane proteins accumulate in enlarged early endosomal compartments. These enlarged endosomes are positive for Rab5 and Hrs, a protein involved in trafficking into the degradative pathway. These experiments suggest that Lgd is a critical regulator of endocytosis that is not present in yeast and acts in the degradative pathway after Hrs (Gallagher, 2006).

The phenotypes observe in lgd mutants are strikingly similar to those that have recently been described for Drosophila members of the ESCRT complexes. These complexes have been identified in yeast but are found in all animals. They are required for protein sorting in the degradative pathway and the formation of multivesicular bodies. Ubiquitinated internalized proteins are recognized by Hrs (Vps27 in yeast), a ubiquitin-binding protein targeted to early endosomes by its FYVE domain. Hrs binds to Vps23, a member of the ESCRT I complex, and these proteins recruit the other members of the ESCRT I complex. ESCRT I activates ESCRT II, leading to the recruitment of ESCRT III, the budding of vesicles into the endosomal lumen, and MVB formation. When MVBs fuse with lysosomes, these internal vesicles and their protein contents are degraded by lipases and hydrolases (Gallagher, 2006).

Although there is no yeast homolog of Lgd, three pieces of evidence suggest that Lgd might act in this pathway: (1) mutations in the Drosophila homologs of vps27 (hrs in flies and mammals), vps23 (erupted in Drosophila; tsg101 in mammals), and vps25 (another ESCRT II complex member) lead to accumulation of ubiquitinated transmembrane proteins in enlarged endosomes, a phenotype that is also observe in lgd mutants. Notch is found in enlarged, Hrs-positive compartments in both lgd and vps25 mutant cells. (2) In lgd mutants, just like in flies mutant for hrs, erupted, or vps25, signaling through transmembrane receptors is ectopically activated. (3) lgd was initially identified as a tumor suppressor gene, and recent papers describing the Drosophila homologs of ESCRT complex members show that they also have tumor suppressor properties (Gallagher, 2006).

Where in the pathway could Lgd act? Due to a paucity of markers for ESCRT complex members in Drosophila, it was not possible to precisely determine the point at which lgd is required. However, the results indicate that lgd acts after hrs in the pathway. Unlike mutants in ESCRT I (vps23, erupted) or ESCRT II (vps25), the Notch pathway is not ectopically activated in hrs mutants. Furthermore, hrs, lgd double mutant experiments suggest that the ectopic activation of Notch in lgd mutants requires the activity of hrs. Consistent with this, in lgd mutant cells, Hrs is recruited to vesicles, and these vesicles contain ubiquitinated proteins. An interpretation is that hrs mutants block Notch trafficking at an earlier step than lgd. In the double mutant, the early block in vesicle trafficking does not allow Notch to reach the later compartment, in which it would accumulate in lgd single mutants, thus preventing ectopic activation of the Notch pathway (Gallagher, 2006).

Is it possible to reconcile the protein-trafficking defect and Notch overactivation observed in lgd mutants? The final step in Notch activation is the Presenilin-dependent S3 cleavage. Since Presenilin has been shown to be required for ectopic Notch activation in lgd mutants, it is proposed that lgd leads to the accumulation of Notch in a compartment where it can be more easily cleaved by the protease. Presenilin localizes to the plasma membrane and to internal membranes and has been shown to be active both at the plasma membrane and in endosomes. Although it cannot be excluded that the S3 cleavage occurs at the cell surface, the data suggest that this proteolytic event can also occur to some level in endosomal compartments. Two reasons can be envisaged to explain the Notch overactivation phenotype in lgd mutants: either Notch is endocytosed to some level even if it has not encountered a ligand, and this pool of endocytosed Notch is activated over time when it accumulates in endosomes. Alternatively, ligand binding triggers the S2 cleavage at the cell surface, and it is the NEXT fragment that accumulates in endosomes and therefore can undergo a more complete S3 cleavage before being degraded in lysosomes. Although full-length Notch is not a good substrate for Presenilin and upregulation of Notch signaling in lgd mutants was thought to be ligand dependant, an accompanying paper (Jaekel, 2006) shows that ectopic Notch signaling in lgd mutants is ligand independent, favoring the first possibility (Gallagher, 2006).

It is puzzling that loss of lgd and loss of ESCRT I/II complex members leads to Notch overactivation but hrs mutations do not. Recent work has shown that accumulation of Notch is not always sufficient to activate Notch signaling, whether it is at the plasma membrane or in late endosomes. In hrs mutants, Notch colocalizes with the syntaxin Avalanche, while in vps25 mutants it does not. This finding indicates that although Notch accumulates in enlarged early endosomes in both cases, there are differences between these endosomes. One difference could be the presence or absence of Presenilin, although this remains to be tested (Gallagher, 2006).

Just as accumulation of Notch does not always lead to ectopic activation of signaling, activation of Notch signaling does not always have the same consequences for the cell. lgd mutant cells activate the Notch target gene Cut, whereas vps25 mutant cells do not. Loss of ESCRT I/II complex members leads to Notch-dependant activation of Unpaired, leading, in turn, to nonautonomous overproliferation, while lgd mutant cells themselves overproliferate. lgd mutant cells retain the capacity to differentiate, while ESCRT I/II mutant cells lose polarity, fail to differentiate, and undergo apoptosis. Clearly, further characterization of lgd and its homologs is required to define its functional relationship with the ESCRT complex (Gallagher, 2006).

All ESCRT complex members identified so far are conserved between yeast and humans. Given that lgd is not conserved in yeast, the phenotypic similarity to vps23 and vps25 mutations is surprising. It is possible that the more complex sorting requirements in multicellular organisms require modifications of the ESCRT machinery. Further study will be required to figure out exactly what evolutionary advantage this modification offers metazoa (Gallagher, 2006).

The tumour suppressor Lethal (2) giant discs is required for the function of the ESCRT-III component Shrub/CHMP4

Recent work indicates that defects in late phases of the endosomal pathway caused by loss of function of the tumour suppressor gene lethal (2) giant discs (lgd) or the function of the ESCRT complexes I-III result in the ligand-independent activation of the Notch pathway in all imaginal disc cells in Drosophila. lgd encodes a member of an uncharacterised protein family, whose members contain one C2 domain and four repeats of the DM14 domain. The function of the DM14 domain is unknown. This study reports a detailed structure-function analysis of Lgd protein, which reveals that the DM14 domains are essential for the function of Lgd and act in a redundant manner. Moreover, this analysis indicates that the DM14 domain provides the specific function, whereas the C2 domain is required for the subcellular location of Lgd. Lgd was found to interact directly with the ESCRT-III subunit Shrub through the DM14 domains. The interaction is required for the function of Shrub, indicating that Lgd contributes to the function of the ESCRT-III complex. Furthermore, genetic studies indicate that the activation of Notch in ESCRT and lgd mutant cells occurs in a different manner and that the activity of Shrub and other ESCRT components are required for the activation of Notch in lgd mutant cells (Troost, 2012).

This study reports the results of a detailed structure-function analysis of Lgd, a member of a recently discovered protein family whose hallmark is the possession of four tandem repeats of the uncharacterised DM14 domain. Although a recent study has reported a similar analysis for human Lgd2 in cell culture (Zhao, 2010), this is the first comprehensive analysis of a member of this uncharacterised protein family in an animal model. For the analysis a new assay system was developed that assured expression of the constructs at the level of endogenous lgd. This was necessary because it was found that the process of protein trafficking is very sensitive to overexpression of Lgd. Thus, data obtained by overexpression of Lgd proteins (e.g., in cell culture) must be interpreted with great caution. This notion can probably be extended to other elements of the endosomal pathway, because dramatic changes have be observed in endosome morphology if other endosomal proteins, such as FYVE-GFP, Rab5-GFP or Rab7-GFP, are expressed with the Gal4 system. Moreover, this study found that overexpression of these proteins suppresses the activation of Notch in lgd cells. These findings indicate that the overexpression of endosomal proteins induces significant changes in protein trafficking through the endosomal pathway (Troost, 2012).

This study found that the DM14 domains are important for the function of Lgd and that they constitute novel modules for direct interaction with a core member of the ESCRT-III complex during protein trafficking. Moreover, this analysis reveals that the DM14 domains provide the specific function of Lgd and function in a redundant manner. Using cell culture, Nakamura (2008) provided evidence that the fourth DM14 domain of Lgd2 is especially important for its function as a scaffold protein that is required for PDK1/Akt signalling activated by the EGF. However, no specific importance of the fourth DM14 domain could be detected in Drosophila. In the assay conditions used, any combination of two of the four domains appears to be sufficient for Lgd function and can rescue the lgd mutant phenotype (Troost, 2012).

However, this notion holds true only if the concentration of Shrub is normal. In situations where the activity of Shrub is reduced (shrub^4-1/+), variants with four domains can provide more activity and assure sufficient interaction to maintain correct endosomal trafficking. This was already observed in animals that are hypomorphic for lgd (lgd^d7/lgd^SH495 shrub^4-1). In other words, four DM14 copies enable the organism to tolerate the lgd shrub double heterozygous situation. Because almost all Lgd-like proteins discovered so far have four copies, it is likely that this ability endows members of the family with a functional robustness that is evolutionarily advantageous. The rescue experiments in the sensitized lgd +/lgd shrub^4-1 backgrounds also suggest that the second DM14 domain is of greatest importance for the function of Lgd in Drosophila. This is in contrast to results of cell culture experiments for human Lgd2 (Nakamura, 2008). However, it is important to point out that most of the evidence for function in mammals is obtained with cell culture experiments, which often involve the overexpression of Lgd orthologues at levels way above endogenous levels. Given the great difficulties in gaining sensible results using the Gal4 system, these data should be interpreted carefully (Troost, 2012).

It has been previously shown that the C2 domain of Lgd can bind to certain phospholipids, such as phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, in an in vitro assay (Gallagher, 2006). Furthermore, cell fractionation experiments using cytosolic extracts from wild-type and the lgd⁰⁸ mutant animals that encode a variant lacking the C2 domain, suggest that a small fraction is associated with the membrane in a C2-dependent manner. These biochemical data are contrasted by microscopy studies, which reported a cytosolic distribution of Lgd without any obvious association with membrane structures. In agreement, this study found that tagged Lgd variants expressed at the endogenous level are localised within the cytosol. Moreover, it was found that a lgd construct, encoding little more than the C2 domain and virtually identical to the Lgd fragment used in the in vitro phospholipid binding assay (Gallagher, 2006), is located in the cytosol similarly to Lgd. The discrepancy between the biochemical and microscopy data might be explained by the possibility that only a small fraction of Lgd (which cannot be detected in antibody staining) is associated with membranes. However, knowing that Lgd interacts with Shrub, it is surprising that no obvious association of Lgd was found even upon depletion of Vps4, although the ESCRT-III complex is locked on the endosomal membrane in this situation. One would expect that the membrane-associated fraction of Lgd should be increased in this situation. Thus, it is believed that Lgd is located within the cytosol. This notion is further supported by the fact that variants of Lgd that lack the C2 domain can rescue the lgd mutant phenotype to a high degree, although they are produced at a much lower level than the other constructs tested and than endogenous Lgd (Troost, 2012).

Three distinct functions were determined for the C2 domain. The first function is that it provides protein stability, because it was found that the constructs encoding variants without the C2 domain give rise to significantly lower amounts of protein than variants with the domain. The second function is the localisation of Lgd within the cytosol. This function provides an explanation for the discrepancy between the in vivo and biochemical studies, because variants without the C2 domains were found to be located in the nucleus. The reason for the mis-localisation of Lgd variants that lack the C2 domain is unclear at the moment. No cryptic nuclear localisation sequence (NLS) has been found within Lgd. Thus, it is possible that it is transported in the nucleus in complex with another protein that contains an NLS (Troost, 2012).

The presented results suggest a third function for the C2 domain, because it was found that LgdδDM14, which cannot provide any specific function in the rescue assay, can out-compete NESLgdδC2 in a C2-dependent manner and thereby prevent the partial rescue of lgd mutants. A likely possibility is that the C2 domain mediates an interaction with other proteins that results in concentration of Lgd at the site of action within the cytosol. In agreement with this possibility, recent reports have shown that the C2 domains of Nedd4L, PKC and PKCe mediate protein-protein interactions. Furthermore, human Lgd2/CC2D1A appears to interact via its C2 domain with the E2 enzyme Ubc13 during NF-kappaB signalling (Zhao, 2010). Therefore, the possibility is favored that the C2 domain of Lgd mediates protein-protein interactions instead of localising Lgd to a distinct membrane. It is possible that the cytosolic interaction prevents Lgd from migrating into the nucleus (Troost, 2012).

Recent results obtained in mammalian cell culture experiments suggest that human Lgd1 and Lgd2 might also act as transcriptional repressors (Hadjighassem, 2009; Ou, 2003). This study found that Lgd requires location within the cytosol for its function. Hence, the current results are not easily compatible with a function as a transcription factor, as suggested for human Lgd1 and Lgd2, and it is believed that a gene regulatory function for Lgd inDrosophila is unlikely (Troost, 2012).

Previous work has established that loss of function of ESCRT-I-ESCRT-III complexes results in non-autonomous and autonomous cell proliferation and activation of the Notch pathway. In addition, the mutant cells lose their epithelial organisation and eventually die. Although loss of function of lgd results in activation of the Notch pathway and overproliferation, these effects are cell-autonomous, and the mutant cells do not lose their polarity and survive well. Thus, the phenotypes of the two groups overlap, but are not identical. Nevertheless, this study has found an intimate relationship between the ESCRT-III component Shrub and Lgd. Both proteins physically interact and this direct interaction is important in vivo, as indicated by the strong genetic interactions uncovered between the two genes. Importantly, it was observed that the time of death for a hypomorphic allelic combination of lgd, which normally results in pharate adults, is earlier than that of lgd null mutants if the activity of shrub is reduced by half. The earlier time of death suggests that the function of shrub is impaired upon loss of lgd function. Thus, it appears that the physical interaction with Lgd is required for the proper function of Shrub. Because the loss-of-function phenotype of shrub is more deleterious and includes more aspects than that of lgd, it is likely that lgd contributes to, but is not absolutely required for, the function of shrub. Either loss of lgd results in the loss of one distinct aspect of Shrub function or it reduces its activity beyond a threshold that is required for complete function. The finding that overexpression of Shrub can rescue the lgd phenotype supports the second possibility. Recent work suggests that Shrub forms long homopolymers on the cytosolic surface of the endosomal membrane. This polymerisation is required for the abscission of vesicles into the lumen of the maturing endosome (Saksena, 2009). In order to polymerise, Shrub has to be converted from a closed cytosolic into the open form (Babst, 2002). After intraluminal vesicle (ILV) formation, Shrub becomes converted into the closed form by Vps4, with consumption of ATP. Because the data suggest that Shrub and Lgd interact in the cytosol, it is possible that Lgd somehow helps to prepare Shrub for the next round of polymerisation on the endosomal membrane (Troost, 2012).

The presented genetic studies suggest an antagonistic relationship between Lgd and several components of the ESCRT complexes with respect to Notch activation. This implies that activation of Notch in lgd cells depends on the function of the ESCRT complexes and therefore indicates that it must occur in a different manner in lgd cells to that in ESCRT-mutant cells. The results suggest that loss of lgd function somehow affects the activity of Shrub, which in turn results in the activation of Notch. It is important to point out that the antagonism between lgd and ESCRT is observed only with respect to activation of Notch signalling. With respect to endosome morphology, they appear to act synergistically because a reduction of shrub function by half results in a dramatic enlargement of endosomes of lgd hypomorphic cells, which normally do not exhibit such a defect. The results therefore reveal a complex relationship between Lgd and the ESCRT function and further work is required to resolve this relationship in detail (Troost, 2012).

Because activation of Notch is not possible without release of the Notch intracellular domain (NICD) into the cytosol, it is assumed that a fraction or all of Notch must somehow remain at the limiting membrane of the endosome and is not incorporated into ILVs in lgd cells. There are three possibilities for how this might be achieved: no ILVs form; Notch might not be efficiently incorporated into the ILVs; or ILVs might back-fuse with the limiting membrane of the maturing endosome. Back-fusion has been documented to occur in vertebrate cells. The current results suggest that loss of lgd function results in a reduction in the activity of Shrub. Therefore, the possibility is favored that the loss of lgd function results in a less efficient incorporation of Notch into the ILVs due to the reduced activity of Shrub (Troost, 2012).

How is Notch that remains in the limiting membrane activated (see Model of Notch activation in lgd cells - Supplemental Figure S4? Activation of Notch in lgd cells is independent of the ligands, but dependent on the γ-secretase complex. The S3 cleavage of Notch mediated by γ-secretase requires previous shedding of its ectodomain. This is normally performed by the ligand-dependent S2 cleavage through Kuz. Thus, it is thought that ectodomain shedding must occur in an alternative, ligand-independent manner in lgd cells. A possibility is that the ectodomain that reaches into the endosomal lumen might simply change its conformation because of the increasing acidification in the lumen. This conformational change might allow Kuz to access its normally hidden cleavage site in Notch to cleave the ectodomain independently of the ligands. Alternatively, the ectodomain might be degraded by the peptidases that become activated in the acidic environment of the late endosome or lysosome. The resulting NEXT-like intermediate could be cleaved by γ-secretase, and the intracellular domain would be released into the cytosol. In the second scenario, activation of Notch could be independent of Kuz. Thus, it is important to determine whether Kuz is required for the ectopic activation of Notch in lgd cells. It is also important to determine whether Notch is activated in the maturing-late endosome or in the lysosome: One possibility to explain the puzzling antagonism observed between Lgd and several components of the ESCRT complexes with respect to Notch activation could be explained by the synergistic endosomal morphology phenotype: if activation of Notch occurs in the lysosome and the synergism between lgd and ESCRT mutants on endosome morphology prevents fusion of the late endosome with the lysosome (e.g., through loss of association with the HOPS tethering complex), Notch activation would be suppressed in double mutant cells (Troost, 2012).

In any case, the data so far available suggest that Lgd is a general component of the endosomal machinery and that the activation of Notch in lgd cells is probably not caused by a specific defect in Notch regulation. It occurs because of a general defect in endosomal trafficking and because of the extraordinary mechanism of Notch activation (Troost, 2012).

So far no function in endosomal trafficking has been described for the mammalian orthologues. However, the current data provide overwhelming evidence that Lgd is involved in endosomal trafficking in Drosophila. In addition, work by Tsang (2006) suggests that the functional relationship between Lgd and Shrub is conserved in humans. That study consists of a yeast-two-hybrid screen in which proteins were sought that interacted with the ESCRT components. Among the identified proteins was human Lgd2/CC2D1A, which interacted with all three Shrub orthologues, CHMP4A, CHMP4B and CHMP4C (Troost, 2012).

DEVELOPMENTAL BIOLOGY

Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis

The Notch signaling pathway plays a central role in animal growth and patterning, and its deregulation leads to many human diseases, including cancer. Mutations in the tumor suppressor lethal giant discs (lgd) induce strong Notch activation and hyperplastic overgrowth of Drosophila imaginal discs. However, the gene that encodes Lgd and its function in the Notch pathway have not yet been identified. This study reports that Lgd is a novel, conserved C2-domain protein that regulates Notch receptor trafficking. Notch accumulates on early endosomes in lgd mutant cells and signals in a ligand-independent manner. This phenotype is similar to that seen when cells lose endosomal-pathway components such as Erupted and Vps25. Interestingly, Notch activation in lgd mutant cells requires the early endosomal component Hrs, indicating that Hrs is epistatic to Lgd. These data suggest that Lgd affects Notch trafficking between the actions of Hrs and the late endosomal component Vps25. Taken together, these data identify Lgd as a novel tumor-suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling (Childress, 2006).

Lgd has been identified as a novel C2-domain protein, and the results indicate that it acts by regulating Notch trafficking. A model is proposed in which Lgd functions as a negative regulator of Notch through endosomal sorting of Notch downstream of Hrs function. Several lines of evidence support this model. The loss of Lgd resulted in the accumulation of Notch in early endosomes, and the results suggest that this triggered a signaling event that was distinct from normal activation of Notch signaling. Furthermore, the data indicate that Notch can be activated in a ligand-independent manner in lgd mutant cells, similarly to other mutations that affect Notch trafficking. Additionally, cells that lack both Hrs and Lgd did not display ectopically activated Notch signaling as measured by Cut expression. Interestingly, hrs lgd double-mutant cells at the wing margin were still able to express margin-specific genes. Therefore, Hrs is not required for normal (ligand-dependent) Notch signaling, but it is required for the ectopic activation of Cut expression found in lgd mutant cells (Childress, 2006).

lgd mutant cells display both similarities and differences compared with cells that are mutant for vps25, a known endosomal-trafficking component. Both mutations induce ectopic Notch signaling resulting in tissue overgrowth, and both mutations alter Notch trafficking. However, lgd mutant cells induce higher levels of Notch signaling than do vps25 mutant cells (Cut was not notably ectopically activated in vps25 mutant cells, do not induce apoptosis, and can survive into adulthood. Also unlike vps25 mutants, lgd mutant cells have no significant defects in cell polarity and do not accumulate increased levels of ubiquitylated proteins. It is thought that Vps25 is an endosomal component used to sort many different molecules, whereas Lgd might act specifically in the Notch pathway. A model is therefore propose where Lgd function is required to target full-length Notch for endosomal degradation or recycling. Removal of Lgd function might leave Notch in an optimal position or modification state for γ-secretase cleavage. The molecular mechanism by which Lgd affects Notch trafficking is currently not known, and no evidence was found of direct binding between Notch and Lgd by immunoprecipitation (Childress, 2006).

It is important to note that the subcellular location of the γ-secretase-complex cleavage of Notch (S3 cleavage) remains controversial. The traditional view is that the cleavage of Notch occurs at the plasma membrane. However, this view conflicts with the evidence that endocytosis is required for Notch signaling in Drosophila. When protein internalization is blocked by shibire mutations, Notch signaling is eliminated. A different view of the location of Notch S3 cleavage was recently developed when the γ-secretase enzyme Presenilin was shown to have a low optimal pH, suggesting that it could be active in the acidic endocytic compartments. It is possible that differentially processed Notch could be activated in separate cellular compartments. In accordance with the model proposed by Hori (2004), Notch activation in the ligand-dependent canonical pathway may occur at the plasma membrane or in endocytic vesicles, whereas Lgd-regulated activation of Notch may occur later, at Hrs-positive endosomes (Childress, 2006).

REFERENCES

Search PubMed for articles about Drosophila lethal (2) giant discs 1

Agrawal, N., et al. (1995). Epithelial hyperplasia of imaginal discs induced by mutations in Drosophila tumor suppressor genes: growth and pattern formation in genetic mosaics. Dev. Biol 169: 387-398. 7781886

Babst, M., (2002). Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3: 271-282. PubMed Citation: 12194857

Bryant, P. J. and Schubiger, G. (1971). Giant and duplicated imaginal discs in a new lethal mutant of Drosophila melanogaster. Dev. Biol. 24: 233-263. 4994924

Bryant, P. J. and Levinson, P. (1985). Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth. Dev. Biol. 107: 355-363. 3918894

Bryant, P. J., Watson, K. L., Justice, R. W., Woods, D. F. (1993). Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila. Dev. Suppl. 239-249. 8049479

Buratovich, M. A. and Bryant, P. J. (1995). Duplication of l(2)gd imaginal discs in Drosophila is mediated by ectopic expression of wg and dpp. Dev. Biol. 168: 452-463. 7729581

Childress, J. L., Acar, M., Tao, C. and Halder, G. (2006). Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis. Curr. Biol. 16(22): 2228-33. PubMed citation: 17088062

Gallagher, C. M. and Knoblich. J. A. (2006). The conserved c2 domain protein Lethal (2) giant discs regulates protein trafficking in Drosophila. Dev. Cell. 11(5): 641-53. Medline abstract: 17084357

Hadjighassem, M. R., (2009). Human Freud-2/CC2D1B: A novel repressor of postsynaptic serotonin-1A receptor expression. Biol. Pschychiatry 66: 214-222. PubMed Citation: 19423080

Hori, K., et al. (2004). Drosophila deltex mediates suppressor of Hairless-independent and late-endosomal activation of Notch signaling, Development 131: 5527-5537. PubMed citation: 15496440

Jaekel, R. and Klein, T. (2006). The Drosophila Notch inhibitor and tumor suppressor gene lethal (2) giant discs encodes a conserved regulator of endosomal trafficking, Dev. Cell 11: 655-669. Medline abstract: 17084358

Klein, T., et al. (2003). The tumor suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development. Dev. Bio. 255: 313-333. 12648493

Nakamura, A., Naito, M., Tsuruo, T. and Fujita, N. (2008). Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt pathway in epiderma, growth factor signaling. Mol. Cell. Biol. 28: 5996-6009. PubMed Citation: 18662999

Ou, X.-M., (2003). Freud-1: a neuronal calcium-regulated repressor of the 5-HT1A receptor gene. J. Neurosci. 23: 7415-7425. PubMed Citation: 12917378

Saksena, S., (2009). Functional reconstitution of ESCRT-III assembly and disassembly. Cell 136: 97-109. PubMed Citation: 19135892

Troost, T., Jaeckel, S., Ohlenhard, N. and Klein, T. (2012). The tumour suppressor Lethal (2) giant discs is required for the function of the ESCRT-III component Shrub/CHMP4. J. Cell Sci. 125: 763-776. PubMed Citation: 22389409

Tsang, H. T. H., (2006). A systematic analysis of human CHMP protein interactions: additional MIT domain-containing proteins bind to multiple components of the human ESCRT III complex. Genomics 88: 333-346. PubMed Citation: 16730941

Watson, K. L., Justice, R. W. Bryant, P. J. (1994). Drosophila in cancer research: the first fifty tumor suppressor genes. J. Cell Sci. Suppl. 18: 19-33. 7883789

Zhao, M., Li, X.-D. and Chen, Z. (2010). CC2D1A, a DM14 and C2 domain protein, activates NF-kappaB through the canonical pathway. J. Biol. Chem. 285: 24372-24380. PubMed Citation: 20529849

date revised: 20 April 2012

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