Notch

Table of contents

Warthog and trafficking of Notch

The warthog (wrt) gene, recovered as a modifier for Notch signaling, was found to encode the Drosophila homolog of rab6, Drab6. Translation of the sequence shows this transcript to have 89% identity to human rab6, 72% to the yeast rhy1 protein, and it has subsequently been cloned as Drab6. Additionally, two putative C. elegans proteins were also found to be homologous (84% and 75%). Vertebrate and yeast homologs of this protein have been shown to regulate Golgi network to TGN trafficking. RAB proteins comprise the largest class of the ras-like GTPase superfamily. Genetic and biochemical studies have shown their involvement in various steps of endocytosis, exocytosis, and transcytosis. Particular rabs are localized to distinct intracellular compartments, and mutant forms of these proteins impair the trafficking of vesicles from one intracellular compartment to another. Rabs have largely been implicated in the fusion or docking of vesicles to acceptor compartments, although some reports have noted rab function in the budding of vesicles from the donor compartment. As GTPases, they act as cyclical switches, alternating between an active GTP-bound state and an inactive GDP-bound state. RabGDI extracts the GDP-bound form from membranes of the acceptor compartment and maintains the rab in this inactive state in the cytosol. Guanine nucleotide exchange factors then promote the exchange of GDP to GTP, converting the rab to an active state, which is presumed to bind to membranes of the donor compartment. Once bound to GTP, hydrolysis of the nucleotide occurs constitutively, providing a timer for the length of rab activation. To slow this constitutive hydrolysis, effector proteins bind to the GTP-bound rab, providing extended time for the complex to target the donor vesicle to the appropriate acceptor compartment. Rab then proceeds through the cycle again. One of these proteins, rab6, has been shown to regulate trafficking from the Golgi to the TGN. In mammalian tissue culture cells, mutations in rab6 lead to morphological changes in the Golgi and a delay in the presentation of proteins to the cell surface. In yeast, null mutations of the rab6 homologues, Ypt6 and rhy1, also show defects in post-ER processing of various proteins. Sequences homologous to rab 6 have also been found in Drosophila, but only structural data have been reported (Purcell, 1999).

To study the function of Drab6 protein in the development of a multicellular organism, three different warthog mutants of Drosophila were analyzed. The first was an R62C point mutation, the second a genomic null, and the third was an engineered GTP-bound form. Contrary to yeast, the Drosophila homologue of rab6 is an essential gene. However, it has limited effects on development beyond the larval stage. Only the mechanosensory bristles on the head, notum, and scutellum are affected by warthog mutations. warthog, enhances the Notch eye phenotype although it does not visibly affect eye development outside of this interaction. It was also noted to have a recessive bristle phenotype independent of its interaction with the aberrant Notch signaling in the eye. In wild-type flies, bristles are part of mechanosensory organs and develop shortly after puparium formation as the trichogen, or shaft cell, sends a cytoplasmic extension from the epidermis into the overlying cuticulin. At the center of this extension is a longitudinal core of microtubules. Around the circumference and positioned near the plasma membrane are regularly spaced bundles of actin filaments. These filaments are hexagonally packed and run parallel to the microtubule core. As development proceeds, continued growth of the shaft occurs in two directions. One is elongation at the distal tip, whereas the second is throughout the width of the shaft as regions of the cytoplasm protrude from between the actin fibers to produce the characteristic ridges seen in a cross-section of the bristle. Five warthog alleles recovered in the screen had considerably shortened bristles as homozygotes or transheterozygotes. This defect was present only for macrochaete of the ocelli, notum, and scutellum, whereas the bristles of the eye, wing, and leg appeared normal. Scanning electron micrographs of warthog bristles show, in addition to the aberrant length, that the morphology of wrt bristles are altered. The wrt bristles do not have finely tapered ends nor do they show the regularly spaced ridges from the membranous protrusions. Instead, the tips are mangled and the surface is either smooth or has very mild and disorganized ruffling. Unexpectedly, a subset of the Drab6 cDNA transformant lines rescue the lethality to produce flies with bristle defects more subtle than the original wrt alleles. Since these same transformant lines are capable of rescuing the bristle defect of the screen alleles with the R62C point mutation, this indicates that bristle development is more sensitive to the quantity or timing of Drab6 expression or function than is lethality (Purcella, 199).

To establish the time period of Drab6 expression critical for viability, homozygotic mutants were monitored at different stages of development. Eggs with these homozygotic genotypes would proceed through embryogenesis to the larval stage, but would not continue to develop into pupae. Therefore, the more severe alleles of warthog are larval lethal. To determine if the lack of embryonic lethality is due to a maternal contribution of wrt, the FLP-FRT system was used to generate females with wrt-/wrt- germlines. All progeny germline mutants develop past embryogenesis, showing that a maternal contribution is not responsible for survival of wrtP2352 through embryonic development. To study the effect of the more severe disruptions in Drab6 function during later stages of development, mosaic clones were induced using the FLP-FRT system. As with the original screen mutants, no defects of eye, wing, or leg development were noted. The defects on macrochaete are more severe and more variable than that seen with the homozygous mutants. Mutation clones also affect the smaller bristles, called microchaete, on the head and thorax. The defects seen in these smaller shafts mirror those seen in the macrochaete of mutant flies; distal tip growth is stunted and the circumferential ridges produced from cytoplasmic protrusions are nearly absent. Surprisingly, the clonal analysis also shows that the phenotypic effect is nonautonomous. Whereas portions of the mosaic clones contained mutant bristles, phenotypically wild-type bristles are also present in patches of mutant tissue, indicating that Wrt protein is not required within the cell producing the shaft of the bristle (Purcell, 1999).

The function of rab proteins in mammalian systems has been elicited by studying the effects of overexpression of wild-type and mutant forms of these proteins. The best characterized forms are those modeled after ras mutations and are known to alter the ability of rabs to cycle between the GDP- and the GTP-bound states. The state of continued GTP binding has been produced by altering the Q of the second conserved GTP-binding domain to a leucine (Q72L in mammalian rab6). This abolishes intrinsic GTPase activity and decreases GAP-stimulated hydrolysis as well. To study the effects of this mutation in the whole organism, a similar mutation was induced in warthog (Drab6-Q71L) . cDNAs of wild-type Drab6, the R62C mutation, and the Q72L mutation were placed under the control of the heat shock promoter to drive expression at different stages of development. Whereas overexpression of the wild-type form and the R62C mutation produces no visible phenotype in the background of wrt+/wrt+, the Q71L mutation alters the direction of bristle growth at any point along the bristle shaft. Overexpression of this GTP-bound mutant produces smoothly curving bristles or bristles with sharp changes in the orientation of growth, followed by continued growth in two opposite directions. Normal morphology appeared distal to the alteration, presumably because of the return of normal Drab6 function after the pulsed overexpression of Drab6 Q71L has passed. Aberrations in the circumferential ridges are also seen, indicating that the membranous protrusions from between the actin bundles are also disrupted. Interestingly, basal expression of the Q71L mutant cDNA without heat shock, is capable of rescuing the bristle phenotype of the R62C alleles, indicating that even small amounts of the Q71L form of Drab6 can rescue the phenotypic effects of the loss-of-function R62C mutation (Purcell, 1999).

What is the relationship between Notch and rab6? The Notch signal transduction pathway is used in many species to modulate the ability of precursor cells to respond to developmental cues. This signal is activated by the binding of the ligand Delta to its receptor Notch to activate downstream proteins. However, the selection for which cells undergo this activation is influenced by the amount of the Notch receptor at the cell surface; Notch is one of only a handful of genes to produce a visible phenotype with either an extra copy of the gene or when missing one copy. Mammalian and yeast forms of Rab6 are involved in Golgi trafficking. In mammalian tissue culture cells, a mutation (Q72L) in rab6 that impairs GTP hydrolysis, leads to a morphological disruption of Golgi structures and a decrease of marker proteins in the late Golgi network. Conversely, a mutation resulting in a GDP-bound form of rab6 (T27N) shows more prominent Golgi structures and an accumulation of marker proteins in the late Golgi network. Both of these rab6 mutations lead to a kinetic inhibition of proteins presented to the cell surface; in pulse-chase experiments, cells that overexpress wild-type or either mutant form of rab6 (Q72L or T27N) eventually secrete the same quantity of extracellular proteins as controls, but the rate of release is markedly decreased. From these tissue culture experiments, mutations in Drab6 would be expected to delay the surface presentation of the Notch receptor. Given that the amount of Notch present on the cell surface is critical for the adoption of different cellular identities, such a delay in transportation of the Notch receptor to the plasma membrane would alter Notch signaling. The phenotypic interaction of the wrt screen alleles was consistent with a decrease in the amount of N available for signaling on the cell surface (Purcell, 1999).

Another explanation for the modification of Notch signaling by wrt is suggested by the observation that rab6 specifically functions at the critical junction of sorting between the amyloidogenic and nonamyloidogenic pathways for the ß-amyloid precursor protein. This role of rab6 in the proper sorting of molecules into different compartments within or from the TGN may account for the interaction between Notch and warthog. Notch undergoes proteolytic cleavage by a furin-like convertase within the TGN to produce a heterodimeric receptor at the cell surface. If rab6 determines which Golgi and post-Golgi enzymes transported proteins encounter, then alterations in warthog function could potentially lead to a missorting of Notch into a transport pathway where the receptor is not cleaved properly (Purcella, 1999 and references).

A rab6-interacting protein, rabkinesin-6, has been shown to bind microtubules and has ATPase activity similar to the plus end motors to which it is homologous; rab6-GTP was postulated to regulate the association and dissociation of rabkinesin-6 to microtubules. However, for warthog, no additive or synergistic interactions were seen when tested with many mutations known to affect bristle structure. More importantly, the nonautonomous phenotype seen in the severe warthog mutants implies the Drosophila homolog of rab6 modifies the surface presentation of other proteins. Nonautonomous phenotypes are typically seen with secreted or transmembrane proteins that signal to neighboring cells. This effect is consistent with results from yeast and mammalian tissue culture experiments that establish the role of rab6 in the proper secretion of other proteins (Purcell, 1999 and references).

Mutations that have previously been studied for rab6 are those engineered based on the GTP- and GDP-bound forms of ras-like molecules. From the screen for modifiers of Notch, a novel mutation was obtained that results in the conversion of an arginine to a cysteine at amino acid 62 (R62C). From biochemical and crystallographic data of other GTPases, the R62C mutation is expected to lie next to a defined GTP-binding domain (DX2G) where the invariant aspartate binds the catalytic Mg2+ through an intervening water molecule. However, in vitro studies reveal that R62C mutant protein is capable of binding and hydrolyzing GTP, suggesting that this point mutation affects Drab6 function through another mechanism, perhaps by altering its interaction with regulatory proteins. This hypomorphic mutation altered rab6 functions differently from the Q71L mutation, which resides next to the same GTP-binding domain. Overexpression Q71L Drab6 disrupts the orientation of bristle growth, whereas overexpression of R62C Drab6 in a wild-type background elicits no effects. Q71L Drab6 is also capable of rescuing the bristle defect of the R62C mutation. Therefore, studying the R62C mutation may reveal new information of Drab6 function (Purcell, 1999 and references).

Perhaps the most interesting aspect of this phenotypic analysis is the limited requirement of a rab6 homolog throughout development. While an essential gene, Drab6 mutations do not affect the development of the eye, wing, and leg, nor the bristle structures within these tissues. This paucity of developmental phenotypes mirrors yeast studies that show null mutations in Ypt6 or rhy1 are not lethal, implying transport redundancy exists as proteins travel to the cell membrane. This redundancy could be the result of more than one rab6 protein, which is supported by the discovery of two putative rab6 homologs in C. elegans. Alternatively, it may be a functional redundancy where parallel but independent trafficking pathways through the Golgi/TGN can compensate for alterations in one another. Recent studies in mammalian systems support the existence of these independent trafficking pathways. The secreted protein ß-APP is processed in a different compartment if rab6 is mutated and a study of cell surface antigen presentation has also shown alterations in rab6 affected one transport pathway but not another (Purcella, 1999 and references).

The bristle phenotype of the warthog mutants, however, reveals there is a limitation to which an organism can compensate for mutations in Drab6, even if redundant or independent pathways exist for transport through the Golgi. This limitation may also be seen only after prolonged Drab6 dysfunction. Overexpression of Drab6 Q71L in a subset of cells within the eye leads to degeneration after two weeks. Having phenotypes associated with this limitation in redundancy through the Golgi/TGN will provide a novel means to dissect Golgi transport mechanisms. Identifying proteins that modify the wrt bristle phenotype will allow an ordered dissection of the protein cascade required for rab6 function. These mutants may also lead to a better understanding of how the cell regulates trafficking of signaling receptors such as Notch. Capitalizing on the interaction between wrt and Notch in sensitized backgrounds, genetic screens may help identify the proteins required for surface presentation of a functional Notch receptor (Purcell, 1999 and references).

Lethal giant discs and notch trafficking

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 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).

Notch signaling at the wing margin is SNARE dependent

The wing of Drosophila has long been used as a model system to characterize intermolecular interactions important in development. Implicit in an understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. The Drosophila wing was used to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and a mutant form of NSF was constructed whose expression was directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, this phenotype was used to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. It is concluded that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways can act as a sensitive reporter of partial membrane-trafficking disruption (Stewart, 2001).

The Syntaxin, VAMP, and SNAP-25 families of proteins are proposed to target and fuse transport vesicles with specific membrane compartments. The SNARE complex is a parallel four-helix bundle with one helix contributed by each of Syntaxin and VAMP and two contributed by SNAP-25 (Sutton, 1998). The formation of a trans-membrane complex, with VAMP on the transport vesicle and Syntaxin and SNAP-25 on the target membrane, is thought to lead to the fusion of the two membranes, resulting in a cis-membrane complex. It follows that the cis-residing protein complexes need to be broken apart to make those proteins available for further trans-complex formation. This complex breakdown occurs under the action of N-ethylmaleimide-sensitive fusion protein (NSF), an ATPase. NSF contains two nucleotide binding domains and demonstrable ATPase activity. Structural analyses have shown that NSF forms a hexamer in vivo. NSF is a homolog of the yeast gene SEC18 and analysis of SEC18 function also reveals its requirement for intracellular membrane transport. NSF-dependent ATP hydrolysis is required to disassemble SNARE complexes, although it is not required for the fusion step. Thus the role of NSF in vesicular transport appears to be primarily one of priming vesicles for fusion and dissociation of SNARE complexes to permit their recycling (Stewart, 2001).

In Drosophila there are two homologs of NSF: dNSF1 and dNSF2 (NEM-sensitive fusion protein 2). dNSF1 is the gene product of comatose and is primarily found in neurons, whereas dNSF2, in addition to being neuronally expressed, is broadly expressed within imaginal discs, salivary glands, and the ring gland (Boulianne, 1995). Thus, dNSF2 is the most likely isoform to contribute to intracellular trafficking in nonneuronal tissue. Despite their proposed role in most intracellular trafficking events, in vivo studies of SNARE proteins have concentrated on two main systems: the budding yeast and calcium-triggered exocytosis in neurons. Relatively little attention has been given to other in vivo contexts in which the SNARE proteins are likely to have important roles. For example, in signaling pathways it is self-evident that transmembrane receptors and ligands need to be delivered to the plasma membrane, although few studies have been devoted to specifically studying the role of SNARE proteins in this process and their potential influence on the strength of intracellular signaling (Stewart, 2001).

The observation that dominant negative NSF protein, NSF^E/Q, causes loss of wing margin implies that SNARE-dependent transport is important for wing margin formation. To test this further mutant alleles of synaptobrevin and syntaxin, two well-characterized SNARE proteins, were used to determine whether they would enhance the wing phenotype. Indeed, all trans-heterozygous combinations of NSF^E/QC96 (mutant NSF expression driven in the wing margin) with synaptobrevin or syntaxin loss-of-function alleles enhance the wing margin phenotype, thus providing further evidence of the involvement of SNARE proteins in wing margin development (Stewart, 2001).

The wing phenotype observed is similar to that observed with mutant alleles of Notch and Wingless signaling pathway genes. To determine whether components of these pathways could be contributing to the NSF^E/QC96 wing phenotype the protein pattern of Wingless in third-instar imaginal wing discs was first examined and a striking effect on the distribution of Wingless was observed. In control discs Wingless appears as a three- to four-cell-wide stripe across the wing disc, whereas in discs expressing the mutant dNSF2 Wingless appears very narrow and patchy. Wg expression was then examined using a Wg-lacZ reporter construct and an incomplete pattern of Wingless expression was found, as was observed for the Wingless protein (Stewart, 2001).

Because Wg is a secreted protein Wg was examined under higher magnification using confocal microscopy to determine directly whether Wg secretion was impaired. In control discs there is punctate Wg staining, indicative of Wg secretion, in the tissue surrounding the narrow stripe of wing margin cells. In the regions of the mutant discs that are immunoreactive for Wg, punctate staining is seen surrounding the positive cells. However, the Wg signal is much stronger in those cells and confocal sectioning of the cells has revealed the accumulation of Wg at the apical region of the wing margin cells. These data indicate that mutant NSF^E/Q impairs, but does not eliminate, Wingless secretion. Because Wingless expression is impaired and its activation is under the control of Notch signaling, the distribution patterns of other proteins involved in the Notch pathway were examined. Notch protein distribution was examined directly using a monoclonal antibody that recognizes the extracellular domain of Notch. At low magnification there is no major difference between mutant and control samples, with the antibody labeling the cell membranes in the wing pouch. However, at higher magnification, in addition to the membrane staining, immunoreactive puncta were also observed within the cells of the mutant wing disc that were not readily observed in the control discs. These puncta likely represent improperly sorted Notch proteins (Stewart, 2001).

The distribution of Cut, Delta, and Achaete, coded for by genes that are downstream of Notch activation in the wing margin signaling pathway, was examined -- all of these markers were disrupted in NSF^E/QC96 larval wing discs. Cut is normally found in a pattern that overlaps with Wg along the presumptive wing margin, whereas in the mutant discs it appears in a broken pattern similar to that of Wg. Delta is normally expressed in two parallel bands along the D/V boundary and this pattern is thought to be the result of the downregulation of Delta in boundary cells by Cut and the upregulation of Delta in flanking cells by Wingless. In NSF^E/QC96 wing discs the expression of Delta is reduced and the two parallel bands appear to be collapsed into a single band along the boundary. Achaete is normally expressed in two broad bands parallel to the D/V boundary in the anterior compartment of the wing disc defining a proneural cluster. In the NSF^E/QC96 discs this pattern is severely disrupted: the number of Achaete-expressing cells is reduced and there is complete absence of Achaete in some areas (Stewart, 2001).

A similar pattern of disruption was found when lacZ reporter constructs were used to examine the expression of neuralized and vestigial, two other genes in the Notch pathway. neu^A101-lacZ is normally detected in sensory organ precursors (SOPs) located in two rows of single cells parallel to the D/V boundary in the anterior compartment of late third-instar wing discs. In the mutant discs this pattern is disrupted and lacking in some areas along the wing margin, while SOPs elsewhere in the disc are unaffected. Similarly, vg^BE-lacZ expression is disrupted. In wild-type discs vg^BE-lacZ expression is seen in the D/V and anterior/posterior (A/P) boundaries, whereas in the mutant discs the expression in the D/V boundary is disrupted (Stewart, 2001).

Interestingly, expression in the A/P boundary remains, although the C96-Gal4 expression pattern overlaps this region. Taken together these results demonstrate that NSF^E/Q affects the distribution and expression of several downstream components of the Notch signaling pathway. To confirm the effect of NSF^E/Q on Notch signaling loss-of-function alleles of several genes in the Notch and Wingless pathways were examined for their ability to enhance the adult wing phenotype caused by NSF^E/Q expression. In that Notch signaling is known to be highly sensitive to haploinsufficiency of interacting gene products, it was reasoned that these loss-of-function alleles should show genetic interaction. Two alleles of Notch and one each of Delta, Serrate, wingless, and fringe were examined and it was found that they all enhanced the wing phenotype in transheterozygous combination with NSF^E/QC96. The severity of the phenotype produced by each allele was similar, although Df(1)N8, a null allele of Notch, did produce a more severe phenotype than did N^nd-3, a hypomorphic allele. With the exception of Df(1)N8, none of these mutants produces a wing-nicking phenotype when examined alone as heterozygotes. Thus, the enhancement of the adult wing phenotype by mutants in the Notch pathway supports the conclusion that NSF^E/Q expression causes a defect in wing margin signaling pathways (Stewart, 2001).

Finally, the ability of UAS constructs of Notch, Delta, and Serrate to rescue the wing phenotype generated by NSF^E/QC96 were tested. Complete rescue could be obtained with both Notch and Delta constructs. Serrate generally appears to rescue less well than do the other constructs because minor nicks in the distal wing persist. Furthermore, no rescue effect was seen when crosses were made to UAS-lacZ lines, indicating that competition for Gal4 protein is not responsible for rescue of the phenotype. The observation that UAS-Notch and UAS-Delta can completely rescue the NSF^E/Q wing phenotype further indicates that the mutation affects intracellular transport and does not create a cell-lethal phenotype because cell lethality should not be rescued by Notch or Delta (Stewart, 2001).

Having established that NSF^E/Q disrupts signaling at the wing margin in a SNARE-dependent manner, and that enhancement of the phenotype can be attributed to haploinsufficiency of known genes, it was asked whether the wings of the NSF^E/QC96 flies could be used as a sensitized background to find novel genes involved in wing margin formation. To this end a small-scale screen was conducted for enhancers and suppressors of the phenotype. In the first set of experiments specific alleles of two genes were tested: big brain and porcupine. These have been shown to be important in Notch and Wingless signaling in other developmental contexts but have not previously been known to be important for wing margin development. In the NSF^E/QC96 background it was found that both mutant alleles of these genes enhance the NSF^E/QC96 wing margin phenotype. This result is the first report of the involvement of these two genes in wing margin development and suggests that NSF^E/QC96 wings provide an ideal sensitized background for conducting forward genetic screens to identify novel genes involved in wing margin development (Stewart, 2001).

In view of current membrane-trafficking models, it is expected that expressing NSF^E/Q impairs the ability of NSF to dissociate cis-SNARE complexes, making fewer SNARE proteins available for functional transmembrane complex formation and thus reducing intracellular transport. These results provide solid evidence that SNARE proteins are important in wing margin formation. This implies that the mutant NSF must suppress but not block all membrane traffic. The disruption of molecular markers, such as Wg, Delta, Achaete, Cut, Vestigial, and Neuralized, indicates that the NSF^E/Q wing phenotype observed is the result of impaired signaling at the developing wing margin. This is consistent with data presented in other studies that manipulated the signaling pathway directly. For example, reduction of Notch activity with N^ts alleles can lead to reduced and patchy Wingless expression. Wingless and Cut expression is also reduced and patchy in Notch mutant wing discs. Stripes of Delta and Serrate that normally flank the D/V boundary collapse into a single stripe along the margin in N^ts alleles exposed to restrictive temperature. In NSF^E/QC96 wing discs, changes in Wingless, Cut, and Delta patterns were observed that are similar to those that occur when Notch activity is directly manipulated; therefore, it seems that NSF^E/Q expression phenocopies genetic mutants of Notch (Stewart, 2001).

Because the Notch and Wingless signaling pathways are so intertwined in controlling wing margin development it is difficult to determine whether the dNSF2 mutants cause a primary defect in one or the other of these proteins, although it seems likely that there are parallel effects on both. The experiments show not only a direct impairment of Wingless trafficking but also that Wg-lacZ expression is disrupted. The latter suggests that an upstream activator of Wingless expression is impaired (although this could be Wingless itself). It was found that Notch subcellular localization is disrupted and that a Wg-independent target of Notch signaling, the vestigial boundary enhancer, is also disrupted. Because this vestigial enhancer element is thought to be under the sole control of Notch this supports the idea that NSF^E/Q has a direct effect on Notch signaling. Thus vg^BE-lacZ expression data strongly suggest direct effects on both Wg and Notch. Moreover, because these molecules are at the top of the hierarchy controlling signaling at the wing margin this provides the likely explanation for the disruption of downstream targets of these genes (Stewart, 2001).

The molecular and genetic interactions that regulate developmentally important signaling pathways are important for defining the final outcome of the signaling cascade. For example, previous studies have identified several molecules, including Fringe, Big Brain, and Numb, that are proposed to influence Notch signals. Because the SNARE proteins interact with many protein partners, some of which are proposed to regulate their availability (e.g., Syntaxinís interaction with rop/nsec-1), these data indicate that regulation of SNARE-dependent transport steps may represent an additional mechanism by which signal transduction pathways can be modulated during development (Stewart, 2001).

Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth caused by increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired

The reproducible pattern of organismal growth during metazoan development is the product of genetically controlled signaling pathways. Patterned activation of these pathways shapes developing organs and dictates overall organismal shape and size. Patches of tissue that are mutant for the Drosophila Tsg101 ortholog, erupted, cause dramatic overexpression of adjacent wild-type tissue. Tsg101 proteins function in endosomal sorting and are required to incorporate late endosomes into multivesicular bodies. Drosophila cells with impaired Tsg101 function show accumulation of the Notch receptor in intracellular compartments marked by the endosomal protein Hrs. This causes increased Notch-mediated signaling and ectopic expression of the Notch target gene unpaired (upd), which encodes the secreted ligand of the JAK-STAT pathway. Activation of JAK-STAT signaling in surrounding wild-type cells correlates with their overgrowth. These findings define a pathway by which changes in endocytic trafficking can regulate tissue growth in a non-cell-autonomous manner (Moberg, 2005). Tsg101 possesses the ability to bind monoubiquitinated substrates. These substrates are predicted to be the ubiquitinated cytoplasmic tails of membrane bound proteins, and this interaction is predicted to deliver cargos to the lysosome via multivesicular bodies (Moberg, 2005 and reference therein).

The Notch receptor has two properties that implicate it in a pathway by which ept mutations non-cell-autonomously promote tissue growth. (1) The restricted activation of Notch in cells along the dorsoventral (D/V) boundary of the eye imaginal disc is required for growth of the entire eye. (2) Ub-dependent endocytosis plays an important role in regulating Notch activity in vivo. In mammalian cells, ubiquitination and endocytosis contributes to Notch1 activation, and, in Drosophila, there is evidence to suggest that the ubiquitin ligase Deltex may be required for endocytosis-dependent Notch activation. Further, alleles of the endosomal sorting gene Hrs, the homolog of yeast Vps27, affect Notch localization in imaginal disc cells, indicating that Notch is a physiological target of the MVB pathway (Moberg, 2005).

In light of these observations, ept mosaic eye discs were stained with an antibody specific to the Notch cytoplasmic domain (anti-N^cyto). Notch protein is detected in wild-type eye discs most prominently in a stripe of cells within the morphogenetic furrow (MF) and is concentrated at the apical cell surface. In contrast, ept cells contain elevated levels of Notch. This increase occurs in ept clones throughout the eye disc, but it is most apparent in clones that lie within or posterior to the MF. Moreover, the Notch in ept cells accumulates in nonnuclear, intracellular puncta that also stain positive for Ub, and for the endosomal protein Hrs. Together, these data indicate that ept mutations block the routing of ubiquitinated cell surface proteins, among them Notch, in an Hrs-positive endosomal compartment (Moberg, 2005).

Notch is normally processed in cells by a series of cleavage events required for receptor maturation and presentation at the cell surface, and for ligand-stimulated activation of the Notch pathway. Because ubiquitination and endocytosis have been shown to affect Notch cleavage, attempts were made to determine if ept mutations also affect Notch processing. Eye-antennal discs composed of ept mutant cells [ept/M(3)] or FRT80B control cells (FRT80B/M(3)) were generated by the eyFLP/Minute technique. Immunoblot of tissue extracts with the anti-N^cyto antibody confirms that Notch levels are increased considerably in eye-antennal discs composed of ept mutant cells, and shows that ept mutant cells are enriched in a ~120 kDa form of Notch. The molecular identity of this fragment has not been determined, but its size appears similar to certain processed forms of Notch. Indeed, while no one form of Notch predominates in wild-type cells, this species appears to be the most abundant Notch species in ept cells (Moberg, 2005).

To examine Notch activation, clones of ept mutant cells were generated in the presence of the Notch-inducible transgene E(spl)mβ-CD2, a Suppressor of Hairless (Su(H))-dependent transcriptional reporter that has been used to detect equatorial Notch activation in the developing eye. Posterior to the MF, CD2 expression is detected in the interommatidial cells, and outlines a single cell from each photoreceptor cluster in a mirror-image pattern along the equator. Thus, in addition to equatorial activation, the reporter detects Notch activation in postmitotic interommatidial cells, and in the R3-R4 cell fate choice. In ept mutant clones, reporter activity is strongly elevated. The degree of activation exceeds that observed in wild-type eye discs, and it does not appear to depend upon the location of ept cells within the disc, occurring on either side of the MF and in the antennal disc. Some ept cells within a single optical section appear not to activate the Notch reporter. However, in most of these cases, CD2, which localizes to cell membranes, can be detected in a focal plane slightly offset from that of the nuclear green fluorescent protein (GFP). Thus, these data show that defects in Notch regulation in ept cells are accompanied by ectopic and excessive activation of the Notch pathway (Moberg, 2005).

The requirement for Notch in eye disc growth has been linked to its ability to induce expression of the eyegone (eyg) gene at the D/V boundary of the eye disc. eyg encodes a Pax6-like transcription factor (Eyg) required for disc growth, and, like Notch, ectopic expression of eyg is able to induce growth nonautonomously. Consistent with its effect on Notch, it was found that ept mutant cells express elevated levels of Eyg compared to surrounding wild-type cells. Thus, Eyg may function downstream of Notch within ept cells to promote the growth of surrounding cells in a manner similar to its normal growth-promoting role at the D/V boundary (Moberg, 2005).

Recent work suggests that the unpaired (upd) gene may be an important growth regulatory target of Notch. upd encodes the secreted ligand (Upd) of the Domeless (Dome) receptor, which signals through the JAK-STAT pathway. JAK-STAT signaling is implicated in many processes during Drosophila development, including the control of cell proliferation, cell motility, stem cell renewal, and planar cell polarity. upd is required for normal growth of the eye, and ectopic expression of upd in the larval eye nonautonomously promotes cell proliferation and produces enlarged and misshapen eyes similar to those observed in ept mosaics. Significantly, Notch is both necessary and sufficient to activate upd transcription along the posterior margin of the eye disc (Moberg, 2005).

When ept mosaic eye discs were stained with an anti-Upd antiserum, a dramatic increase was observed in the level of Upd protein in ept mutant cells compared to adjacent wild type cells. Consistent with a transcriptional link between Notch and upd, Upd protein accumulation appears coincident with expression of the Notch reporter, and ept mosaic eye-antennal discs contain clones of cells expressing very high levels of upd mRNA. Together, these observations suggest that Notch, perhaps acting via Eyg, promotes ectopic upd expression in ept mutant cells (Moberg, 2005).

Clonal overexpression of upd induces localized tissue outgrowths and deregulates the division of surrounding cells. This mitogenic activity is linked to induction of cyclin D, and to accelerated progression through the G1 phase of the cell cycle. ept mutant clones can produce phenotypes quite similar to clonal overexpression of upd. In one example of an ept clone, lower half of the disc appeared morphologically normal, while the other half, despite being composed largely of wild-type cells, was misshapen and enlarged. This localized effect correlated with proximity to a large ept mutant clone expressing Upd. Similar hyperplastic growth was associated with clones of upd-expressing cells in the antennal disc. The patterns of BrdU incorporation in ept mosaic eye discs are disorganized, and the number of BrdU-labeled nuclei increases in proximity to Upd-expressing ept mutant cells in the eye and antenna. This aberrant cell proliferation occurs in GFP-positive wild-type cells. Hence, the growth-promoting activity of ept mutations is likely mediated by a diffusible extracellular signal like Upd (Moberg, 2005).

Receipt of the Upd signal via Domeless initiates a signaling cascade that activates a transcription factor encoded by the stat92E gene. stat92E encodes the Drosophila ortholog of the mammalian signal transducers and activators of transcription (STAT) family of transcriptional regulators, which function in diverse processes such as immunity and oncogenesis, and is the only member of this gene family in Drosophila. Heterozygosity for a stat92E loss-of-function allele (stat92E⁰⁶³⁴⁶) strongly suppresses the nonautonomous eye overgrowth associated with mosaicism for ept mutations, such that ept-mosaic;stat92E⁰⁶³⁴⁶/+ eyes are comparable in size to control FRT80B mosaic eyes. Thus, nonautonomous overgrowth elicited by ept mutations is sensitive to the genetic dosage of the Upd-responsive transcription factor stat92E. In light of the effect on Upd, these data strongly indicate that the growth-promoting activity of ept mutant cells requires Upd-dependent activation of the JAK-STAT pathway in adjacent tissue (Moberg, 2005).

ept mutant clones in mosaic eye discs are small and survive poorly into adulthood. It is possible that this is the result of cell competition, a process by which slow-growing cells in the vicinity of wild-type cells are eliminated. If so, then the poor survival of ept cells might be rescued by eliminating competing cells. Therefore the growth characteristics were examined of ept/M(3) discs, which are composed almost entirely of cells lacking Tsg101 function. ept/M(3) animals reach the larval 'wandering' stage 4 days later than control larvae, and, when they do, they are enlarged. A small fraction of these animals pupate and die before becoming pharate adults. The remainder die as giant larvae containing high levels of Upd (Moberg, 2005).

Allowing ept mutant cells to grow in epithelia lacking wild-type cells also uncovers a context-dependent cell-autonomous overgrowth phenotype. Rather than surviving poorly as they do in mosaic discs, ept/M(3) eye discs overgrow into large masses that lack normal disc morphology. These masses are composed of folded and convoluted sheets of cells fused together, and they often include a distended sac-like structure. The overgrowth phenotypes of ept/M(3) animals and discs do not reflect an increased rate of growth: control L3 larvae are the same size as ept/M(3) larvae of the same temporal age, and the ept/M(3) eye discs, while mispatterned, are not obviously increased in size. Thus, the ept/M(3) masses are the result of an extended larval phase, and a failure of the disc to stop growing when it reaches the appropriate size. Thus, cells lacking Tsg101 may be unable to respond to signals that normally sense and restrict organ size (Moberg, 2005).

The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking

Cell-cell signaling coordinates proliferation of metazoan tissues during development, and its alteration can induce malignant transformation. Endocytosis regulates signaling by controlling the levels and activity of transmembrane receptors, both prior to and following ligand engagement. Vps25 , a component of the endosomal sorting complex required for transport (ESCRT) machinery that regulates endocytic sorting of signaling receptors, has been identified as an unconventional type of Drosophila tumor suppressor. vps25 mutant cells in the eye disc undergo autonomous neoplastic-like transformation, but they also stimulate nonautonomous cell proliferation. Endocytic trafficking defects in vps25 cells cause endosomal accumulation of the signaling receptor Notch and enhanced Notch signaling. Increased Notch activity leads to ectopic production of the mitogenic JAK-STAT pathway ligand Unpaired, which is secreted from mutant cells to induce overproliferation of the surrounding epithelium. The data show that defects in endocytic sorting can both transform cells and, through heterotypic signaling, alter the behavior of neighboring wild-type tissue (Vaccari, 2005).

A model is presented for tissue transformation in vps25 mosaic epithelia. In wild-type epithelial cells, Notch is endocytosed and degraded via MVB sorting in endosomes. In vps25 mutant cells, Notch is endocytosed but fails to be degraded due to impaired MVB sorting; thus, it accumulates in enlarged endosomes. vps25 mutant cells also fail to polarize, to exit the cell cycle and to differentiate; they are later eliminated by apoptosis. Due to ectopic Notch activation, vps25 mutant cells produce and secrete Upd. Via the JAK-STAT pathway, the ectopic Upd promotes extra growth of the neighboring wild-type epithelium. This heterotypic signaling process echoes aspects of the tumor-host interactions observed during malignant transformation of mammalian tissues (Vaccari, 2005).

Class E vps proteins have been studied in cultured vertebrate cells, but the early lethality of mutant mice and cell cycle arrest seen in tissue-specific inactivation have hampered functional analyses in mammals. As in yeast and mammals, loss of ESCRT-II function in flies causes accumulation of ubiquitinated proteins in an enlarged endosomal structure, indicating that the cell biological role of ESCRT-II is conserved across phylogeny. Trapping of Notch in an early endosomal compartment in hrs mutants, and in an Hrs-positive compartment in vps25 mutants, is also consistent with the ordering of class E protein functions in yeast. ESCRT-II physically interacts with both ESCRT-I and ESCRT-III, and identical phenotypes on endosomal organization and sorting are seen in yeast mutant for any ESCRT complex member. Thus, the phenotype described here probably represents that of general ESCRT absence in flies (Vaccari, 2005).

It was surprising to find strong differences between the Drosophila mutant phenotypes of vps25 and hrs. In yeast, deletion of the hrs homolog vps27 causes an endosomal and cargo accumulation phenotype that is indistinguishable from deletion of ESCRT complex members. However, the immunoreactivity to Notch, ubiquitin, and the early endosomal marker Avalanche (Avl), which is largely regular in hrs cells (Jekely, 2003), is highly irregular in vps25 cells. Loss of vps25, which acts downstream of hrs in the endosomal sorting pathway in yeast (Babst, 2002b) thus causes a more severe disruption in endosomal organization than hrs. It remains possible that other Drosophila gene products can partially substitute for Hrs or that the N-terminally truncated protein produced by the single hrs allele (Lloyd, 2002) retains some function. The distinct endosomal phenotypes of hrs and vps25 are significant given their different affects on signaling pathways and dramatically disparate functions in controlling both autonomous and nonautonomous proliferation (Vaccari, 2005).

The autonomous overproliferation of vps25 mutant cells strongly resembles that of tissues mutant for other Drosophila neoplastic tumor suppressor genes, including the endocytic syntaxin-encoding avl. In addition to the immense increase of cell numbers seen in mutant eye discs, vps25 cells resemble avl cells in misdistribution of polarized proteins and cellular junctions, and both genes are required for the endocytic trafficking and degradation of Notch. However, their effects on Notch localization and Notch pathway activity are quite different. In vps25 cells, Notch is trapped internally in the early endosome, and high levels of Notch activity are seen. In avl tissue, where Notch is trapped at the cell surface, ectopic pathway activation is not seen despite the elevated pools of Notch protein. Similarly, ectopic Notch pathway activation is not seen in cells mutant for other characterized neoplastic tumor suppressor genes (D. Bilder, unpublished data cited in Vaccari, 2005). While a requirement for Notch in the cell-autonomous overproliferation of vps25 mutant tissue has not been directly tested, the avl phenotype suggests that altered activities of other mistrafficked membrane proteins, perhaps including the apical membrane determinant Crumbs, are responsible for the persistent proliferation of mutant cells (Vaccari, 2005).

By contrast, the nonautonomous tissue growth induced by vps25 (but not avl) cells is spurred by Notch-mediated production of the extracellular ligand Upd. Overexpression of Upd, either alone or in response to ectopic Notch expression, can cause hyperproliferation of cells anterior to the morphogenetic furrow resembling the phenotype of vps25 mosaic clones. Moreover, Upd overexpression phenotypes, like vps25 mutant phenotypes, are suppressed by STAT92E heterozygosity. Importantly, Notch also acts through Upd to regulate eye disc tissue growth during wild-type development. Thus, activation of Upd by Notch in vps25 mutant eye cells is an appropriate cellular response in an inappropriate developmental context that leads to tumorous tissue growth (Vaccari, 2005).

In mammalian immune cells, ectopic Notch pathway activity can lead to lymphomas, while, during normal development, Notch activation induces production of interleukin-4 and related molecules, which, like Upd, signal through the JAK-STAT pathway. Intriguingly, a recent report describes a critical role for Interleukin-8 expression in neovascularization of Ras-transformed human cells. While a number of cytokines are among the secreted factors that can be produced in tumors, there are no examples in which mammalian Upd orthologs specifically have been shown to modify the tumor environment of Notch-induced malignancies, for instance by promoting angiogenesis or recruiting stroma. Nevertheless, it is speculated that heterotypic signaling by Upd-like factors might alter the proliferation rates of untransformed cells relative to tumor cells, ultimately favoring tumor expansion. Further investigations will be required to establish whether the vps25 mutant phenotype represents a novel inductive mechanism relevant to mammalian tumor-host interactions (Vaccari, 2005).

Notch and its ligands are distributed widely throughout development, yet Notch activity is highly localized to specific times and places. Many posttranslational mechanisms are involved in restricting Notch activity, including ligand presentation, modification of the receptor by sugars, and proteolytic processing to create active receptor forms. Each of these processes both provides a potential point of regulation and ensures that inappropriate activation does not occur, which is critical due to the potent effects of Notch-mediated signals. The vps25 phenotype described here highlights another mechanism that prevents inappropriate activation: endocytic sorting of receptor that is being cleared from the cell surface. In wild-type cells, Notch is continuously internalized and degraded in the lysosome to maintain steady-state levels of surface expression and therefore receptor availability. For this process to accurately control signaling, the cell must ensure that the unliganded Notch is not activated during internalization. The results indicate that prevention of Notch activation requires MVB sorting, and they suggest that rapid transit through the endosomal environment is required to prevent this inappropriate activation (Vaccari, 2005).

How could the endosomal accumulation of Notch in vps25 cells lead to ectopic Notch signaling? The terminal proteolytic cleavage in Notch activation is mediated by the membrane bound γ-secretase activity provided by Presenilin and its associated proteins. The site of γ-secretase activity is controversial, with some evidence pointing toward the cell surface and other evidence pointing to an endosome. Interestingly, ubiquitination of Notch has recently been linked to both its internalization and its activation. A partially processed form of mammalian Notch requires ubiquitination for efficient γ-secretase cleavage and activation, while several Drosophila ubiquitin ligases seem to influence an endosomal sorting decision specifying degradation rather than activation of unliganded Notch. In this regard, it is notable that although hrs and vps25 mutant cells both contain elevated levels of ubiquitinated proteins, only vps25 mutants show ectopic Notch signaling. The differences in Notch signaling activity could in theory arise from differences in the amount of Notch trapped in the different endocytic mutants. However, very high amounts of Notch are present in avl mutant cells, which do not show ectopic Notch signaling. Therefore, the possibility is favored that ectopic Notch activity may be due to the locus of endocytic trapping, which differs between avl, hrs, and vps25 mutant cells. Possible mechanisms for inappropriate activation include coaccumulation of Notch and its ligands, prolonged exposure to γ-secretase, or eventual dissociation of the heterodimer in the endosomal environment. These possibilities are not mutually exclusive, and the altered organization of the vps25 endosome in addition to the absence of flux through the compartment is likely to contribute to inappropriate activation of Notch signaling. Future studies will discriminate among these possible mechanisms of ectopic Notch activation (Vaccari, 2005).

While this discusssion has concentrated on the Notch pathway, it is clear that many molecules are trapped in vps25 endosomes and that vps25 mutations are phenotypically pleiotropic due to alterations in a number of signaling pathways. For instance, STAT92E suppression of vps25 phenotypes is less complete than STAT92E suppression of overproliferation mediated by Upd alone, suggesting that additional factors contribute to vps25-induced tissue overgrowth. One candidate that merits exploration is the MAPK signaling cascade, since vps25 mutants enhance gain-of-function alleles of EGFR (Elp) and MAPK (Sem). The latter evidence is consistent with the persistent MAPK signaling described in several class E mutant tissues, including hrs in flies and TSG101 in mammals (Vaccari, 2005).

Thus, the complexity of the vps25 mutant phenotype emphasizes that endosomal sorting is a point of contact between diverse signaling pathways, and a likely regulatory nexus for normal development and for pathology. Since human tumors benefit from the coordinated disruption of multiple signaling pathways, subversion of endosomal sorting may be one susceptible route toward malignant transformation. Increasing evidence implicates defects in trafficking of specific receptors in the ontogeny of mammalian tumors. Moreover, the ESCRT-I complex member TSG101 was originally isolated for a tumor suppressive function in cultured cells, although such a role in vivo has not been established. The accessibility of Drosophila tissues, along with the availability of mutations that block specific steps of endocytic traffic, will help to elucidate how endocytosis affects metazoan signaling and the consequent effects on cell proliferation during development as well as tumorigenesis (Vaccari, 2005).

The Drosophila gene vps25 possesses several properties of a tumor suppressor. (1) vps25 mutant cells activate Notch and Dpp receptor signaling, inducing ectopic organizers in developing eyes and limbs and consequent overproliferation of both mutant and nearby wild-type cells. (2) As the mutant cells proliferate, they lose their epithelial organization and undergo apoptosis. Strikingly, when apoptosis of mutant cells is blocked, tumor-like overgrowths are formed that are capable of metastasis. vps25 encodes a component of the ESCRT-II complex, which sorts membrane proteins into multivesicular bodies during endocytic trafficking to the lysosome. Activation of Notch and Dpp receptor signaling in mutant cells results from an endocytic blockage that causes accumulation of these receptors and other signaling components in endosomes. These results highlight the importance of endocytic trafficking in regulating signaling and epithelial organization and suggest a possible role for ESCRT components in human cancer (Thompson, 2005).

In a genetic screen for genes controlling tissue growth, a mutation was recovered in vps25 that caused tissue outgrowths in eyes, wings, and legs of adult Drosophila when clones of mutant cells were induced during larval stages. Interestingly, these tissue overgrowths were not associated with overproliferation of the vps25 mutant clone, which instead occupied only a tiny proportion of the overgrown tissue. The size of control and vps25 mutant clones can be compared in adult eyes, where they are marked by an orange eye color encoded by the white⁺ transgene carried on the Piggybac transposon. These results show that growth of vps25 mutant clones is impaired cell-autonomously, but that these clones nonetheless stimulate growth of surrounding tissue non-cell-autonomously. The basis for these phenotypes was examined (Thompson, 2005).

The ability to stimulate growth of surrounding tissue is one property of an 'organizer'. Cells that form organizers release intercellular signals that can act on the cell itself and on nearby cells to drive cell proliferation. Indeed, the eye and limb outgrowths caused by vps25 mutant clones are similar to those obtained when ectopic dorsoventral (DV) axis organizers are formed in these tissues. However, the mechanisms by which DV organizers are established are different in eyes, wings, and legs (Thompson, 2005).

In the developing eye imaginal disc, the DV organizer is established at the boundary between the dorsal and ventral compartments by activation of Notch signaling. Notch signaling induces expression of the secreted signal Unpaired (Upd, a cytokine), which mediates the function of the DV organizer in driving eye growth. When clones mutant for vps25 were induced in the eye imaginal disc, Upd was ectopically expressed within the clones, indicating that vps25 mutant clones establish an ectopic DV organizer. These results suggest that Notch signaling is ectopically activated in vps25 mutant clones (Thompson, 2005).

In the developing wing imaginal disc, the DV organizer is, like the eye, established along the DV compartment boundary by the activation of Notch signaling. In this case, however, the target of Notch signaling is the secreted signal Wingless (Wg). When vps25 mutant clones were induced in the wing disc, Wg expression was ectopically activated, once again indicating that these clones have activated Notch signaling that establishes an ectopic DV organizer, leading to tissue outgrowths (Thompson, 2005).

In the developing leg, the DV axis is organized by Dpp and Wingless signals that are expressed near the anterior-posterior compartment boundary in response to Hedgehog signals. Wingless is restricted ventrally, while Dpp is stronger dorsally, due to mutual antagonism between the two signals. Clones mutant for vps25 mimic those expressing an activated form of the Dpp receptor Thickveins. Tkv signaling is upregulated in vps25 mutant cells and is responsible for generating ectopic DV organizer activity and consequent ventral leg outgrowths (Thompson, 2005).

Since ectopic Notch and Dpp signaling is sufficient to explain how vps25 mutant clones produce tissue outgrowths, attempts were made to analyze how Notch and Dpp signaling could have become activated in vps25 mutant cells. vps25 encodes a component of the ESCRT-II complex, one of three protein complexes discovered in yeast to mediate a critical step during endocytic trafficking of transmembrane proteins to the lysosome. Downregulation of transmembrane signaling receptors by endocytosis and degradation in the lysosome has long been suspected to be of pivotal importance in determining the level of signaling activity in cells. On their journey to the lysosome, signaling receptors are first delivered to endosomes and then sorted from the outer membrane of the endosome into internal vesicles by an inward vesiculation event of unusual topology that gives rise to the multivesicular body (MVB). The contents of the MVB can then be degraded upon fusion with the lysosome (Thompson, 2005).

The precise function of ESCRT complexes is to generate the internal vesicles of MVBs, loaded with transmembrane proteins bound for the lysosome. A key signal that determines entry of membrane proteins into MVBs is ubiquitylation. In both yeast and Drosophila, loss of ESCRT activity interferes with MVB biogenesis and causes accumulation of ubiquitylated proteins on enlarged endosomes. vps25 mutant cells also exhibit these classical MVB sorting defects, with large amounts of ubiquitylated proteins detected on endosomes. Among the proteins that accumulate on endosomes in vps25 mutant cells are Notch and the Dpp receptor, Tkv. These results show that downregulation of Notch and Tkv receptors is prevented in vps25 mutant cells due to an endocytic blockage at the point of entry into MVBs (Thompson, 2005).

Merlin and Expanded function cooperatively to modulate EGFR and Notch endocytosis and signaling

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues. While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. This paper examines the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton. Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors, and loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. It is proposed that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis (Maitra, 2006).

Merlin's tumor suppressor function is conserved from humans to flies, but the cellular basis for this function remains unclear. Genetic studies in Drosophila suggest that Mer regulates signaling pathways that control proliferation, and cell biological experiments indicate that Merlin may play a role in endocytic processes. In addition, Merlin physically interacts with Expanded, a distantly related member of the FERM superfamily, and these proteins colocalize in the apical junctional region of epithelial cells. Furthermore, genetic studies have shown that while mutations of each gene produce modest overproliferation phenotypes in the eye and wing, double mutant Mer; ex cells display severe overgrowth and differentiation defects that are not seen in either mutation alone. Thus, Mer and ex are partially redundant in regulating proliferation and differentiation (Maitra, 2006).

Given these observations, it was reasoned that the difficulty in identifying precise cellular functions for Merlin might stem from its redundancy with Expanded and that this difficulty could be overcome by examining tissues from double mutant animals and double mutant cell clones generated by somatic recombination. Overproliferation of Mer; ex wing imaginal discs is more extreme than that observed with either mutation alone. Surprisingly, however, Mer⁴; ex⁶⁹⁷ eye-antennal imaginal discs have severely reduced eye primordia with a substantial reduction in or total absence of photoreceptors, although the antennal portion is normal or slightly larger than normal and occasionally is duplicated. Apoptosis does not appear to be enhanced in double mutant eye-antennal discs, suggesting that loss of the eye primordium is not due to cell death. Thus, loss of Mer and ex function has a tissue-specific defect in the developing eye that is very different from its effects on proliferation in the wing imaginal disc (Maitra, 2006).

Why does the combined loss of two tumor suppressors cause reduction rather than hypertrophy of eye tissue? Previous studies have shown that initiation of the morphogenetic furrow, which organizes development of the eye, is regulated by a complex network of signals at the posterior and lateral margins of the eye-antennal disc. Mutations that affect these signals not only block furrow initiation, but also may significantly reduce the size of the eye field and disrupt photoreceptor differentiation. For example, ectopic Wingless expression either at the posterior and lateral margins or throughout the eye primordium results in dramatic losses of eye tissue that closely resemble the Mer; ex phenotype just described. Similar effects are seen from reduction in Decapentaplegic (DPP) or Hedgehog signaling in the same cells (Maitra, 2006).

If Merlin and Expanded affect initiation of the morphogenetic furrow rather than differentiation of photoreceptors, then Mer; ex double mutant somatic clones should block ommatidial development only when present at the posterior or lateral margins of the eye field. Indeed, Mer; ex clones could differentiate photoreceptors, but only when located in the middle of the eye field. In contrast, clones in contact with the posterior or lateral margin of the eye fail to produce photoreceptors. It is inferred from these observations that one or more of the signaling pathways that control initiation of the morphogenetic furrow are likely disrupted in Mer; ex double mutant cells (Maitra, 2006).

Given that Merlin is associated with the plasma membrane and may function in endocytic processes, it was asked if Merlin and Expanded play a role in regulating localization and/or abundance of transmembrane receptors that function in eye development. For these studies, Mer; ex somatic mosaic cell clones were examined to allow side-by-side comparisons of wild-type and mutant cells in the wing and eye imaginal discs. Immunofluorescence staining with specific antibodies then allowed comparison of the steady-state levels of receptors between adjacent wild-type and mutant cells. Intriguingly, Notch, the EGF receptor, Patched, and Smoothened all displayed increased antibody staining in double mutant cells relative to their wild-type neighbors. Notch, which is primarily localized to the apical junctional domain in wild-type cells, showed not only increased junctional staining in mutant cells, but also more diffuse staining. Similarly, preparations with anti-EGFR display more abundant membrane-associated and cytoplasmic staining in mutant than in wild-type cells. Patched staining, which is less obviously junctional than Notch or EGFR, appeared more punctate in Mer; ex cells. Thus, simultaneous loss of Merlin and Expanded results in increased abundance of receptors for multiple signaling pathways, though the precise localization defect seems to be specific to each receptor. Two adhesion-related receptors, E-cadherin and Fat, a cadherin superfamily member, were examined; both are similarly upregulated in Mer; ex cells. However, Coracle, a membrane-associated cytoplasmic protein, is not affected. In addition, the localization of markers for apical-basal polarity, including DLG, PATJ, and aPKC, was unaffected in the double mutant cells, indicating that epithelial polarity is not disrupted. In contrast to the double mutant cells, clones lacking just Merlin show no apparent difference in receptor localization or abundance, and ex^e1 cells display only a slight increase in staining. Taken together, these results indicate that Merlin and Expanded are required to reduce the steady-state abundance of a variety of signaling and adhesion receptors in developing epithelia (Maitra, 2006).

Membrane trafficking was examined in Mer; ex double mutant cells. Antibodies were used against the extracellular domain of Notch (anti-ECN) to label protein on the surface of living cells in imaginal discs bearing somatic mosaic clones. Side-by-side comparisons of wild-type and Mer; ex mutant cells show increased cell-surface Notch labeling, consistent with what was observed with fixed tissue and indicating that there are increased levels of receptor at the plasma membrane in mutant cells. In addition, in double mutant cells, the junctional band of Notch staining is broader, indicating that Notch localization to the junctional region also may be affected. Similar differences in junctional staining were observed with the same antibody on fixed and permeabilized tissues, indicating that surface labeling of live cells does not affect Notch localization (Maitra, 2006).

To ask if the increased abundance is due to a defect in turnover, a pulse-chase approach was used to label Notch receptor at the plasma membrane and then its removal from the cell surface was followed. To restrict analysis to Notch that remains at the cell surface, tissues were fixed but not permeabilized at the end of the chase period. A progressive loss was observed of Notch staining at the cell surface during the chase period that appeared more rapid in wild-type than in mutant cells, suggesting a defect in trafficking off the plasma membrane. Quantitative fluorescence analysis was used to determine the relative quantities of Notch on wild-type and mutant cells at the various chase time points. The results indicate that the ratio of cell-surface Notch fluorescence in mutant versus wild-type cells increases significantly between 0 and 10, 30, or 60 min postlabeling. Therefore, Notch protein is cleared more rapidly from the surface of wild-type than mutant cells (Maitra, 2006).

It is worth noting that current models for Notch receptor activation require cleavage and release of its extracellular domain in response to ligand binding. Because an antibody was used that recognizes this domain, it follows that these studies examined only ligand-independent trafficking of the receptor. In support of this inference, the pattern of Notch internalization in pulse-chase experiments was unaffected in Delta⁻ clones. These observations suggest that Merlin and Expanded function in steady-state, ligand-independent clearance of receptors from the plasma membrane, rather than internalization and degradation that occurs in response to ligand binding (Maitra, 2006).

Increased receptor abundance may be expected to result in increased signaling output, if receptor quantity is a limiting factor. In addition, even if overall receptor quantity is not limiting, alterations in subcellular localization or the dynamics of receptor trafficking may have dramatic effects on receptor function. To ask if loss of Merlin and Expanded result in increased output from signaling pathways that regulate eye development and cell proliferation, markers specific for downstream activation of the EGFR, Wingless, and Notch signaling pathways were used. First, double mutant clones were stained with an antibody that recognizes the phosphorylated, activated form of MAP kinase (anti-dpERK), a downstream effector of the EGFR pathway. In addition to the normal anti-dpERK pattern in the wing imaginal disc, increased staining was observed in Mer; ex clones relative to their wild-type neighbors, suggesting upregulation of EGFR pathway activity. Similarly, output from the Wingless pathway was monitored by looking at expression of Distalless, a target of Wingless signaling and it was found to be dramatically higher in the double mutant wing clones. In contrast, similar experiments with the mAb323 antibody to E(spl) bHLH proteins, a marker for Notch pathway activity, did not show upregulation of Notch signaling. This result is consistent with the observation that overexpression of Notch in a wild-type genetic background has little or no phenotype. To examine this further, a genetic context was analyzed in which Notch receptor quantities are known to be limiting, that is, in animals that are heterozygous for a null Notch mutation. Such animals display a dominant, haploinsufficient phenotype characterized by notching along the wing margin. To ask if reduction in Merlin and Expanded in this context can cause upregulation of Notch pathway output, animals triply heterozygous for Notch, Merlin, and expanded were generated and it was found that the characteristic Notch wing phenotype was strongly suppressed (Maitra, 2006).

Taken together, these results are consistent with the observation that the steady-state level of multiple receptors is elevated in Mer; ex cells and indicate that, depending on the precise developmental or genetic context, loss of Merlin and Expanded can result in increased output from the corresponding signaling pathways. In Mer; ex eyes, upregulation of Wingless signaling may be a primary contributor to the observed defect in ommatidial development. Previous studies have shown that ectopic Wingless signaling produces remarkably similar eye phenotypes, and preliminary data suggest that inhibiting Wingless signaling partially suppresses the Mer; ex eye phenotype. In the wing, the dramatic overproliferation of Mer; ex cells may be the combined result of upregulation of several pathways, including EGFR and Wingless (Maitra, 2006).

Merlin and Expanded are associated with the apical junctional region in imaginal epithelia and with endocytic vesicles in cultured cells. Results shown in this study indicate that loss of these proteins affects abundance, cell-surface localization, and endocytic trafficking of Notch, EGFR, and other signaling and adhesion receptors in epithelial cells. Recent studies of endocytic trafficking in receptor/ligand regulation suggest aspects of endocytosis that could relate to Merlin and Expanded function. For example, it is possible that Merlin and Expanded function at the plasma membrane to recruit or anchor transmembrane proteins at sites on the membrane from which they are endocytosed or in the sorting between recycling endosomes and lysosomal degradation by promoting receptor degradation. Both possibilities are consistent with observations of increased receptor levels at the plasma membrane in Mer; ex mutant cells and colocalization of Merlin and Expanded with Notch in punctate structures at the plasma membrane. In addition, a partial colocalization was observed of Merlin and Expanded with Rab 11, a marker for recycling endosomes, and with EEA-1, which labels early endosomes. Intriguingly, it has been suggested that the closely related ERM protein Ezrin functions to promote recycling rather than degradation of the β2-adrenergic receptor via its interactions with filamentous actin. Understanding the exact relationship of Merlin and Expanded to endocytosis and recycling of receptors, as well as their possible relationship to ERM proteins in this process, will require further analysis (Maitra, 2006).

A recent study has proposed that Merlin and Expanded function upstream of Hippo in the Warts signaling pathway, which regulates proliferation. Merlin and expanded mutants display similar phenotypes to those seen in hippo mutants. However, there are significant phenotypic differences between Mer; ex and hippo mutations, most notable of which is that hippo mutations have not been reported to block induction of eye morphogenesis. In addition, there is no evidence to suggest that the Hippo pathway regulates output of the EGFR, Wingless, or Notch signaling pathways. Thus, the relationship of Merlin and Expanded to the Hippo pathway may be more complicated than the linear pathway proposed. One possibility is that Hippo activation is a downstream consequence of Merlin and Expanded's effects on output of multiple signaling pathways (Maitra, 2006).

More than a decade after its molecular characterization, the precise cellular functions of Merlin in regulating cell proliferation remain unclear. Based on the current studies, it is proposed that Merlin's tumor suppressor phenotype results from defects in endocytic trafficking of signaling receptors and accompanying hyperactivation of associated signaling pathways. Recent studies highlight the importance of endocytosis in regulation of signaling pathways. Based on the results presented in this study, it is suggested that proper regulation of membrane trafficking also may have important implications for understanding the cellular basis of tumor suppression in flies and mammals (Maitra, 2006).

Protein degradation and Notch signaling

In eukaryotic cells, degradation of many proteins involves their covalent modification by conjugation with ubiquitin. Ubiquitinated proteins can be rapidly degraded by a large multisubunit complex called the 26S proteasome. This complex is present in the nucleus and in the cytosol of all cells. The 26S proteasome consists of a 20S core particle capped by two 19S regulatory complexes. The 20S proteasome is a barrel-shaped cylinder composed of four stacked rings of seven subunits each. The two external rings are composed of seven alpha subunits (alpha1-7), and the two inner rings comprise seven beta subunits (beta1-7) that catalyze the hydrolysis of polypeptide substrates (Schweisguth, 1999).

In Drosophila, the DTS7 and DTS5 dominant temperature-sensitive (DTS) mutations affect the beta2 and beta6 proteasome subunit genes, respectively (Saville, 1993; Smyth, 1999). DTS5 and DTS7 heterozygous flies develop perfectly at the permissive temperature (25°C), but die as undifferentiated pupae with failures in head eversion at the restrictive temperature (29°C). The DTS5 and DTS7 mutations behave genetically as antimorphic mutations: they correspond to substitutions in residues that are conserved from flies to vertebrates (Saville, 1993; Smyth, 1999). The structure proposed for the yeast 20S proteasome indicates that beta2 directly interacts with beta6 in the adjacent ring, and it predicts that the amino acids mutated in the DTS5 and DTS7 mutant subunits alter the beta2-beta6 interface. Consistent with this possibility, the DTS5 and DTS7 mutations display synthetic lethality, even at 18°C (Schweisguth, 1999 and references therein).

Because DTS5 and DTS7 mutants develop normally at the restrictive temperature until early pupal stages, the potential role of proteasome activity in regulating cell determination was examined in the adult sense organ lineage. Bristle mechanosensory organs are composed of four different cells that originate from a single precursor cell, pI. In the notum, pI cells appear around 8-14 hr after puparium formation. Each pI divides asymmetrically along the anteroposterior (a-p) axis of the fly body to generate two secondary precursor cells: a posterior pIIa cell and an anterior pIIb cell. pIIb divides prior to pIIa, perpendicular to the plane of the epithelium, to generate a small subepithelial glial cell (that will later migrate away from the sense organ), and a pIIIb cell. pIIa then divides asymmetrically, along the a-p axis to produce the shaft and socket cells. Finally, pIIIb divides to generate the neuron and the sheath cell. At the pI, pIIa, and pIIIb divisions, distinct fates are conferred on sister cells by the unequal activation of Notch signaling that results from the asymmetric segregation of Numb. Because Numb is unequally segregated during the pIIb division, it is conceivable that Notch signaling also participates in the pIIIb/glial cell fate decision (Schweisguth, 1999 and references therein).

In Drosophila, dominant-negative mutations in the beta2 and beta6 proteasome catalytic subunit genes have been identified as dominant temperature-sensitive (DTS) mutations. At restrictive temperature, beta2 and beta6 DTS mutations confer lethality at the pupal stage. The role of proteasome activity has been investigated in regulating cell fate decisions in the sense organ lineage at the early pupal stage. Temperature-shift experiments in beta2 and beta6 DTS mutant pupae occasionally result in external sense organs with two sockets and no shaft. This double-socket phenotype is strongly enhanced under conditions in which Notch signaling is up-regulated. Furthermore, conditional overexpression of the beta6 dominant-negative mutant subunit leads to shaft-to-socket and to neuron-to-sheath cell fate transformations, which are both usually associated with increased Notch signaling activity. Finally, expression of the beta6 dominant-negative mutant subunit lead to the stabilization of an ectopically expressed nuclear form of Notch in imaginal wing discs. This study demonstrates that mutations affecting two distinct proteasome catalytic subunits affect two alternative cell fate decisions and enhance Notch signaling activity in the sense organ lineage. These findings raise the possibility that the proteasome targets an active form of the Notch receptor for degradation in Drosophila (Schweisguth, 1999).

The neuron-to-sheath and shaft-to-socket cell fate transformations, as well as the genetic interactions between Notch signaling components and the DTS5 and DTS7 mutations, indicate that decreasing proteasome activity enhances Notch signaling activity in the sense organ lineage. Notch signaling appears to involve the ligand-induced processing of the receptor at the membrane, followed by nuclear translocation of a processed intracellular fragment called NICD. In the nucleus, NICD is thought to act as a transcriptional regulator as part of a complex with Su(H). Therefore, the proteasome may participate in the degradation of a signal transduction component, such as Su(H) and/or NICD. Increased accumulation of Su(H) has previously been shown to also result in shaft-to-socket cell fate transformations. Thus, it is conceivable that a reduction in proteasome activity may lead to the stabilization of Su(H), resulting in double-socket bristles. However, accumulation of Su(H) in socket cells has been found to be lower in the multiple socket cells of sca-DTS5 pupae than that observed in socket cells of wild-type pupae. Hence, accumulation of Su(H) does not appear to correlate with shaft-to-socket fate transformation. Furthermore, a role for Su(H) in specifying the neuron/sheath decision has not yet been established. Together, these observations suggest that Su(H) may not be responsible for the neuron-to-sheath and shaft-to-socket cell fate transformations resulting from a loss of proteasome activity. Alternatively, the proteasome might act in an indirect manner to activate an antagonist of Notch signaling, such as Numb, or Hairless. For instance, an unstable inhibitor of either Hairless or Numb might be degraded by the proteasome. However, no such inhibitors have been characterized to date. Finally, the stabilization of NICD might be responsible for the bristle phenotypes seen in the DTS5 and DTS7 mutant flies. Consistent with this possibility, an activated form of Notch, Nintra, was stabilized in DTS5-expressing cells. This finding shows that the intracellular domain of Notch is either a direct or indirect target of the proteasome. Whether Notch, or an activated form of Notch such as NICD, is ubiquitinated and directly degraded by the proteasome will require additional biochemical experiments (Schweisguth, 1999).

The model of the intracellular processing of Notch predicts that immunoreactivity against the intracellular part of Notch should be detectable in the nucleus after receptor activation. However, endogenous nuclear Notch has not yet been observed in Notch-activated cells by immunodetection methods. This negative result has been interpreted to mean that NICD accumulates to levels below immunodetection thresholds. In support of this interpretation, transfection studies have indicated that the minimal amount of nuclear Notch sufficient to activate a target gene is too small to be detected by standard immunocytochemistry. Consistent with this postulated low level of nuclear accumulation, the presence of a PEST sequence at the Notch C terminus suggests that nuclear Notch may turn over rapidly. However, endogenous Notch immunoreactivity is not detected in the nucleus of ptc-DTS5- or sca-DTS5-expressing cells at the restrictive temperature, indicating that inhibition of protein degradation is not sufficient to allow for the accumulation of detectable amount of NICD in the nucleus (Schweisguth, 1999).

Although there is, as yet, no direct evidence for the ligand-induced ubiquitination of processed Notch receptors, studies in nematodes suggest that the effect of the proteasome on Notch could be direct. Sel-10, a putative component of an E3 complex that might be involved in target recognition for ubiquitination, has been shown to down-regulate Lin-12 signaling in Caenorhabditis elegans and to bind to the intracellular parts of nematode Lin-12 and human Notch-3. The sequence conservation of the sel-10 genes in C. elegans, Drosophila (Berkeley Drosophila Genome project/HHMI EST project, unpublished data reported by Schweisguth, 1999) and humans further suggests that the regulation of Lin-12/Notch signaling by ubiquitin-dependent degradation of the processed receptors may be evolutionarily conserved. An attractive hypothesis is that the proteolytic degradation of activated Notch is required to switch off Notch signal transduction (Schweisguth, 1999 and references therein).

Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-ß signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).

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