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).

Crumbs and Notch Trafficking

Crumbs (Crb) is a conserved apical polarity determinant required for zonula adherens specification and remodelling during Drosophila development. Interestingly, crb function in maintaining apicobasal polarity appears largely dispensable in primary epithelia such as the imaginal discs. This study shows that crb function is not required for maintaining epithelial integrity during the morphogenesis of the Drosophila head and eye. However, although crb mutant heads are properly developed, they are also significantly larger than their wild-type counterparts. In the eye, this is caused by an increase in cell proliferation that can be attributed to an increase in ligand-dependent Notch (N) signalling. Moreover, in crb mutant cells, ectopic N activity correlates with an increase in N and Delta endocytosis. These data indicate a role for Crb in modulating endocytosis at the apical epithelial plasma membrane, which is shown to be independent of Crb function in apicobasal polarity. Overall, this work reveals a novel function for Crb in limiting ligand-dependent transactivation of the N receptor at the epithelial cell membrane (Richardson, 2010).

This demonstrates a novel function for crb in the proper control of head and eye size during Drosophila development. This function is not restricted to the fly head and eye, but also extends to other tissues such as the wing. In the case of the eye, the data indicate that this is linked to a function for Crb in limiting ligand-dependent transactivation of N. In support of this model, a significant increase in endosomes positive for NICD, NECD and Dl was observed in crb mutant eye discs, that correlates with excessive cell proliferation. The eye overgrowth phenotype associated with the loss of crb function is correlated with an increase in NECD/Dl co-endocytosis. The data further indicate that this increase is dependent on the S2 cleavage of N. There is currently no evidence for the requirement of the S2 cleavage to promote endocytosis of N with Dl in cis. Moreover, Dl in cis is thought to inhibit N activation. It is therefore concluded that during eye development, Crb limits ligand-dependent transactivation of the N receptor (Richardson, 2010).

Mutations in the crb gene are associated with a failure to properly polarise the ectoderm along the apicobasal axis in the gastrulating embryo. In human, three Crb orthologues, CRB1, 2 and 3, have been described to date. Interestingly, CRB1 can be differentially spliced to produce the CRB1b isoform, which contains only the extracellular domain, whereas CRB3 lacks the conserved extracellular domain and comprises just the TM and intracellular domains. This suggests independent function for the extracellular and intracellular domains. The function of crb in establishing apicobasal polarity can be rescued in the Drosophila embryo using its intracellular domain anchored to the plasma membrane via the TM domain. Interestingly, crb function in the developing pupal photoreceptor has been linked to stalk membrane endocytosis through the connection of Crb to βHeavy-spectrin. This idea is supported by the finding that overexpression of Crb in either the gastrulating fly ectoderm or fly pupal photoreceptor leads to an increase the length of the apical and stalk membranes, respectively. Interestingly, when examining crb mutant adult photoreceptors, clathrin-coated-like pits were detected in the region of the stalk membrane, which are not normally readily detectable in the WT. Importantly, overexpression of the extracellular domain of Crb linked only to its TM domain is sufficient to cause a striking increase in the length of the stalk membrane in the developing fly photoreceptor. Consistent with this finding is the observation that this transgene can rescue the overgrowth phenotype in crb mutant heads, a phenotype that is correlated with an increased in N/Dl endocytosis. Moreover, the data indicate that Crb function in regulating N activity does not depend on the ability for Crb to interact with βHeavy-spectrin, Moesin, Yurt, Sdt, Patj, or Lin7. This suggests that the extracellular domain of Crb might bind to a component of the extracellular matrix or with itself. It is therefore probable that in the WT, both the extracellular and intracellular domain could synergise to limit endocytosis at the apical membrane (Richardson, 2010).

During Drosophila eye development, N activation at the D/V boundary is thought to promote cell proliferation within the eye primordium via activation of the JAK-STAT pathway. Consistent with this model, overexpression of the NICD in the developing eye discs leads to overgrowth of the eyes. Indeed, flies heterozygous for N, Dl and Ser have smaller eyes than WT flies. However, the width of the corresponding head capsules remains unchanged, arguing that N activity is not required during the head capsule growth. These data suggest that crb function in this epithelium might not be related to N activity, but is instead linked to that of another growth signalling pathway. The data in the eye strongly argue that loss of crb function causes ectopic activation of the N signalling pathway. Moreover, the overgrowth of crb mutant eye tissue can be suppressed by inhibiting the N pathway. Finally, this analysis of crb mutant clones during oogenesis, together with the inhibition of the S2 cleavage, indicates that crb limits ligand-dependent transactivation of N (Richardson, 2010).

Trafficking, and in particular endocytosis, plays a major role in modulating the N signalling pathway. A steady level of N at the cell surface is achieved by a balance between its activation on the way to the plasma membrane and its endocytosis and degradation. Ligand activation also requires the endocytosis and recycling of the ligand back to the cell surface in the signal-sending cell. Upon ligand binding, the transendocytosis of NECD bound to its ligand into the signal-sending cell allows the S2 cleavage of N, and recent work indicates that proteolytic cleavage of N by the γ-secretase complex occurs in the endocytic compartment. In normal conditions, the S2 cleavage of N is required for its subsequent S3/S4 cleavage by the γ-secretase complex. The increase in the number of NECD/Dl endosomes observed in the absence of crb function cannot be explained by Crb's link to the γ-secretase complex and indicates that in the developing eye disc, crb is also required to limit ligand-dependent transactivation of N (Richardson, 2010).

How does Crb act to regulate ligand-dependent N signalling activity? Structural analysis of Crb, N and Dl shows that these proteins all contain extracellular domains that contain multiple EGF-like repeats. This raises the possibility that the extracellular domain of Crb could interact with N and/or Dl via their EGF-like repeats, thus preventing N-Dl binding. Such specificity towards the N pathway is supported by the observation that there is no significant increase in Hrs-positive endosomes in the absence of crb function. Interestingly, other EGF repeat-containing proteins have been shown to inhibit N signalling; Dlk, for example, interacts and inhibits Notch1 in mammalian cells (Baladron, 2005). Alternatively, Crb might limit the rate of endocytosis of N or Dl. One outcome of this could be that Crb limits Dl activation by regulating its endocytosis and subsequent recycling back to the plasma membrane in order for it to become competent for signalling. Another possibility is that Crb might limit the endocytosis of NECD and Dl into the signal-sending cell, which is proposed to be required for the S2 cleavage of N. An increase in either of these endocytic events due to Crb loss-of-function could result in ectopic N activity. It is difficult to differentiate signal-sending versus signal-receiving cells in the context of the early developing eye epithelium, which prevents determination of whether crb function is required in one of these cell types or in both. In addition, it will be interesting to determine how exactly the extracellular domain of Crb modulates endocytosis at the apical membrane, and whether, as suggested by the present study, this might target specific signalling pathways such as the N pathway. Finally, given that crb itself is a transcriptional target of the N pathway, its ability to limit the activity of the γ-secretase complex, together with its function in limiting ligand-dependent transactivation of N, is likely to provide a very robust negative-feedback loop mechanism to regulate N activity during organogenesis (Richardson, 2010).

Ligand-independent trafficking of Notch by Axin and Apc

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of β-catenin. This study shows that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module (Muñ-Descalzo, 2011).

Cells expressing ArmS10, a form of Arm that is insensitive to phosphorylation by GSK3, do not overgrow and remain integrated in the epithelium. Clones of cells mutant for Axin, a central element of the Arm destruction complex, exhibit very high levels of Arm, some of which can be found in the nucleus, and exhibit overgrowths and round edges suggestive of defects in cellular recognition. These phenotypes are related to, but distinct from, those caused by expression of ArmS10 and support the contention that Axin exerts controls on the activity of Arm that are additional to those mediated through its role as a scaffold for GSK3. The effects of Axin loss of function are reminiscent of those caused by expression of ArmS10 in cells with compromised Notch function. Since these effects are caused by the loss of the ligand-independent traffic of Notch, this study tested whether Axin exerts some effect on the traffic of Notch (Muñ-Descalzo, 2011).

Clones of cells mutant for Axin did not show alterations in ligand-dependent Notch signalling, although they exhibited a mild but reproducible increase in Notch protein on the apical side, and overexpression of Axin reduced the amount of Notch present at the cell surface. These observations suggest that Axin regulates the amount of Notch at the cell surface. To test whether this control is exerted by targeting the endocytosis and traffic of Notch, label and chase experiments were performed with Notch. Under the experimental conditions and focusing the analysis in the pouch of the wing imaginal disc, labelled Notch disappeared from the cell surface within 10 minutes of the chase and could be found in punctate intracellular structures, presumably vesicles associated with endocytic traffic. Performing the same assay in the absence of Axin revealed that the endocytosis and traffic of Notch is impaired in Axin mutant cells, and after 30 minutes a substantial amount of Notch could still be detected on the cell surface. This suggests that Axin is involved in, or can influence, the traffic of Notch. Performing the same experiment in discs overexpressing Axin, a decrease was observed in the amount of Notch over time. Altogether, these results suggest that Axin contributes to the removal of Notch from the cell surface and to targeting it for degradation (Muñ-Descalzo, 2011).

Regulation of the activity of Arm by Notch is mediated by its ligand-independent traffic as shown by the activity of chimeric receptors in which the extracellular domain of Notch has been substituted by the extracellular domain of CD8 (CeN) or Torso (TN; Tor - FlyBase). Since Wingless signalling promotes the traffic and degradation of these receptors and cells lacking Axin have elevated levels of Wnt signalling, this study examined what would happen to the stability of CeN in this situation. Surprisingly, the levels of CeN remained largely unchanged in clones of cells mutant for Axin, suggesting that in the absence of Axin, despite high levels of Wnt signalling, CeN cannot be degraded . This could be because Axin is required for the degradation of CeN or because this degradation is dependent on Wnt and Dsh but not on Axin. A contribution of Axin is favoured by the observations that overexpression of Axin reduces, and Axin loss of function increases, Notch levels (Muñ-Descalzo, 2011).

A functional relationship between Axin and Notch is also highlighted by the observation that, in tissue culture, simultaneous reductions of Notch and Axin induce very high levels of Arm activity. However, in vivo, simultaneous loss of both Notch and Axin leads to a suppression of the growth induced by the loss of Axin alone, a phenotype that is associated with extensive cell death and perhaps reflects a synergy of the roles of each protein in apoptosis. For this reason, to test the synergy between the two proteins in determining Arm activity in vivo, a NotchRNAi construct was expressed that reduces, but does not abolish, Notch function in clones of cells mutant for Axin. Under these conditions, there is no apoptosis and larger outgrowths than those promoted by the loss of Axin alone were observed. These phenotypes indicate a synergistic effect of the mutations and suggest that Axin is involved in the modulation of Notch while it traffics through the cell (Muñ-Descalzo, 2011).

Apc, a second element of the Arm destruction complex, is encoded in Drosophila by Apc1 (Apc - FlyBase) and Apc2, which play redundant roles in the regulation of Wnt signalling. In order to test whether Apc is also involved in the traffic of Notch, clones of cells mutant for Apc1 and Apc2 were generated in wing imaginal discs and the traffic of Notch was assessed. In these clones, cells exhibited very similar phenotypes to those of Axin mutants in terms of growth, overall shape and levels of Arm. In addition, they exhibited altered traffic of Notch. However, instead of being clearly localised in vesicles or in the cell membranes, as in the case of Axin mutant cells, Notch protein appeared as a 'fuzzy' stain throughout the cytoplasm of the Apc1/2 mutant cells that was not associated with any subcellular structure. Axin and Apc have been shown to play functionally related, but distinct, roles in the regulation of Arm/δ-catenin and these differences might extend to their effects on Notch (Muñ-Descalzo, 2011).

The function of Axin and Apc is to provide a scaffold for the phosphorylation of Arm/β-catenin by Sgg/GSK3. Since, in mammalian systems, GSK3 has been shown to phosphorylate Notch and there are reports of interactions between Notch and Sgg in Drosophila, tests were performed to see whether Sgg has an effect on the traffic of Notch. Clones of cells mutant for sgg displayed elevated levels of Arm but no discernible effects on the endocytosis and traffic of Notch. This is consistent with the observation that Sgg is not required for the effects of Notch on Wnt signalling (Muñ-Descalzo, 2011).

In addition to their interactions with Wnt signalling, Axin and Apc display interactions with other signalling pathways and, in the case of Apc, with the cytoskeleton. These additional interactions might contribute to the differences between the effects of activated Arm and the loss of function of Axin and Apc. Notwithstanding this, the results reveal a function of Axin and Apc in the traffic of Notch. Previous studies have shown that compromising the traffic of Notch elevates the activity of an activated form of Arm. In Axin or Apc1,Apc2 mutant clones, in addition to the elevation of active Arm, the traffic of Notch is compromised and probably contributes to the increase in Arm activity. In this situation, the levels and activity of Arm would be higher than those resulting from the expression of an activated form of Arm alone. There is evidence that Axin functions in the regulation of Arm activity in a manner that is independent of its role as a scaffold for GSK3. Some of these effects could be mediated through its role in the endocytosis and traffic of Notch, which also could traffic with a GSK3-independent form of Arm (Muñ-Descalzo, 2011).

These results underscore the inadequacy of the notion that Wnt signalling flows through a linear pathway to target the destruction complex and promote β-catenin transcriptional activity. Although this framework helps to explain some of the effects associated with Wnt signalling, it is inconsistent with the observation that, in many instances, changes in the concentration of Arm/β-catenin are insufficient to promote transcriptional activity. While the axis Wnt-Dsh-Axin/Apc-β-catenin is the backbone of Wnt signalling, it is clear that there are additional elements that are not simply modulatory add-ons. In this regard, the interactions between Wnt and Notch signalling are a recurrent theme in developmental biology and disease and might not reflect a simple functional convergence in specific processes at the transcriptional level. The results presented in this study reinforce the notion that Wnt and Notch configure a molecular device (Wntch), in which the mutual control of their activities serves to regulate the assignation of cell fates with the effect of Notch providing a buffer to fluctuations in the resting levels of Arm (Muñ-Descalzo, 2011).

Ral GTPase promotes asymmetric Notch activation in the Drosophila eye in response to Frizzled/PCP signaling by repressing ligand-independent receptor activation

Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. This study shows that in response to planar cell polarity (PCP) signals, Ras-related protein (Rala) modulates asymmetric Notch signaling in the Drosophila eye. Specification of the initially equivalent R3/R4 photoreceptor precursor cells in each developing ommatidium occurs in response to a gradient of Frizzled (Fz) signaling. The cell with the most Fz signal (R3) activates the Notch receptor in the adjacent cell (R4) via the ligand Delta, resulting in R3/R4 cell determination and their asymmetric positions within the ommatidium. Two mechanisms have been proposed for ensuring that the cell with the most Fz activation sends the Delta signal: Fz-dependent transcriptional upregulation in R3 of genes that promote Delta signaling, and direct blockage of Notch receptor activation in R3 by localization of an activated Fz/Disheveled protein complex to the side of the plasma membrane adjacent to R4. This study discovered a distinct mechanism for biasing the direction of Notch signaling that depends on Ral. Using genetic experiments in vivo, it was shown that, in direct response to Fz signaling, Ral transcription is upregulated in R3, and Ral represses ligand-independent activation of Notch in R3. Thus, prevention of ligand-independent Notch activation is not simply a constitutive process, but is a target for regulation by Ral during cell fate specification and pattern formation (Cho, 2011).

The results presented support the model for Ral function in which Ral transcription is upregulated in response to Fz activation. As Fz is activated more in the equatorial cell than the polar cell, Ral is enriched in the equatorial cell. Ral activity represses ligand-independent Notch activation, and thus biases the equatorial cell to become R3. One way in which ligand-independent Notch activation occurs is an accident when normal Notch trafficking is disrupted. Notch receptor undergoes endocytosis and endosomal trafficking continually and mutations that block trafficking of late endosomes to the lysosome block Notch degradation and result in endosomal accumulation of Notch and ligand-independent activation. One possibility is that the endosomal environment promotes production of Nicd by Presenilin cleavage. Ligand-independent Notch activation may also occur normally in the lysosomal membrane. Ral GTPase activity might block ligand-independent Notch activation by regulating Notch trafficking to the lysosome, or by inhibiting another process, such as endosomal acidification, Nicd production or nuclear translocation. The punctate appearance of Ral protein suggests the possibility that Ral may play a role in endosomal trafficking. Although further experiments are required to determine the precise mechanism of Ral function, this study has shown that Ral, a protein that prevents ligand-independent Notch activation, is a target for regulation during pattern formation. Fz/PCP signaling upregulates Ral expression to ensure that ligand-independent Notch activation does not tip the scales in favor of pre-R3 becoming the signal receiving cell. Moreover, it was shown that prevention of ligand-independent Notch activation is not simply a constitutive process, but one that is modulated to ensure specific developmental outcomes (Cho, 2011).

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, NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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. neuA101-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, vgBE-lacZ expression is disrupted. In wild-type discs vgBE-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 NSFE/Q affects the distribution and expression of several downstream components of the Notch signaling pathway. To confirm the effect of NSFE/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 NSFE/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 NSFE/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 Nnd-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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/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 NSFE/QC96 background it was found that both mutant alleles of these genes enhance the NSFE/QC96 wing margin phenotype. This result is the first report of the involvement of these two genes in wing margin development and suggests that NSFE/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 NSFE/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 NSFE/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 Nts 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 Nts alleles exposed to restrictive temperature. In NSFE/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 NSFE/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 NSFE/Q has a direct effect on Notch signaling. Thus vgBE-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., Syntaxin’s 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-Ncyto). 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-Ncyto 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).

The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila

This study identifies Rabconnectin-3alpha and beta (Rbcn-3A and B) as two regulators of Notch signaling in Drosophila. In addition to disrupting Notch signaling, mutations in Rbcn-3A and B cause defects in endocytic trafficking, where Notch and other membrane proteins accumulate in late endosomal compartments. Notch is transported to the surface of mutant cells, and signaling is disrupted after the S2 cleavage. Interestingly, the yeast homolog of Rbcn-3A, Rav1, regulates the V-ATPase proton pump responsible for acidifying intracellular organelles. Similarly, Rbcn-3A and B appear to regulate V-ATPase function. Moreover, mutants were identified in VhaAC39, a V-ATPase subunit, and it was shown that they phenocopy Rbcn-3A and Rbcn-3B mutants. These results demonstrate that Rbcn-3 affects Notch signaling and trafficking through regulating V-ATPase function, which implies that the acidification of an intracellular compartment in the receiving cells is crucial for signaling (Yan, 2009).

In a genetic screen in the follicular epithelium, mutations were identified in the fly homologs of the WD40 proteins Rbcn-3A and B, and a V-ATPase V0 d subunit, VhaAC39. It was shown that interfering with V-ATPase function leads to a block in Notch signaling (Yan, 2009).

Mammalian Rbcn-3A and B form a complex that interacts with both Rab3-GAP and Rab3-GEP, but the biological function of this complex has remained elusive. Interestingly, the closest homolog of Rbcn-3A in yeast is the protein Rav1, a component of the RAVE complex. The RAVE complex was shown to interact with the V1 subcomplex of the V-ATPase and promote its activity by regulating the assembly of the peripheral V1 and membrane V0 subcomplexes to form the V-ATPase holoenzyme. V-ATPases are evolutionarily conserved ATP-driven proton pumps responsible for the acidification of several intracellular compartments, including endosomes, lysosomes, secretory vesicles, and the Golgi apparatus. Inhibition of V-ATPase function leads to a failure in luminal acidification of these different compartments. Likewise, in Rav1 mutants in yeast, V-ATPase-dependent vacuolar acidification is disrupted. Similarly, Rbcn-3A and B mutant follicle cells in Drosophila fail to acidify intracellular compartments. A comparable lack of acidic compartments was seen in follicle cells mutant for the VhaAC39 gene, which encodes one of the two V-ATPase V0 d subunits. The V0 d subunit was suggested to regulate the coupling of ATP hydrolysis and proton translocation and is therefore indispensable for V-ATPase activity (Yan, 2009).

In eukaryotic cells, V-ATPase-dependent acidification of organelles is necessary for protein sorting, trafficking, and turnover. For instance, hydrolases responsible for protein degradation in the lysosome have an optimal activity at a low pH. In addition, several trafficking steps along the endocytic pathway have been shown to rely on V-ATPase function. Mutations in V-ATPase components or pharmacological inhibition of V-ATPase activity results in an accumulation of membrane proteins in endocytic compartments and in some cases blocks transport between the late endosome and the lysosome. Likewise, Rav1 mutants in yeast show an accumulation of endosomes and a delay in vacuolar transport and degradation. Consistent with these data, in Rbcn-3 mutant cells Notch and other integral membrane proteins accumulate in enlarged late endocytic compartments. An identical phenotype was seen upon disruption of VhaAC39 (Yan, 2009).

The RAVE complex was identified in yeast, but so far a similar complex has not been described in higher eukaryotes. By demonstrating a striking resemblance between Rbcn-3 and VhaAC39 mutant cells with respect to intracellular acidification and protein trafficking, evidence is provided for the existence of a similar complex in higher eukaryotes. This conclusion is supported by the observation that HA-tagged Rbcn-3B can be immunoprecipitated with at least two components of the V1 subcomplex, the B subunit Vha55 and the H subunit VhaSFD. In addition to Rav1, the yeast RAVE complex contains two other components: Rav2 and Skp1. Skp1 is a highly conserved SCF ubiquitin ligase that forms multiple distinct complexes involved in a wide array of cellular processes. Rav2 on the other hand has no obvious homologs in Drosophila or other higher eukaryotes. Conversely, no clear Rbcn-3B homolog exists in yeast. In rat, the Rav1 homolog Rbcn-3A forms a complex with Rbcn-3B, and based on the identical phenotypes of Rbcn-3A and B mutants in Drosophila they likely act in a complex in flies as well. An interesting possibility is therefore that Rbcn-3B performs the function of Rav2 in the Drosophila RAVE complex (Yan, 2009).

Interestingly, Rbcn-3 and VhaAC39 mutants were isolated in a screen because of their phenotypic similarity to mutations in Notch pathway components, and indeed it was confirmed that both Rbcn-3 and VhaAC39 are critical factors required for Notch signaling. During Drosophila oogenesis, Notch signaling is required for multiple processes. In particular, at stage 6 of oogenesis, Delta expressed in the germline signals to Notch in the follicle cells to initiate the switch from mitosis to endocycle. The loss of Rbcn-3 or VhaAC39 in follicle cells phenocopies defective Notch signaling with respect to the mitosis-endocycle switch. In addition, defects were observed in other Notch-dependent processes during oogenesis in the absence of Rbcn-3 or VhaAC39, including fused egg chambers and anterior-posterior polarity defects. It was found that Rbcn-3 also affects Notch signaling in eye discs. The results showing that disrupting either a V-ATPase subunit (VhaAC39) or a V-ATPase regulator (Rbcn-3) both lead to a loss of Notch signaling provide evidence for a role of the vacuolar proton pump in the regulation of Notch signaling (Yan, 2009).

How could the vacuolar proton pump regulate Notch signaling? Since the loss of Notch signaling is evident upon disruption of Rbcn-3 or VhaAC39 function in the follicle cells, the signal-receiving cells with respect to Notch signaling, it is clear that V-ATPase function must be required at the level of Notch or in a downstream signaling event. In the absence of Rbcn-3 or VhaAC39, Notch accumulates strongly in enlarged late endosomal compartments, consistent with the known role for V-ATPases in endocytic trafficking and lysosomal degradation. However, it is highly unlikely that the accumulation of Notch in this late endosomal compartment is responsible for the observed block in signaling. Defects in early endocytic trafficking of Notch have been correlated to aberrant Notch signaling. Mutations in proteins that affect the first steps in endocytosis, such as Clathrin heavy chain (Chc), the fly dynamin Shi, the Rab5 GTPase, and the syntaxin Avalanche lead to a loss of Notch activity. In these mutants, Notch accumulates at the cell surface and fails to reach the early endosome. In contrast, mutations in some proteins that affect sorting at the MVB, such as the endosomal sorting complex required for transport (ESCRT) components Tsg101 and Vps25, cause ectopic activation of the Notch pathway. In these mutants, Notch accumulates with ubiquitinated cargo in enlarged MVB. However, mutations in proteins acting at the late endosome, such as the Phosphatidylinositol 3-Phosphate 5-kinase Fab1, cause Notch to accumulate in the late endosome after deubiquitination has occurred. Significantly, whereas these late endosomal mutants show Notch accumulation in late endosomal compartments comparable to that seen in Rbcn-3 and VhaAC39 mutants, they do not perturb Notch signaling. This indicates that the accumulation of Notch in late endosomes upon disruption of V-ATPase activity cannot explain the loss of Notch signaling (Yan, 2009).

In addition to a role in endocytosis and lysosomal degradation, V-ATPases function in the secretory pathway, both at the level of protein sorting in the Golgi and in the fusion of secretory vesicles with the plasma membrane. It has also been observed that acidification of vesicles is important for recruiting certain cytosolic coat proteins. It was therefore possible that the loss of Notch signaling in Rbcn-3 or VhaAC39 mutant cells was caused by a defect in the trafficking of Notch to the cell surface. No evidence was found for a requirement of Rbcn-3 or VhaAC39 in exocytosis; Notch and other membrane proteins still reach the cell surface in the absence of Rbcn-3 or VhaAC39. Nevertheless, it remains possible that VhaAC39 or Rbcn-3 mutants show subtle defects in exocytic trafficking or in the posttranslational modification of Notch in the secretory pathway, which may then be responsible for a loss of signaling (Yan, 2009).

It is possible that the loss of Notch signaling in VhaAC39 and Rbcn-3 mutant cells is due to a defect in the trafficking and/or activity of another pathway component. A possible candidate is γ-secretase. γ-secretase-mediated S3 cleavage of NEXT, the Notch product generated upon ligand-induced cleavage by ADAM metalloproteases, results in the generation of the active form of Notch, NICD. It was shown that the expression of NICD, but neither full-length Notch nor NEXT, can rescue defective Notch signaling in Rbcn-3 mutant follicle cells. Furthermore, NEXT ectopically expressed in wing imaginal discs signals much less efficiently in the absence of Rbcn-3B. These results are consistent with a requirement for V-ATPase activity at the level of or downstream of the S3 cleavage. The γ-secretase complex consists of four core transmembrane proteins. The complex is assembled in the ER and shuttles between the ER and the Golgi. A small fraction of the γ-secretase complex is transported to the plasma membrane and endosomes where it is thought to mediate the cleavage of its substrates such as NEXT. It is conceivable that disrupting V-ATPase activity results in the aberrant trafficking of the γ-secretase complex, thus preventing the S3 cleavage of Notch (Yan, 2009).

Alternatively, it has been reported that γ-secretase activity is optimal at a low pH. Recent evidence also indicates that S3 cleavage of Notch can generate heterogeneous fragments that differ by a few amino acids with different stabilities, thus exhibiting different signaling potencies. Therefore, a loss of V-ATPase activity and the resulting alkalization of intracellular organelles could affect the generation or the release of the S3 cleavage product. In addition, a recent study suggested that mutations in the Aquaporin Big brain, which is required for Notch signaling, also show a reduced luminal acidification (Yan, 2009).

In summary, these results have demonstrated that regulating V-ATPase activity is fundamental to Notch signaling in Drosophila (Yan, 2009).

The vacuolar ATPase is required for physiological as well as pathological activation of the Notch receptor

Evidence indicates that endosomal entry promotes signaling by the Notch receptor, but the mechanisms involved are not clear. In a search for factors that regulate Notch activation in endosomes, mutants were isolated in Drosophila genes that encode subunits of the vacuolar ATPase (V-ATPase) proton pump. Cells lacking V-ATPase function display impaired acidification of the endosomal compartment and a correlated failure to degrade endocytic cargoes. V-ATPase mutant cells internalize Notch and accumulate it in the lysosome, but surprisingly also show a substantial loss of both physiological and ectopic Notch activation in endosomes. V-ATPase activity is required in signal-receiving cells for Notch signaling downstream of ligand activation but upstream of γ-secretase-dependent S3 cleavage. These data indicate that V-ATPase, probably via acidification of early endosomes, promotes not only the degradation of Notch in the lysosome but also the activation of Notch signaling in endosomes. The results also suggest that the ionic properties of the endosomal lumen might regulate Notch cleavage, providing a rationale for physiological as well as pathological endocytic control of Notch activity (Vaccari, 2010).

Cell-cell signaling via the Notch receptor is used throughout development to regulate multiple cell behaviors, and inappropriate activation of Notch is emerging as a common hallmark of an increasing number of cancers. Thus, resolving the mechanisms by which Notch signaling is regulated is of great importance and of widespread interest in order to understand human development as well as to devise effective anticancer therapies. In response to ligand engagement, the Notch receptor is activated by S3 cleavage, a γ-secretase-mediated intramembrane proteolysis that liberates the Notch intracellular domain from its transmembrane anchor, allowing the soluble form to travel to the nucleus, where it regulates the transcription of a variety of important target genes (Vaccari, 2010).

Mounting evidence, particularly in Drosophila, has pointed to the unexpected involvement of the endosomal system in regulating activation of the Notch receptor in signal-receiving cells. Importantly, endosomal regulators can either increase or decrease Notch signaling depending on the specific site of the endocytic pathway at which they act. For example, factors that positively regulate traffic from the cell surface to endosomes, including Dynamin (Shibire -- FlyBase), the ubiquitin ligase Deltex, the syntaxin Avalanche (Avl; Syntaxin 7), the GTPase Rab5, the Rab5 effector Rabenosyn-5, and the Sec1/Munc18 family protein Vps45, are required to promote signaling. By contrast, Endosomal sorting required for transport (ESCRT) complex components, the C2-domain protein Lethal (2) giant discs 1 (Lgd), and the ubiquitin ligase Su(dx), which subsequently sort cargo within the endosome towards lysosomal degradation, are required to prevent excess signaling. Much of this evidence points to early endosomes as important sites of signaling activation. However, the molecules and mechanisms that restrict activation of the Notch receptor to this endosomal compartment remain unknown (Vaccari, 2010).

To identify genes involved in endocytic control of the Notch receptor, a collection of Drosophila mutants enriched for endocytic regulators was screened to identify those that display altered Notch localization. Eye imaginal discs consisting predominantly of mutant cells were generated and immunostained using Notch antibodies. It was found that discs mutant for the single allele complementation group MENE2L-R6 accumulate high levels of Notch as compared with wild-type (WT) discs. Clones of MENE2L-R6 mutant cells generated in the context of a mosaic eye disc also displayed substantial accumulation of Notch, which was found in large intracellular puncta. MENE2L-R6 discs were consistently smaller than WT discs and also displayed aberrant morphology, compromised epithelial polarity, and upregulated Matrix metalloprotease 1 (Mmp1) production. Together, these data suggest that R6 disrupts a gene that regulates Notch trafficking as well as the proper growth and organization of imaginal disc tissue (Vaccari, 2010).

The R6 mutant by complementation was mapped to a deficiency that removes the chromosomal region 34A3. Tests with lethal transposon insertions within the deficiency region revealed that R6 fails to complement P[PZ]l(2)01510, which is inserted in the first intron of the predicted gene CG3762. CG3762 is also known as Vha68-2, the most widely expressed of the three Drosophila genes that encode the A subunit of the multiprotein vacuolar ATPase (V-ATPase) proton pump (Allan, 2005). The A and B subunits form a hexamer that binds nucleotide (ATP) in the peripheral V1 sector; nucleotide binding and hydrolysis are essential for transmembrane proton conductance through the integral membrane V0 sector. Sequencing of Vha68-2 in R6 mutants revealed a nonsense mutation in the region encoding the C-terminal domain. Based on the crystal structure of the related A-ATPase subunit A, the truncated C-terminal domain of the R6 mutant protein, even if expressed, is likely to lack part of the region that contacts subunits D and F, which are involved in torque generation. Since torque generation is essential for proton transport and thus endomembrane acidification, the ability of Vha68-2 mutant cells to incorporate the vital dye Lysotracker, which accumulates in acidified lysosomes, was tested. Compared with WT cells or cells mutant for the endosomal component Hrs, it was found that Vha68-2 mutant cells incorporate very low levels of Lysotracker, indicating impairment of the ability to acidify endocytic organelles (Vaccari, 2010).

Failure of Lysotracker incorporation into Vha68-2 mutant cells is not due to absent endocytic structures, as staining with endosomal markers and electron microscopy revealed that both early and late endosomes, as well as lysosomes, are present in mutant cells. In addition, ultrastructural analysis of the morphology of the endocytic compartment revealed that Vha68-2 mutant cells contain, compared with WT, more multivesicular bodies (MVBs), a degradative endosomal organelle. These MVBs were also enlarged and contained a high number of internal luminal vesicles (ILVs). Consistent with a role for acidification in activating low-pH lipid hydrolases, these data suggest that Vha68-2 is required for lysosomal degradation of ILVs. Overall, the Lysotracker incorporation and the ultrastructural analyses indicate that Vha68-2 causes a substantial loss of proton transport activity (Vaccari, 2010).

Recent evidence suggests that the route and rate of Notch traffic through endocytic compartments can be regulated to either potentiate or diminish signaling activity. The results of this study identify a physiological mechanism to account for this effect on signaling. A set of genes that are required for endosomal Notch signaling ncode subunits of the V-ATPase, the molecular machine that creates a proton motive force to acidify most compartments of the endosomal system. The data thus establish a novel role of V-ATPase that is relevant to both physiological and pathological Notch signaling. While this work was under review, an article describing a similar role for V-ATPase regulators and a different subunit of the V-ATPase in Notch signaling was published (Yan, 2009). These independent results confirm those findings and expand them by reporting the ultrastructure of V-ATPase mutant cells, extending the role to tumor contexts that depend on excess Notch signaling activity and, importantly, providing evidence for the early endosome as the site of the requirement for V-ATPase activity. Interestingly, the aquaporin Big brain (Bib) was recently shown to promote Notch signaling at a similar step of Notch processing, and bib mutant clones, like V-ATPase mutant clones, show defects in endosomal acidification. It is possible that some other, as yet uncharacterized, V-ATPase function unrelated to its well-established role in proton pumping might be required for Notch signaling activation. Alternatively, the common acidification and Notch signaling phenotypes of V-ATPase and bib mutant cells might indicate that Notch signaling normally requires endosomal acidification (Vaccari, 2010).

The best-understood requirement for V-ATPase-dependent acidification is in promoting lysosomal degradation by ensuring the proper targeting, release and activation of lysosomal proteases. Indeed, this study found that Notch and other cargoes are trapped in a lysosome-like compartment in V-ATPase-deficient cells, where they accumulate rather than being degraded. The cargo-trapping phenotype resembles that seen in the presence of chemical inhibitors of lysosomal acidification, such as chloroquine, as well as in cells mutant for regulators of lysosomal genesis such as the HOPS components Dor and Car and the PIKFYVE Fab1. However, in striking contrast to these latter mutants, in which Notch signaling is largely unaffected, a substantial loss of Notch signaling is seen in V-ATPase-deficient cells. This indicates that the requirement for V-ATPase in Notch signaling must precede lysosomal entry (Vaccari, 2010 and references therein).

How could acidification of earlier endosomal compartments promote Notch signaling? One possibility is that the role of acidification reflects the requirement of endosomal transport for Notch activation. In mammalian cells, V-ATPases have been implicated in the recruitment of proteins that modulate traffic between early and late endosomes. This study found a reduced rate of endocytosis in V-ATPase mutant cells, and it is possible that a dampened flux of Notch through the endocytic pathway contributes to reduced Notch signaling. However, it is believed that this role is unlikely to account for the V-ATPase mutant phenotype for the following reasons. First, although its endocytic rate may be reduced, Notch can clearly reach both early and late endosomal compartments in mutant cells; this mild quantitative reduction in traffic contrasts with the more substantial and qualitative loss of Notch signaling. Second, in mammalian cells, loss of endosomal acidification reduces progression between early and late endosomes. Since the requirement for Notch signaling is in entry into early endosomes, Notch activation would seem to be less sensitive to this step. Moreover, because Notch reaches late endosomal compartments in V-ATPase mutant cells, in Drosophila, at least, redundant mechanisms to energize endocytic traffic must exist. Third, the striking failure of Notch trapped within Hrs-positive early endosomes in V-ATPase ESCRT double mutants to signal suggests that even when endocytic traffic is blocked at the endosomal sorting step, Notch activation still requires V-ATPase activity. The absence of Notch activation in vps22 Vha55 cells is particularly striking because even when most Notch cannot reach endosomes in cells double mutant for the ESCRT component TSG101 (erupted) and the endosomal syntaxin avl (Syx7), the minor fraction of Notch that does reach endosomes is efficiently cleaved and strongly activates ectopic Notch signaling. This suggests that the role of V-ATPase in Notch activation goes well beyond trafficking. The data also reveal that it is not mere access to the early endosome that is required for Notch signaling, but rather that a specific physiological feature of that endosome is central to the signaling activation mechanism (Vaccari, 2010).

An attractive alternative is that V-ATPase-dependent acidification creates an endosomal environment that is conducive for productive Notch S3 cleavage and, therefore, signaling. In this scenario, Notch transits through the early endosome in V-ATPase mutant cells, but is not efficiently activated because of altered γ-secretase function. Such a role is consistent with genetic experiments, which show that a membrane-tethered Notch truncation that is automatically processed to an active form by γ-secretase in WT cells cannot signal in cells that lack V-ATPase function. Although it is appealing to speculate that an acidic pH could promote overall cleavage by γ-secretase, which requires low luminal pH for optimal activity, experiments testing the acid dependence of other γ-secretase substrates have failed to show a reduction in overall S3-type processing. Although these assays are limited in their ability to reflect the in vivo situation, an interesting possibility suggested by mammalian studies is that pH might alter the precision of the S3 cleavage site, producing NICD forms that are quickly degraded and cannot effectively signal. Additional possibilities exist, such as that rather than creating a cleavage-promoting environment per se, the V-ATPase could serve as a pH sensor that couples the generation of a low pH to the recruitment of γ-secretase regulators in order to restrict cleavage to an endosomal site. Finally, acidification could directly affect the correct maturation and localization of the γ-secretase enzyme. Future work will distinguish between these possibilities (Vaccari, 2010).

Regardless of the specific mechanism governing the role of V-ATPase in Notch cleavage, overall the current data suggest that drugs that impair V-ATPase function and consequently reduce endosomal acidification might be used to curtail pathologic overactivation of Notch, such as that observed in cancers characterized by Notch overexpression. Considering that such a goal is presently being pursued by the use of γ-secretase inhibitors, the use of already established V-ATPase inhibitors, such as Bafilomycin A1, either alone or in combination with γ-secretase inhibitors, could represent a promising therapeutic avenue (Vaccari, 2010).

Common and distinct genetic properties of ESCRT-II components in Drosophila

Genetic studies in yeast have identified class E vps genes that form the ESCRT complexes required for protein sorting at the early endosome. In Drosophila, mutations of the ESCRT-II component vps25 cause endosomal defects leading to accumulation of Notch protein and increased Notch pathway activity. These endosomal and signaling defects are thought to account for several phenotypes. Depending on the developmental context, two different types of overgrowth can be detected. Tissue predominantly mutant for vps25 displays neoplastic tumor characteristics. In contrast, vps25 mutant clones in a wild-type background trigger hyperplastic overgrowth in a non-autonomous manner. In addition, vps25 mutant clones also promote apoptotic resistance in a non-autonomous manner. This study genetically characterize the remaining ESCRT-II components vps22 and vps36. Like vps25, mutants of vps22 and vps36 display endosomal defects, accumulate Notch protein and--when the tissue is predominantly mutant--show neoplastic tumor characteristics. However, despite these common phenotypes, they have distinct non-autonomous phenotypes. While vps22 mutations cause strong non-autonomous overgrowth, they do not affect apoptotic resistance. In contrast, vps36 mutations increase apoptotic resistance, but have little effect on non-autonomous proliferation. Further characterization reveals that although all ESCRT-II mutants accumulate Notch protein, only vps22 and vps25 mutations trigger Notch activity. It is concluded that the ESCRT-II components vps22, vps25 and vps36 display common and distinct genetic properties. These data redefine the role of Notch for hyperplastic and neoplastic overgrowth in these mutants. While Notch is required for hyperplastic growth, it appears to be dispensable for neoplastic transformation (Herz, 2009).

Appropriate cell/cell signaling requires both coordinated activation and inactivation of cell surface signaling receptors. Usually, the receptors are activated by ligand binding upon which they induce an intracellular response including ubiquitination of the receptor which provides the signal for receptor internalization by endocytosis. Endocytosis also controls the steady-state levels of cell surface receptors independently of ligand occupation. After endocytosis, the cell surface receptors are present at the early endosome. Because the intracellular domain of activated signaling receptors is exposed to the cytosol, the receptors are still able to signal. In fact, signaling from the endosomal location appears to be the preferred mode of several signaling pathways as it brings the receptor in close proximity to intracellular signaling complexes. To fully inactivate the signaling receptors, a second form of internalization at the limiting membrane of the early endosome is necessary to form the multi-vesicular body (MVB). In the MVB, the receptors are completely detached from the cytosol and stop signaling. Finally, the MVB fuses with lysosomes for proteolytic degradation (Herz, 2009).

Genetic studies in yeast have identified fifteen class E vps (vacuolar protein sorting) genes required for MVB formation (Raymond, 1992). These genes encode the components of four ESCRT (Endosomal Sorting Complex Required for Transport) protein complexes. Hrs (Vps27) and STAM (Hse1) form ESCRT-0, which initiates the recruitment of the signaling receptor (the cargo) to the early endosome and delivers it to ESCRT-I. From there, the cargo is transferred to ESCRT-II and then to ESCRT-III. At ESCRT-III, the receptors are internalized into MVBs. Loss of class E vps function in yeast leads to accumulation of ubiquitinated proteins on the limiting membrane of enlarged endosomes. Biochemical studies in mammalian cells have revealed a similar function for endosomal protein sorting (Herz, 2009).

The phenotypic consequences of loss of class E vps genes in the context of a multi-cellular organism have just recently been unveiled. In Drosophila, mutants in hrs, erupted (ept, encoding the ESCRT-I component vps23) and vps25 (a component of ESCRT-II) have recently been described. These mutants are characterized by enlarged endosomes which contain increased protein levels of Notch, Delta, EGFR, Patched, Smoothened, and Thickveins (the Drosophila TGF? type 1 receptor). Despite these common endosomal defects, hrs, ept and vps25 display different phenotypes at the organismal level. While hrs mosaics do not display any obvious adult phenotypes, ept and vps25 mosaics are characterized by overgrown adult eyes and heads, and overgrown larval imaginal discs due to hyperplastic proliferation. Hyperplastic proliferation refers to increased proliferation and overgrowth; however, hyperplastic cells still maintain epithelial polarity and will eventually stop proliferating. Interestingly, this hyperplastic growth does not occur in ept and vps25 mutant tissue itself. Instead, it occurs in wild-type cells immediately abutting the mutant tissue. This non-autonomous hyperplastic proliferation is caused by increased Notch activity at the ept and vps25 endosomes which stimulates neighboring cells to undergo proliferation by activating the Jak/STAT pathway. Increased Notch activity has not been observed in hrs mutants despite the accumulation of Notch protein, explaining the lack of hyperplastic overgrowth in hrs mutants (Herz, 2009).

In addition to non-autonomous hyperplastic growth in genetic mosaics, ept and vps25 mutations can cause neoplastic overgrowth. Neoplastic cells lose epithelial polarity and fail to stop proliferating giving rise to significant overgrowth. ept and vps25 mutants show neoplastic overgrowth if almost the entire imaginal disc is mutant. Neoplastic overgrowth can also be induced in vps25 mosaic tissue, if apoptosis is blocked in vps25 mutant cells. Under both conditions, neoplastic growth occurs in an autonomous manner, i.e. in the mutant tissue. These findings were significant for a better understanding of tumor formation caused by inactivation of Tsg101 (tumor susceptibility gene 101), the human vps23 homolog, which has been implicated in cervical, breast, prostate and gastrointestinal cancers (Herz, 2009).

In addition, although vps25 mutant cells undergo apoptosis, before they die they can increase the apoptotic resistance of neighboring cells through up-regulation of the apoptosis inhibitor Diap1 (Drosophila Inhibitor of Apoptosis Protein 1) (Herz, 2009).

Except for vps25, a genetic analysis of the ESCRT-II components for endosomal protein sorting in metazoan organisms has not been reported. This study characterizes and compares the mutant phenotypes of the individual components of the ESCRT-II complex, vps22 (also called larsen, vps25 and vps36 in Drosophila. The ESCRT-II complex is a heterotetramer composed of two Vps25 subunits, and one subunit each of Vps22 and Vps36. This study shows that mutant cells of the three ESCRT-II components display endosomal defects and accumulate Notch protein. Moreover, imaginal discs predominantly mutant for the three ESCRT-II components show characteristics of neoplastic tissue growth. However, despite these common defects, the phenotypic consequences of loss of vps22, vps25 and vps36 in mosaic animals are distinct. vps22 and vps25, but not vps36 mosaics show non-autonomous hyperplastic growth. In contrast, vps25 and vps36, but not vps22 mosaics strongly increase apoptotic resistance. These differences are caused by selective Notch activation. vps22 and vps25 clones display high Notch signaling activity, while vps36 clones do not, suggesting that hyperplastic growth depends on Notch signaling. However, neoplastic growth may be independent of Notch signaling. Thus, despite their intimate physical relationship, the individual ESCRT-II components are genetically not equivalent (Herz, 2009).

Endosomal defects caused by mutations in the ESCRT-II components vps22, vps25 and vps36 in Drosophila are similar. These mutant endosomes accumulate ubiquitinated proteins and signaling receptors including Notch and its ligand Delta. They also show neoplastic characteristics. However, despite these common endosomal defects, at the organismal level, vps22, vps25 and vps36 mosaic animals display distinct phenotypes. vps22 mosaics are characterized by strong non-autonomous proliferation, but not an increase in apoptotic resistance. vps36 mosaics exhibit the reverse phenotype, i.e. increased apoptotic resistance and no or only weak non-autonomous proliferation. vps25 mosaics combine both phenotypes. Thus, this analysis shows that although these components are part of the same structural complex, they are not genetically equivalent and display distinct genetic properties (Herz, 2009).

While the vps22 allele used in this study is a clear null allele, one might argue that the vps36 allele is not a null and that the observed differences are due to the hypomorphic nature of vps36. However, such an assumption does not explain why vps36 is a strong suppressor of GMR-hid, while a null allele of vps22 that causes a strong overgrowth phenotype, completely fails to suppress GMR-hid. In addition, the common phenotypes (endosomal defects giving rise to enlarged endosomes, accumulation of Notch protein, apoptosis and the neoplastic phenotype) are very similar between vps22 and vps36. Thus, it does not appear that the phenotypic differences observed between vps22 and vps36 are due to allelic strength of the mutants. Rather, they appear to be caused by intrinsic differences of the endogenous genes (Herz, 2009).

It has previously been shown that inappropriate Notch signaling is required for non-autonomous proliferation in vps25 mosaics. The current data confirm this notion for vps22 mosaics. vps22 and vps25 mutants contain increased Notch activity and heterozygosity of Notch suppresses the non-autonomous overgrowth phenotype. In contrast, vps36 mosaics do not activate Notch signaling and hence do not cause non-autonomous overgrowth. Thus, Notch activity is required for non-autonomous hyperplastic overgrowth (Herz, 2009).

It is puzzling that despite their intimate physical association in the ESCRT-II complex, loss of vps22, vps25 and vps36 affects Notch signaling differently. One possibility to explain these differences is that these mutants form distinct endosomal microenvironments which may affect signaling from the endosome differently. The resolution of labeling technologies may not be sufficient to pick up these differences in the endosomal microenvironment, but the fact that genetic differences are observed suggests that microenvironmental differences may exist. There is precedence for such a conclusion. Although hrs mutants contain abnormal endosomes leading to accumulation of Notch protein, they do not trigger Notch activity and hence no significant growth defects. Further support of the idea that Notch needs to be in a particular microenvironment at the early endosome in order to be activated comes from a study that analyzes that act upstream of the ESCRT machinery in the endosomal pathway, namely shibire, avalanche and Rab5. Mutations in these genes also result in accumulation of Notch protein, but do not activate the pathway (Herz, 2009).

Class E vps genes have also been reported to function outside of endosomal protein sorting. As such they are involved in virus budding, transcriptional control, cell cycle progression, mRNA localization and apoptosis. Therefore, it is possible that the observed genetic differences of the ESCRT-II components may be caused by distinct requirements in addition to and independently of endosomal function and possibly independently of the ESCRT-II complex and the remaining ESCRT machinery. Future work will be necessary to dissect the roles of the ESCRT-II components in processes unrelated to endosomal processing (Herz, 2009).

While inappropriate Notch signaling correlates well with non-autonomous hyperplastic growth, it does not correlate with autonomous neoplastic growth. Imaginal discs entirely mutant for vps22, vps25 and vps36 all display overgrowth and loss of cellular architecture, hallmarks of neoplastic behavior. The neoplastic phenotype has been attributed to either increased Notch signaling or to mis-localization of the apical transmembrane protein Crumbs. However, vps36 mutant discs display a very robust neoplastic phenotype, but do not activate the Notch signaling pathway significantly, suggesting that activation of Notch is not required for neoplastic growth in vps36 mutant discs. This observation is consistent with previous findings that mutations in the neoplastic tumor suppressor genes avalanche and Rab5 do not activate Notch signaling. This study has not analyzed a genetic requirement of crumbs for the neoplastic phenotype in vps22, vps25 and vps36 mutants, but that would be an interesting experiment in the future (Herz, 2009).

It is clear that the endosomal defects in ESCRT-II mutants not only affect Notch signaling. Other membrane proteins are also affected which may contribute to the neoplastic phenotype. For example, in the case of hrs and vps25, other signaling receptors such as EGFR, Tkv, Ptc and Smo accumulate at endosomes. However, it was also shown that these accumulated proteins are largely derived from the pool of unliganded receptors, suggesting that the endosomal defect affects receptor turnover which does not necessarily cause receptor activation. The only receptor known to be activated at the endosome in a ligand-independent manner is Notch. Future work will be necessary to dissect the role of Crumbs and other signaling pathways for developing the neoplastic phenotypes (Herz, 2009).

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 exe1 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).

Clathrin heavy chain and Notch down-regulation by endocytosis

The clathrin heavy chain is a fundamental element in endocytosis and therefore, in the internalization of several cell-surface receptors through which cells interact with their environment. The only non-lethal mutant allele of the clathrin heavy chain identified to date in metazoans, the Drosophila Chc4, involves the substitution of a residue at the knee region of the molecule that impairs clathrin-dependent endocytosis. This study investigated the consequences of this endocytic defect in Drosophila retinal development and found that it produces an inhibition of programmed cell death in the retinal lattice, followed by widespread death of interommatidial pigment cells once retinal development has been completed. Through genetic interactions and transgenic analyses, Chc4 phenotypes were shown to be cauesed by a Notch receptor gain-of-function, providing a dramatic example of the importance of Notch down-regulation by endocytosis. An increase in Notch signaling is also observed in Drosophila wings in response to the mutant clathrin, suggesting that Notch levels are controlled by clathrin-dependent endocytosis. The implications of these findings are discussed for current models on eye-development and for the role of endocytosis in Notch signaling (Peralta, 2009).

The clathrin heavy chain (CHC) is a highly conserved polypeptide comprised of five functionally distinct domains: a globular N-terminal domain; the proximal leg; the knee domain; the distal leg; and a C-terminal domain (Schmid, 1997). The functional unit of CHC is the triskelion, that is formed by three CHC molecules bound at their C-terminal ends, and by three clathrin light chains (CLC) associated to their proximal legs. The clathrin coat is formed through interactions between the proximal and distal legs of different triskelions, while the N-terminal domain is responsible for binding clathrin-interacting proteins that contain a clathrin-box related sequence (Ybe, 1999). The knee is the only domain of CHC that is not known to participate in any protein-protein interactions (Peralta, 2009 and references therein).

The dominant nature and temperature sensitivity of the hypomorphic Chc4 allele, make it a particularly useful tool to study the biology of the many processes in which clathrin-dependent endocytosis participates. The change of a highly conserved alanine at position 1082 to threonine is responsible for the Chc4 mutation. This alanine lies within a region of the CHC known as the knee that separates the proximal from the distal leg. This region is believed not to interact with other proteins, and is thought to be responsible for much of the flexibility required by CHC to adapt to the bending of the clathrin lattice. Nevertheless, it still remains to be determined if the change in size or charge introduced by the mutant threonine residue in the knee region affects the flexibility of Clathrin legs, and whether this may translate into changes in the rate of assembly or disassembly of the coat that could explain the phenotypes of the Chc4 mutants (Peralta, 2009).

The Chc4 mutation halves the endocytic capacity of Garland cells, a type of nephrocyte with a high rate of endocytosis (Kosaka, 1983). The assembly of clathrin in forming the coat can explain the semi-dominant nature of this hypomorphic mutation (Bazinet, 1993), particularly since the mutant and wild-type CHC molecules are mixed in the triskelion of a heterozygous individual, and triskelions with different compositions are mixed in the coat. This might also explain the variable penetrance displayed by the mutation and by the different transgenic lines expressing wild-type or mutant forms of CHC. This variability may reflect the sensitivity to changes in the proportion and/or amount of mutant and wild-type forms of Clathrin (Peralta, 2009).

The reduced endocytic capacity of the mutant becomes lethal at higher temperatures (28°C), although it is sufficient to allow a small percentage of escapers to complete development at lower temperatures. In these escapers, the retina, the sperm production (Fabrizio, 1998), and the digestive system appear to be particularly affected (Peralta, 2009).

Although endocytosis is required at many steps during larval retinal development, the first phenotype in the eye that can be detected in the Chc4 mutant at 25°C is the partial inhibition of interommatidial PCD during pupal retinal development. The correct spacing of the ommatidia depends on the elimination of roughly one third of the interommatidial cells by PCD, leaving nine interommatidial pigment cells (IOPCs) to isolate each ommatidium. The mechanisms that direct interommatidial PCD are not completely understood, but while Notch and the roughest-irregular chiasma C receptor are required for apoptosis, the EGFR-Ras pathway provides a survival signal for IOPCs. Contrary to certain aspects of this model, yet in support of an increase in Notch signaling explaining the Chc4 phenotype, the expression of Notch (NFL) in IOPCs inhibits PCD in the pupal retina, rather than augmenting apoptosis. The same inhibition of PCD was obtained when a constitutively active form of Notch is expressed, or its downstream target, Suppressor of Hairless, as indicated previously. Moreover, an increase in PCD is seen when a dominant-negative form of Notch is expressed . In the pupal retina, Notch is mainly expressed by IOPCs and to a lesser extent by primary pigment cells (PPCs), and the Notch receptor is endocytosed and directed toward the Hrs degradation pathway. Indeed in a mutant in which endocytosis is intensified Notch loss of function phenotypes are enhanced in the Drosophila wing, constituting additional evidence that endocytosis negatively influences Notch signaling. It remains to be explained why the absence and the excess of Notch signals in interommatidial cells, produce a similar phenotype, (i.e. inhibition of PCD and excess of IOPCs). It can only be speculated that the response of the cells to the absence of such signaling might be mediated by a mechanism other than that which responds to changes in the strength of its signal (Peralta, 2009).

Once PCD is completed and the final number and identity of all the retinal cells have been established, all the IOPCs in the Chc4 mutant retina start to die, such that the retina is totally devoid of IOPCs upon completion of this process. The resulting loss of the ommatidial arrangement probably leads to a severe reduction in the capacity to form images, which relies on the precise orientation of the optical elements in the Drosophila neural superposition eye, a type of compound eye where the image is formed in the brain through parallel processing of the signals from multiple ommatidia. The mutant clathrin is responsible for this pigment cell death because it could be rescued by specifically expressing a wild-type copy of CHC in them. The induction of pigment cell death by disrupting endocytosis with a dominant-negative form of dynamin further confirms that this process is dependent on endocytosis (Peralta, 2009).

As in the case of the PCD phenotype, the IOPC death phenotype displayed by the mutant can also be reproduced by the sole expression of Notch (NFL) in wild-type pigment cells. Indeed, NFL enhances the mutant IOPC phenotype when expressed in Chc4 mutant cells, resulting in a more rapid disappearance of IOPCs. Conversely, expression of a dominant-negative form of Notch (NECN), provokes the suppression of the mutant phenotype. These results suggest that although the defect in Chc4 affects endocytosis, the ultimate cause of the IOPC death phenotype was the excess of Notch signaling that ensued. Genetic analysis supports this notion, as two Notch alleles, Nfa-g62 that causes specific loss of Notch function in the pupal retina, and the temperature-sensitive allele Nts1, both completely suppress the demise of IOPCs caused by the Chc4 mutation (Peralta, 2009).

Once the correct number of IOPCs have been specified, Notch lacks an obvious role in late pupal development, as demonstrated by the absence of retinal defects when Notch activity is lost in late pupas after PCD. At this time, the survival signal mediated by EGFR that protects IOPCs during PCD also disappears, consistent with the failure of overexpression of the EGFR negative regulator Argos to produce an effect, similar to the activated form of armadillo from whose deleterious effects on retinal cells are protected by EGFR activity until about P40%. However, the presence of Notch is required for the IOPC death induced by the Chc4 mutation. If Chc4 mutants fail to down-regulate Notch at this time, an increase in the receptor would be expected at the membrane of IOPCs and PPCs. Indeed, Notch is more prominent at the apical membranes between IOPCs and PPCs, and even between PPCs and cone cells (CCs) in Chc4 mutants. At present there is no evidence that the levels or localization of Delta are altered in the Chc4 mutant retina, raising the possibility that membrane-bound Notch receptor could to be the main target for the Chc4 reduced endocytosis (Peralta, 2009).

It has been shown that there is a strict requirement for Delta ligand endocytosis in the signal-sending cell, while an equivalent requirement for Notch receptor endocytosis in the signal-receiving cell has not been demonstrated yet, although a requirement for dynamin in both cells raises that possibility. Surprisingly, Notch signaling continues to take place in Chc4 during the unaffected larval retinal development, and as required for PPC specification in pupal development. It is believed that is due to Chc4 hypomorphic nature that retains a significant part of its endocytic capacity. If clathrin is responsible for the endocytosis of the empty receptor, it would provide a mechanism for Notch down-regulation consistent with current observations. A lesser decrease in endocytosis would first increase the amount of empty receptor at the membrane, increasing sensitivity and therefore signaling, when ligand is available. A greater reduction in endocytosis would eventually reduce Notch signaling by interference with ligand endocytosis, while a block in endocytosis would prevent Notch from signaling (Peralta, 2009).

One would expect the regulation of Notch receptor levels through clathrin-dependent endocytosis to be a general phenomenon, even if the retina constitutes a particularly sensitive system to such alterations. Indeed, it was found that Notch signaling in the wing is also affected by the Chc4 mutation, although it was necessary to sensitize the system to detect these defects. This sensitization was achieved by either halving normal Notch levels using a null mutation in heterozygosis, or by expressing an antimorphic mutated form of the receptor. The reproduction of the Notch gain-of-function phenotype of Chc4 in the wing suggests that endocytic down-regulation of the Notch receptor is a general mechanism by which cells can control Notch responsiveness (Peralta, 2009).

Although the results shed light over the role of clathrin-dependent endocytosis in Notch down-regulation, the effect of Chc4 on pupal retinal PCD indicates that Notch induces survival among interommatidial cells. This finding apparently contradicts two well established facts about retinal PCD: that lack of Notch reduces cell death, and that the EGFR-Ras pathway is responsible for the survival of interommatidial cells in the last instance. However, all these results are not necessarily incompatible. Throughout retinal development one of the main roles of Notch signaling is to maintain cells in an undifferentiated state, while the EGFR-Ras pathway is responsible for differentiating all the cell types in the retina in successive waves of signaling. At the end of cell specification all extra cells must be lost through apoptosis. If, as the results suggest, Notch promotes the maintenance of the undifferentiated state and opposes the apoptotic pathway, cells with a stronger Delta-Notch signal will avoid being culled by apoptosis, and eventually the Ras/Notch signal ratio will be sufficiently favorable to specify them as IOPCs. In this model, stronger Notch signaling (as in Chc4 or by expressing NFL, NICD, or Su(H)) will increase the number of IOPCs by reducing the number of cells that die. Conversely less Notch signaling (as following NECN expression), will reduce the number of IOPCs by increasing the number of cells that die. However, the absence of Notch in this model forces the cells with a low EGFR signal towards the IOPC fate, where activation of the Ras pathway inhibits apoptosis through a different mechanism, the activation of caspase inhibitors. This model allows inhibition of PCD both by an increase in Notch and by the lack of Notch, although through a different pathway. A prediction of this model is that in the absence of both Notch and EGFR, all the cells should be killed by the apoptotic pathway. Indeed this result should be indistinguishable from eliminating just the Ras pathway, as this prevents the interommatidial cells from differentiating as IOPCs, leaving apoptosis as the only possible outcome. These experiments have already been performed indicating that EGFR/Notch double mutants show extensive cell death that is no different from the EGFR mutants alone, therefore placing EGFR downstream of Notch, as in the current model (Peralta, 2009).

One intriguing aspect of these results is that Notch appears to work in opposite directions in both Chc4 retinal phenotypes. During PCD Chc4-induced excess Notch signaling causes an increase in cell survival, while afterwards it appears to provoke cell death. Currently no explanation is available for this disparity, since the final cause for the IOPC death is not known. It is possible that since, Notch is neither required nor appears to have a function in IOPCs after PCD, its abnormal activation by Chc4 might provoke some aberrant situation that causes the cells to die. Indeed evidence is available to suggest that this could be the case. While investigating the death of the IOPCs, the baculovirus caspase inhibitor P35 was expressed in the interommatidial cells using the GAL454 driver. While PCD was completely inhibited, P35 failed to significantly impair IOPC death in Chc4 retinas, suggesting that the cells did not die through apoptosis. However surprisingly, expression of P35 in control pigment cells also caused the IOPCs to die, despite inhibiting PCD, a retinal phenotype identical to Chc4 and excess of Notch, that also inhibit PCD. P35-induced IOPC death phenotype can be appreciated at its early stages in the original work reporting P35 expression in the Drosophila retina with a different driver. Since Notch appears to inhibit the death pathway, and P35 does precisely that, it is speculated that the underlying cause of IOPC death is the inhibition in them of the apoptotic machinery. Some aspect of pigment granules synthesis might require non-apoptotic caspase activity, as it has been reported for sperm production. This explanation would be consistent with Chc4 other major phenotype: male sterility due to defects in sperm production (Peralta, 2009).

The functions of auxilin and rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. This study use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, it was found that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, Drosophila female germline cells, the one cell type known to signal without clathrin, was found to not require auxilin to signal. Third, it was fond that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling (Banks, 2011).

There are three major results of this work. First, it was found that Rab11 is not required for several Notch signaling events in the developing Drosophila eye that require epsin and auxilin. Thus, as in the female germline cells, ligand recycling, at least via a Rab11-dependent pathway, is not necessary for Notch signaling in the eye disc. Second, the one Notch signaling event presently known to be clathrin-independent is also auxilin-independent. This result reinforces the idea that rather than performing some obscure function, the role of auxilin in Notch signaling cells is to regulate clathrin dynamics. Finally, overexpression of both clathrin heavy chain and epsin were found to rescue to nearly normal the severely malformed eyes and semi-lethality of aux hypomorphs. Presumably, vesicles uncoated of clathrin fuse with the sorting endosome, and so it seems reasonable to assume that uncoating clathrin-coated vesicles containing ligand is preprequisite for trafficking ligand through endosomal pathways. Thus, if ligand endocytosis is prerequisite to recycling, efficient production of uncoated vesicles would be required. In aux mutants with severe Notch-like mutant phenotypes, clathrin vesicle uncoating is inefficient. It is presumed that this remains so even when clathrin and epsin are overexpressed, yet the eye defects and lethality are nearly absent. Thus, it is reasoned that auxilin is required not for efficient production of uncoated vesicles per se, but for the other product of auxilin activity -- free clathrin (and possibly also free epsin). Taken together, these results argue strongly that at least in some cell types, the fundamental role of Notch ligand endocytosis is not ligand recycling (Banks, 2011).

Is it possible that the fundamental mechanism of Notch signaling is so completely distinct in different cell types, that ligand endocytosis serves only to activate ligand via recycling in some cellular contexts, and only for exerting mechanical force on the Notch receptor in others? While formally possible, this is not parsimonious. Thus, a model is favored where the fundamental role of ligand endocytosis is to exert mechanical force on the Notch receptor. In addition, some cell types will also require ligand recycling. As no altered, activated form of ligand has yet been identified, while ligand transcytosis has been well-documented, the most likely role of recycling is to relocalize ligand on the plasma membrane prior to Notch receptor binding (Banks, 2011).

Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors

The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. This study shows that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. It is concluded that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred (Rusten, 2006).

Cell growth, survival, proliferation, and differentiation are controlled by signals that activate their cognate receptors on the cell surface. Important examples include the soluble ligand Wnt (and its Drosophila homologue Wingless) and the membrane bound ligand Delta, which bind to G protein-coupled receptors and Notch receptors, respectively, on receiving cells. During development and normal physiology, the levels of the ligands and their receptors are tightly controlled in time and space (Rusten, 2006).

Receptor density at the cell surface is an important determinant of signaling responses, and there are both slow and fast mechanisms attenuating receptor levels. Transcriptional down-regulation is a slow and long-lasting mechanism, whereas posttranslational modification and/or internalization represent fast ways to reduce the amounts of functional receptors on the cell surface. Internalization of many receptors, including Notch and Wnt receptors, is followed by their transport from endosomes to lysosomes, where they become degraded, resulting in a transient reduction in the ability of cells to receive signals. Adding to the complexity of signaling regulation is the fact that ligand-bound receptors may also signal from endosomal membranes, and their signaling output from endosomes may differ from the output triggered from the plasma membrane (Rusten, 2006).

The key roles of the endocytic pathway in cell signaling are highlighted by the analyses of mutants interfering with endocytic trafficking. Such an example is provided by Hrs, a protein that sorts ubiquitinated receptors into intraluminal vesicles of multivesicular bodies (MVBs), destined for degradation in lysosomes. Drosophila hrs mutants show impaired sorting of receptors into MVBs, causing their accumulation in early endosomes. In hrs mutants, Dpp (a transforming growth factor-β homologue) and epidermal growth factor receptor signaling is enhanced, presumably because the activated receptors have a prolonged residence time in the limiting membrane of endosomes. Likewise, mutations of two subunits of the endosomal sorting complex required for transport (ESCRT)-I and -II, Tsg101 and Vps25, which are thought to function immediately downstream of Hrs, cause endosomal accumulation of receptors and tumor-like overproliferation in a cell nonautonomous manner due to increased Notch signaling. This supports the view that proper endocytic traffic has an important antitumorigenic function (Rusten, 2006).

Hrs is recruited to endosome membranes by binding the phosphoinositide (PI) phosphatidylinositol (PtdIns) 3-phosphate [PtdIns(3)P], formed by phosphorylation of PtdIns by a class III PI 3-kinase. PtdIns(3)P is specifically localized to endosomal membranes and not only recruits Hrs but also several other proteins containing FYVE or PX domains (Ellson, 2002; Stenmark, 2002). Class III PI 3-kinase and PtdIns(3)P are thus crucial regulators of endocytic trafficking, mediating endosome fusion as well as degradative sorting, recycling, and retrograde trafficking to the biosynthetic pathway (Lindmo, 2006a). PtdIns(3)P is metabolized by dephosphorylation and by lysosomal lipases. In addition, this PI can be phosphorylated in the 5-position of the inositol headgroup, giving rise to phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. The kinase catalyzing this phosphorylation, Fab1, was first characterized in yeast. Saccharomyces cerevisiae fab1 mutants have abnormally enlarged vacuoles and show impaired trafficking of the ubiquinated cargo carboxypeptidase S to the vacuole lumen (Odorizzi, 1998). Fab1 is evolutionarily conserved, and overexpression of a kinase-dead mutant of the mammalian Fab1 homologue PIKfyve in cultured cells has been reported to inhibit fluid-phase transport of endocytic markers but not recycling/degradation of endocytosed receptors or sorting of procathepsin D (Ikonomov, 2001; Ikonomov, 2003). Moreover, PIKfyve has been found to be phosphorylated by the PI 3-kinase–regulated protein kinase, PKB, after insulin stimulation (Berwick, 2004), and PIKfyve colocalizes with a highly motile subpopulation of vesicles containing insulin-responsive aminopeptidase (Rusten, 2006).

These findings indicate that Fab1/PIKfyve plays a role in controlling specific membrane trafficking processes, but its functions in signal termination and in the physiology of a multicellular organism are not known. To address this, Drosophila fab1 mutants were generated and their phenotype was studied with respect to survival, growth, membrane trafficking and cell signaling. It was found that the activity of Drosophila Fab1 is essential for development and cell volume control and that its inactivation leads to endosomal accumulation of Wingless and Notch. Remarkably, this accumulation is not accompanied by increased signaling, indicating that Fab1, unlike Hrs and ESCRT-I and -II, is not involved in receptor silencing (Rusten, 2006).

That endosomal sorting of ubiquitinated cargoes is of great physiological importance is illustrated by studies of Drosophila mutants of the two ESCRT subunits, Tsg101 and Vps25. Loss of these proteins yields endosomal accumulation of receptors and ubiquitin, similarly to hrs mutants. Importantly, loss of Tsg101 and Vps25 in clones of cells causes a tumor-like overproliferation of adjacent tissue due to increased Notch-mediated signaling. No such effects were observed with fab1 mutant clones consistent with the finding that Notch signaling (as well as Wg and Dpp signaling) is unaffected in fab1 mutants. Thus, Fab1, unlike Hrs and ESCRT-I and -II proteins, does not seem to play any role in receptor silencing, even though it is important for receptor degradation. This is reminiscent of the ESCRT-III subunit hVps24, which mediates degradation but not silencing of the epidermal growth factor receptor. Moreover, it is interesting to note that impaired Hrs, Tsg101, or Vps25 function causes a strong accumulation of ubiquitinated proteins in endosomes, whereas this was not observed in fab1 mutant clones. These results, together with the fact that Fab1 mainly localizes to later endocytic structures than Hrs, suggest that Fab1 functions later than Hrs and ESCRT-I/-II in endocytic trafficking, at a point beyond receptor deubiquitination and signal termination (Rusten, 2006).

Studies in yeast and mammalian cells have suggested a role for Fab1 in endocytic membrane homeostasis, although its exact functions are not known. Indeed, confocal and EM revealed the accumulation of larger late endosomes in fab1 mutant Drosophila cells, consistent with previous studies in fab1 yeast and overexpression of kinase-dead PIKfyve in mammalian cells. The findings that the enlarged vacuoles in fab1 yeast mutants and late endosomes in kinase-dead PIKfyve-overexpressing cells contain few internal vesicles have suggested the possibility that Fab1 could mediate formation of such vesicles (Odorizzi, 1998; Ikonomov, 2001). In agreement with this, in fab1 mutant Drosophila cells enlarged endosomes were frequently observed with few or no intraluminal vesicles. However, in the fab1 mutants highly enlarged MVBs were frequently observed that were filled with numerous normal-sized intraluminal vesicles. This indicates that the increased endosome size in the absence of Fab1 cannot be explained by an inhibited formation of intraluminal vesicles, in contrast to what has been reported for Hrs. A more likely explanation is that late endosomes expand in fab1 mutants because of inhibited retrograde membrane flux to the biosynthetic and early endocytic pathways (Rusten, 2006).

Cell and organ size is controlled by genetic, hormonal, and environmental inputs. In particular, insulin signaling is important for growth, and the functions of the downstream class I PI 3-kinases in growth signaling are well characterized. The striking growth phenotypes observed in fab1 mutants indicate that PtdIns(3)P 5-kinase also regulates cell size. Interestingly, however, whereas PI 3-kinases promote growth, the current findings indicate that Fab1 has an inhibitory effect on cell size. Garland cells were strongly enlarged in fab1 mutants, suggesting a function of Fab1 in negative cell size regulation. In addition, fab1 deficiency led to a thickening of legs and enlargement of wings and heads, demonstrating a role for Fab1 in attenuating organ size. Overexpression of Drosophila Fab1 did not cause any overt growth-inhibitory effects, consistent with the finding that overexpression of Fab1 in yeast does not yield any increase in PtdIns(3,5)P2 levels, presumably because regulatory components are limiting. No strong genetic interactions were detected between fab1 and mutants in components of the insulin signaling pathway, suggesting that the increased cell size in fab1 mutants may not be due to up-regulation of this pathway. Instead, there was a striking correlation between cell size and endosome overgrowth in fab1 mutant larvae. Thus, the increased cell and organ size in fab1 mutants may be due to the volume expansion of endosomes. It is therefore proposed that Fab1, through its effects on endosome morphology, functions in negative regulation of cell volume. Further work will reveal whether Fab1 also regulates cell size by additional mechanisms (Rusten, 2006).

The Abelson tyrosine kinase regulates Notch endocytosis and signaling to maintain neuronal cell fate in Drosophila photoreceptors

The development of a functional organ requires coordinated programs of cell fate specification, terminal differentiation and morphogenesis. Whereas signaling mechanisms that specify individual cell fates are well documented, little is known about the pathways and molecules that maintain these fates stably as normal development proceeds or how their dysregulation may contribute to altered cell states in diseases such as cancer. In Drosophila, the tyrosine kinase Abelson (Abl) interfaces with multiple signaling pathways to direct epithelial and neuronal morphogenesis during embryonic and retinal development. This study shows that Abl is required for photoreceptor cell fate maintenance, as Abl mutant photoreceptors lose neuronal markers during late pupal stages but do not re-enter a proliferative state or undergo apoptosis. Failure to maintain the differentiated state correlates with impaired trafficking of the Notch receptor and ectopic Notch signaling, and can be suppressed by reducing the genetic dose of Notch or of its downstream transcriptional effector Suppressor of Hairless. Together, these data reveal a novel mechanism for maintaining the terminally differentiated state of Drosophila photoreceptors and suggest that neuronal fates in the fly retina retain plasticity late into development. Given the general evolutionary conservation of developmental signaling mechanisms, Abl-mediated regulation of Notch could be broadly relevant to cell fate maintenance and reprogramming during normal development, regeneration and oncogenic transformation (Xiong, 2013).

This study reports a novel requirement for the Abl nonreceptor tyrosine kinase in maintaining the terminally differentiated state of Drosophila photoreceptors. The failure to maintain expression of neuronal markers correlates with impaired trafficking and ectopic signaling by the Notch receptor. Consistent with the idea that aberrant Notch activation might drive photoreceptor dedifferentiation, Notch signaling has been shown to inhibit neuronal differentiation in many developmental contexts in all animals, and Notch is normally absent in pupal photoreceptor cells. Thus it is proposed that ectopic activation of Notch signaling provides a molecular mechanism coupling Abl loss to a program of neuronal dedifferentiation in the fly retina (Xiong, 2013).

Dedifferentiation describes a regressive process whereby a differentiated somatic cell loses its mature identity and reverts to an earlier multipotent developmental state. The observation that some Abl mutant photoreceptors not only lose neuronal marker expression, but also turn on pigment cell marker expression, raises the possibility that the former photoreceptor neurons might transdifferentiate toward a new pigment cell-like state. Genome-wide gene expression analysis of Abl mutant cells should provide a more precise molecular definition of this transition and of the final state of these cells. Experiments to determine whether the dedifferentiated or partially transdifferentiated Abl mutant cells can be redirected toward other cell fates or to re-enter the cell cycle will provide additional insight into the extent of their multipotency and plasticity (Xiong, 2013).

Previous work has shown that Abl is largely dispensable for photoreceptor cell fate specification, but then plays crucial roles throughout the elaborate morphogenetic programs that lead to rhabdomere formation and determine the spatial organization of ommatidial cells within the epithelium. Whether altered Notch signaling and dedifferentiation result directly from failed morphogenesis, or whether they reflect independent requirements for Abl later in development is an open question. The fact that ectopic expression of an activated Notch transgene at mid-pupal stages, after completion of the morphogenetic program, leads to loss of neuronal marker expression, suggests the two can be uncoupled. However Notch trafficking defects are apparent in Abl mutant photoreceptors well before morphogenesis is complete, raising the possibility that these cellular events could be tightly intertwined. Examination of neuronal marker expression and Notch signaling at mid-late pupal stages in apical polarity mutants might help elucidate the extent of molecular coupling between morphogenesis and photoreceptor fate maintenance (Xiong, 2013).

A second open question concerns the molecular mechanisms by which Abl regulates Notch trafficking to activate downstream signaling. Prior work has implicated endocytic trafficking in regulating both ligand-independent and ligand-dependent Notch signaling. Supporting the argument for the former mechanism in Abl mutant photoreceptors, it was found that Delta levels are below detection threshold at the mid-pupal stages when Notch accumulation, and presumably signaling, is highest. However, previous studies have reported that ligand-independent activation of Drosophila Notch in mutants affecting late endosome/ multivesicular body sorting results in overproliferation rather than dedifferentiation of retinal cells. One possible explanation to reconcile a model of ligand-independent Notch activation with these observations is that the endocytic pathway genes potentiate early functions of Notch in regulating cell proliferation, whereas loss of Abl affects later roles. Another non-mutually exclusive explanation is that the endocytic defects observed in Abl clones might be highly specific to Notch trafficking, an argument substantiated by lack of effect on two other cell-surface proteins Delta and Egfr, whereas the phenotypes resulting from loss of a general component of the endocytic machinery might reflect a complex disruption of multiple signaling pathways. Alternatively, the ectopic Notch signaling that results from loss of Abl could reflect a ligand-dependent response, either to undetectably low levels of Delta or to an alternate ligand such as Serrate. Analysis of the endocytic route taken by Notch in Abl mutant photoreceptors, genetic exploration of ligand-dependence versus independence, and investigation of Abl interactions with specific Notch regulators like Nedd4, Deltex and Cbl should help distinguish between the different models (Xiong, 2013).

In conclusion, these results reveal a novel requirement for the Abl tyrosine kinase in preventing Notch activation to maintain the terminally differentiated state of Drosophila photoreceptor cells. The discovery that Abl, a key morphogenetic regulator, is also required for cell fate maintenance, suggests a new molecular strategy for coordinating tissue morphogenesis with differentiation. The extent to which Abl-mediated maintenance of the differentiated cell state might be relevant to other tissues and developmental or pathogenic contexts will be an important direction for future investigation (Xiong, 2013).

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).

Uninflatable and Notch control the targeting of Sara endosomes during asymmetric division

Cell fate decision during asymmetric division is mediated by the biased partition of cell fate determinants during mitosis. In the case of the asymmetric division of the fly sensory organ precursor cells, directed Notch signaling from pIIb to the pIIa daughter endows pIIa with its distinct fate. Previous studies have shown that Notch/Delta molecules internalized in the mother cell traffic through Sara endosomes and are directed to the pIIa daughter. This study shows that the receptor Notch itself is required during the asymmetric targeting of the Sara endosomes to pIIa. Notch binds Uninflatable, and both traffic together through Sara endosomes, which is essential to direct asymmetric endosomes motility and Notch-dependent cell fate assignation. The data uncover a part of the core machinery required for the asymmetric motility of a vesicular structure that is essential for the directed dispatch of Notch signaling molecules during asymmetric mitosis (Loubery, 2014).

The Notch signaling pathway plays multiple roles in organisms ranging from flies and worms to mammals. A powerful model system to elucidate the cell biology of Notch signaling is the Drosophila sensory organs. Each sensory organ precursor (SOP) cell divides asymmetrically to produce a pIIa cell and a pIIb daughter cell, which perform directed Notch signaling: pIIb signals to pIIa. Four independent endocytic mechanisms control asymmetric signaling in the SOP. These include asymmetric endocytic events mediated by the E3 ubiquitin ligase Neuralized, recycling endosomes, and the endocytic adaptors α- and γ-adaptin together with Numb (Loubery, 2014).

During SOP cytokinesis, a fourth mechanism involves a population of endosomes marked by the adaptor protein Sara. Sara endosomes contain as cargo a pool of endocytosed Notch and Delta molecules. Notch and Delta reach the Sara endosome 20 min after their endocytosis in the SOP; this pool is dispatched into pIIa during cytokinesis. In contrast, the pools of Notch in endosomal populations upstream (Rab5 early endosomes) or downstream (Rab7 late endosomes) of Sara endosomes are segregated symmetrically. The specific pool of Notch in Sara endosomes is relevant for signaling: it is cleaved in a ligand- and gamma-secretase-dependent manner to release the transcriptionally active Notch intracellular domain (NICD) in pIIa (Loubery, 2014).

A key question is what machineries control the asymmetric targeting of these endosomes. Is the cargo (the ligand Delta or its receptor Notch) playing a role on the specific targeting of these endosomes? To unravel the machinery regulating the behavior of Sara endosomes during SOP mitosis, candidate factors from previously reported proteomics approaches or genetic screens were tested for Notch signaling. Thus, Uninflatable was identified as a factor involved in the asymmetric dynamics of Sara endosomes (Loubery, 2014).

MARCM homozygous mutant clones were generated for a null allele of Uninflatable (Uif2B7) an the trafficking of Delta, Notch, and the Notch effector Sanpodo through Sara endosomes was monitored. To look at the motility of the endogenous population of Sara endosomes, the cohort of internalized Delta molecules 20 min after its endocytosis was followed in the SOP by means of a pulse-chase antibody uptake assay. Delta, Notch, and Sanpodo traffic normally through Sara endosomes in the absence of Uif, and these endosomes are targeted to the cleavage plane (the central spindle) in cytokinesis (Loubery, 2014).

In Uif mutants or RNAi knockdown conditions, iDl20'/Sara endosomes fail to be asymmetrically dispatched to pIIa after their targeting to the central spindle. These results indicate that Uif is not required to bring Notch to the Sara endosomes or to target the endosomes to the central spindle. However, once in the spindle, Uif is essential for the specific dispatch of Sara endosomes from the spindle into the pIIa cell (Loubery, 2014).

This function of Uninflatable is specific to the asymmetric segregation of Sara endosomes. To gain mechanistic insights into the mechanism of action of Uif, this study has analyzed the density of microtubules in the central spindle and has shown that Uninflatable does not regulate the organization of the microtubular cytoskeleton. In contrast, it was found that Uif controls the residence time of Sara endosomes on the central spindle: in control SOPs, Sara endosomes depart from the central spindle with a decay time of 103 ± 21 s, whereas upon Uif downregulation this decay time goes up to 175 ± 42 s. These data indicate that Uif is not involved in the organization of the spindle, but rather in the motility properties of the endosomes, particularly their last step of departing from the central spindle and end up in pIIa (Loubery, 2014).

Consistent with the role of Uif in the asymmetric targeting of Sara endosomes, Uif contributes to Notch-dependent cell fate assignation in the SOP lineage. To address this, the composition of SOP lineages was examined in homozygous Uif2B7 MARCM clones or upon Uif RNAi. In wild-type animals, the SOP lineage consists of four different cells: two external cells (the shaft and the socket) originating from pIIa and two internal cells (the sheath and the neuron) from pIIb, which can be identified by immunostaining. In Uif mutant clones, instead of a sheath and a neuron per SOP lineage, two sheath cells can be frequently observed in the notum, indicating a symmetric division in the pIIb lineage. Similarly, upon Uif downregulation in the postorbital SOPs, duplications of sockets were observed, which is diagnostic of symmetric divisions in the pIIa lineage. These data uncover a role for Uninflatable in Notch-dependent asymmetric cell fate assignation that is mediated by the asymmetric dispatch of the Sara endosomes (Loubery, 2014).

The Uif phenotype during asymmetric endosomal targeting and cell fate assignation prompted us to look whether Uif is a cargo of Sara endosomes. To detect the endogenous protein, anti-Uif antibodies were generated. To look at Uif trafficking in vivo, transgenic flies were generated expressing a Uif-GFP protein, which can provide activity to rescue the lethality of a Uif lethal mutation at least partly (Loubery, 2014).

Uif-GFP is strongly colocalized with both Sara-GFP and iDelta20'. Since a cargo of Sara endosomes is Notch itself (73% ± 2.7% of the vesicular population of Notch molecules is in Sara endosomes), the presence of Notch cargo was examined in Uif vesicles: 44% ± 4.7% of Uif-positive vesicular structures contain Notch. Therefore, a population of Uninflatable and Notch traffics through Sara endosomes during SOP asymmetric mitosis (Loubery, 2014).

The fact that Uninflatable controls the asymmetric dispatch of the Sara endosomes, which contain internalized Notch and Uninflatable, prompted a look at a possible molecular interaction between Uninflatable and Notch. Uif- and Notch-expressing plasmids were cotransfected in S2 cells and immunoprecipitation experiments were performed by using anti-Uif-coupled beads, followed by immunoblotting with a clean anti-Notch antibody that was purified from a hybridoma cell line (DSHB #C17.9C6). Uif was shown to immunoprecipitate Notch. This coimmunoprecipitation can be reproduced from lysates of S2 cells expressing Notch and Uif tagged with the PC peptide tag and anti-PC-coupled beads; as a control, other transmembrane proteins such as Tkv-GFP are not coimmunoprecipitated with Uif-PC. Together, these results indicate a specific molecular interaction between Notch and Uif (Loubery, 2014).

Uninflatable is a transmembrane protein that, like Notch, contains an array of epidermal growth factor (EGF) repeats. It has been shown that Notch is engaged in protein-protein interactions through its EGF repeats with other factors containing EGF repeats. These include its ligand Delta, but also a number of noncanonical Notch ligands, secreted or membrane proteins lacking the DSL domain characteristic of canonical Notch ligands (Dlk-1, Dlk-2, DNER, Trombospondin, LRP1, EGFL7, and Weary). Consistently, it has recently been reported that a synergistic genetic interaction between Uif and Notch depends on Notch EGF repeats. Therefore, studies were performed to discover which EGF repeats of Uif could be involved in the molecular interaction with Notch. A coimmunoprecipitation experiment was performed in S2 cells coexpressing Notch and an N-terminal, truncated form of Uif tagged with PC (UifΔCter-PC) that lacks the four EGF domains flanking the transmembrane domain but still contains the other 17 EGF repeats and other extracellular domains. While full-length Uif-PC coimmunoprecipitates Notch, UifΔCter-PC does not. This indicates that the interaction between Uif and Notch may be mediated by the four EGF domains of Uif flanking its transmembrane domain (Loubery, 2014).

Although Uif binds and colocalizes with Notch, it does not play a role in core Notch signaling: embryos deprived of maternal and zygotic Uif in germline clones do not show a Notch signaling phenotype, whereas they display loss of inflation of the trachea as previously reported. Consistently, loss of Uif in wing mosaics does not cause a defect of Notch-dependent expression of Wingless at the wing margin. This indicates that Uninflatable interaction with Notch is not essential during core Notch signaling, but rather during the asymmetric dispatch of Notch-containing Sara endosomes during asymmetric cell division. This prompted the possibility that Notch itself is required for the asymmetric motility of the endosomes (Loubery, 2014).

To study whether Notch plays a role during the asymmetric dispatch of Sara endosomes, the trafficking was studied of a Notch-GFP fusion expressed at endogenous levels. The idea was to confirm previous observations using a Notch antibody uptake assay to follow Notch expressed at endogenous levels. To achieve this, a reporter transgenic fly strain was set up in which Notch-GFP fusion is driven by the Notch endogenous promoter and is expressed at endogenous levels. In this fusion, GFP is inserted in the middle of the Notch-intra domain. Since in protein fusions GFP is frequently cleaved out, whether the fusion protein is intact was examined. This would be particularly important in this case, since a cleavage event would lead to a truncated Notch-intra peptide (Loubery, 2014).

In these transgenic Notch-GFP flies, GFP is very efficiently cleaved out (74% of total GFP is cleaved, leading to truncated Notch-intra peptides that can only partially support Notch function and thereby cause a highly penetrant mutant phenotype. This precludes the usage of this reagent as a bona fide marker for Notch. In particular, the cytosolic GFP signal cannot be used as a readout of signaling as previously reported: a nuclear accumulation of the GFP signal in these flies does not solely reflect the accumulation of Notch-intra-GFP, but rather the overall accumulation of different GFP-containing fragments (Loubery, 2014).

Whether, in these conditions, the pool of membrane associated GFP-Notch traffics through Sara endosomes and is asymmetrically dispatched to the pIIa cell was studied. Only 11% ± 1.3% of the total GFP signal in these flies is membrane associated (plasma membrane and intracellular vesicular structures). The rest, representing the vast majority (89%), corresponds to cytosolic and nuclear cleaved GFP (Loubery, 2014).

In Notch-GFP flies, 3.1% of the total GFP signal is associated with intracellular vesicular structures. These correspond to various intracellular vesicular compartments, including Notch in the secretory pathway, as well as in early endosomes, Sara endosomes, recycling endosomes, and late endosomes. To measure the size of the specific pool of Notch in Sara endosomes, a Notch antibody internalization assay was performed, and internalized Notch was chased 20 min after its endocytosis (iNotch20'). As previously established, 73% ± 2.7% of Notch-GFP vesicles are positive for iNotch20'. Of this iNotch20'-positive pool, 79% would be targeted to pIIa . This is consistent with only 65% ± 3.1% of the total pool of Notch-GFP being dispatched to pIIa (Loubery, 2014).

Whether Notch itself plays a role on the asymmetric targeting of Sara endosomes was addressed. Notch was depleated in the SOP by expressing a previously validated Notch dsRNA, and the behavior of Sara endosomes was examined. Upon Notch knockdown in the SOP, iDl20'/Sara endosomes are still targeted to the central spindle, but the subsequent directed dispatch to pIIa is defective. This indicates that Notch itself contributes to the endosomal recruitment of the machinery that endows the Sara endosomes with their asymmetric behavior (Loubery, 2014).

It has been shown that the targeting of Notch to Sara endosomes does not depend on Uninflatable; it was then determined whether the recruitment of Uninflatable on Sara endosomes depended on Notch. Interestingly, it was found that, conversely, the targeting of Uif to Sara endosomes is not controlled by Notch. This implies that these two molecules use different machineries to get to the endosome, where they can interact and are both required for the asymmetric motility of the endosome (Loubery, 2014).

Since the Notch receptor itself is required for the asymmetric targeting of Sara endosomes, it was asked whether Notch signaling plays a role in the process. Notch signaling was blocked by inactivating the ligand Delta through overexpression of Tom in the SOP cell; Tom overexpression leads to inactivation of the Ubiquitin ligase Neuralized and thereby blocks endocytosis-dependent activation of Delta. In the absence of Notch signaling, targeting of Sara endosomes to the central spindle and their asymmetric dispatch to the pIIa cell remains intact. This indicates that although the Notch receptor is essential for the asymmetric targeting of Sara endosomes, Notch signaling is not (Loubery, 2014).

This report has started to unravel the machinery that mediates asymmetric endosome motility during asymmetric cell division. Both Notch and Uninflatable were shown to play a key role in the last step of the asymmetric motility of endosomes: the final, specific stride of the Sara endosomes from the central spindle into the anterior pIIa cell. This is based on the following four key sets of observations (Loubery, 2014).

First, it was confirmed that a functional Notch-GFP fusion expressed at endogenous level does traffic through Sara endosomes, which are indeed dispatched asymmetrically during SOP mitosis. Second, Notch binds Uninflatable, and both colocalize in Sara endosomes. Third, neither Notch nor Uninflatable is essential for the targeting of Notch/Delta/Uif to the Sara endosomes or the targeting of those endosomes to the central spindle, but they are essential for the final dispatch from the central spindle into the pIIa cell. Although Notch is necessary for this process, Notch signaling is not. Fourth, Uninflatable is not an integral component of the Notch signaling pathway, but it plays a role during asymmetric Notch signaling in the SOP, and therefore mutant Uif conditions lead to a lineage identity phenotype. It remains to be elucidated what machineries downstream of Notch/Uninflatable implement the control of the final step toward pIIa and what is asymmetrical in the cytoskeleton so that this final step occurs toward pIIa and not pIIb (Loubery, 2014).

Table of contents

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

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