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 (Endosomal Sorting Complexes Required for Transport-II) complex, one of three protein complexes discovered in yeast to mediate a critical step during endocytic trafficking of transmembrane proteins to the lysosome (reviewed in Katzmann, 2002 and in Raiborg, 2003). 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 (Jekely, 2003; Katzmann, 2002; Lloyd, 2002). 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).
Is upregulation of Notch and Tkv sufficient to explain why Notch and Dpp signaling is activated in vps25 mutant cells? In the case of Tkv, it is known that receptor overexpression can have strong effects upon signaling, but the leg outgrowths observed require more explanation. The answer is likely to lie in the regulatory wiring of the leg imaginal disc, where expression of the Dpp ligand can be induced by activated Tkv signaling itself. This forms a positive feedback loop that drives signaling to high levels. This mechanism explains how vps25 mutant cells in the leg disc can induce expression of the Dpp ligand and generate leg outgrowths (Thompson, 2005).
In the case of Notch, simple upregulation of receptor levels has relatively mild effects on signaling in vivo. However, Notch signaling is unique in that its activating ligands and other signal transducing components are also transmembrane proteins. Indeed, upregulation of the Notch ligand Delta is found in vps25 mutant cells. Whereas overexpression of Delta alone causes cis-inhibition of Notch signaling, co-overexpression with Notch causes activation. Hence, accumulation of all of these Notch signaling components within the same endosomal compartment provides a likely explanation for why Notch signaling is activated in vps25 mutant cells (Thompson, 2005).
The effect of interfering with MVB sorting in Drosophila was first examined with mutants in hrs, which encodes an ancillary factor that recognizes ubiquitylated membrane proteins and delivers them to ESCRT complexes (Jekely, 2003; Lloyd, 2002). In yeast, all components of the ESCRT complexes and their ancillary factors (such as Hrs/Vps27) have a similar 'class E' vacuolar protein sorting (vps) mutant phenotype (reviewed in Katzmann, 2002). In Drosophila, both hrs and vps25 mutants cause MVB sorting defects, but vps25 appears to have a much stronger phenotype, particularly with respect to Notch signaling. This raises the question of whether regulation of Notch signaling is a general function of the ESCRT complexes or a specific function of Vps25 (Thompson, 2005).
This question was addressed by using a cell culture assay for Notch signaling. In these cells, transfection of the full-length Notch receptor produces a low level of signaling activity. When expression of vps25, another ESCRT-II component (vps22), or an ESCRT-III component (vps32) is knocked down by RNA interference, Notch signaling increases. In contrast, knockdown of hrs does not increase Notch signaling, which is actually decreased. Note that the effects observed in this reporter assay are a specific outcome of Notch signaling, because reporter activity depends strictly on the presence of the Notch receptor, presenilin activity (which can be blocked with the inhibitor DAPT), and the presence of binding sites for the nuclear effector of Notch, Su(H), in the reporter gene. Thus, loss of Vps25 activates Notch signaling by the normal presenilin-dependent Notch cleavage and nuclear signaling mechanism. It is concluded that Vps25 behaves like other core components of the ESCRT machinery, while Hrs is not essential for all aspects of ESCRT complex function. These findings may be explained by the ability of another MVB sorting factor, the ESCRT-I component Vps23/Tsg101, to directly recognize ubiquitylated membrane proteins, independently of Hrs (Katzmann, 2002). Indeed, mutants in Drosophila Tsg101 (Moberg, 2005) have also been found to activate Notch signaling in vivo (Thompson, 2005).
A serious obstacle to the foregoing analysis was that vps25 mutant clones survive poorly in proliferating imaginal disc epithelia. However, mutant clones survive relatively well after arrest of cell proliferation, which occurs in cells behind the morphogenetic furrow of the eye disc and in the follicular epithelium of the ovary. This finding is interesting, because it suggests that the survival defect of a vps25 mutant cell is not simply a general one, but instead is a consequence of cell proliferation (Thompson, 2005).
This result can be explained by considering the phenomenon of 'cell competition', in which faster-growing cells are able to eliminate neighboring slower-growing cells by inducing their apoptosis. If cell competition is responsible for the survival defect of vps25 mutant clones, then clonal growth should be restored by either (1) reducing the competitiveness of neighboring cells, or by (2) blocking apoptosis within the vps25 mutant clone. Both of these predictions were tested in the eye imaginal disc. Slowing the growth of neighboring cells by making them heterozygous for a Minute mutation (encoding a subunit of the ribosome) or expression of the caspase inhibitor, p35, in vps25 mutant cells completely restores growth of the vps25 mutant clone. Strikingly, these mutant clones cause massive tissue overproliferation, producing tumorous discs several times the size of normal eye discs (Thompson, 2005).
The phenotype of these tumorous discs was examined more closely, choosing smaller examples that can be visualized whole with a 40× objective lens. At this magnification, it is clear that some wild-type tissue remains, indicating that tissue overgrowth is caused by secreted signals that are released by vps25 mutant cells and consequently drive proliferation in both mutant and nearby wild-type cells. It is also clear that vps25 mutant cells lose their epithelial organization, forming three-dimensional masses of cells with a rounded, mesenchymal appearance surrounded by distorted wild-type epithelia. In addition, mutant cells are unable to differentiate, as shown by the absence of Elav, a marker of arrested, differentiating cells in the eye. These tumor-like growths are formed not only in eye discs, but also in wing and leg discs when apoptosis is blocked in vps25 mutant clones (Thompson, 2005).
Finally, whether such tumorous discs had the potential to metastasize was tested. A simple test for metastasis in Drosophila is to implant the tumor into the abdomen of an adult female host and examine whether migration of cells to secondary sites can occur. Portions of eye discs containing clones of vps25 mutant cells expressing p35 and RFP were transplanted, and, after 1 week, dead animals in which RFP cells had migrated to other parts of the fly were found in 2/15 cases. Such events did not occur when control discs were transplanted. These results suggest that vps25 mutant cells in which apoptosis has been blocked are capable of growth and metastasis to secondary sites (Thompson, 2005).
This work highlights the potential of Drosophila imaginal disc epithelia as a simple in vivo model system in which forward genetic screening approaches can be applied to identify genes controlling the behavior of clones of cells in tissues. The possibility that this system will deliver discoveries that are relevant for human cancer has been freshly appreciated. The transition from autonomous single-cell life to multicellular organisms defines the origin of metazoans and must have required the invention of controls on cell behavior that subsumed the fate of single cells to the needs of the organism as a whole. It is therefore likely that many of these controls will be shared between all animals. Cancer reflects a reversion toward an autonomous state by cells that have subverted the normal controls on cell behavior. It is clearly of interest to identify mutations that can trigger such transformations, and isolation of mutants in vps25 provides an illustrative example (Thompson, 2005).
Mutation of vps25 endows cells with two key properties involved in the malignant transformation of epithelial cells: activation of signals that drive cell proliferation and loss of epithelial organization. In addition, mutation of vps25 also renders cells sensitive to apoptosis. As far as proliferation is concerned vps25 mutant cells induce ectopic DV organizer activity in developing eye, wing, and leg imaginal disc epithelia. The mutant cells secrete intercellular signals that drive proliferation in both mutant and surrounding wild-type cells. Thus, mutation of vps25 hijacks the normal program of growth control in these tissues. The mechanism by which organizers are established differs in the three tissues, but mutation of vps25 is able to induce all three types of organizer activity. The mutant cells can achieve this because they upregulate both Notch and Dpp signaling, which is sufficient to induce ectopic DV organizers in these tissues (Thompson, 2005).
The root cause of activated Notch and Dpp signaling lies in an endocytic blockage at the point of entry into MVBs, where Vps25 is normally required as part of the ESCRT-II complex to sort transmembrane proteins into the internal vesicles of MVBs for later degradation in the lysosome. In vps25 mutant cells, transmembrane proteins accumulate to high levels in enlarged endosomes, and among these proteins are Notch and the Dpp receptor, Tkv. While upregulation of receptors can increase signaling, the dramatic phenotypes observed in vps25 mutants suggest that induction of ligand is also involved. In the case of the secreted Dpp ligand, its transcription is induced by an indirect positive feedback mechanism that is normally involved in establishing the DV organizer in leg discs. The case of Notch ligands is different, as they are broadly expressed transmembrane proteins that appear to accumulate with their receptor and, very likely, other transmembrane components of the signaling machinery in the enlarged endosomes of vps25 mutant cells, leading to productive signaling (Thompson, 2005).
Note that the cell culture system is devoid of known Notch ligands. Thus, the signaling detected upon overexpression of the full-length Notch receptor is likely to reflect a backgound level of presenilin-mediated cleavage that is independent of ligand. Knockdown of vps25 enhances this background cleavage, and this effect can be reversed by addition of the presenilin inhibitor DAPT. It is concluded that blockade of MVB sorting causes Notch to accumulate with presenilin in the same endosomal compartment, which can lead to ligand-independent cleavage and signaling in this assay (Thompson, 2005).
Metastasis begins when a tumor cell loses its epithelial organization, allowing it to exit the epithelium. In the proliferating imaginal disc epithelia, vps25 mutant cells lose their epithelial organization and drop basally from the epithelium, but this behavior is accompanied by apoptosis of the mutant cells. As a consequence, mutant cells are eventually eliminated from the growing tissue, but neighboring wild-type cells that have overproliferated are left behind as a memory of the organizer activity of mutant cells (Thompson, 2005).
Is apoptosis a consequence of the loss of epithelial organization? This study has shown that loss of epithelial organization and apoptosis occur only when mutant cells are induced in proliferating tissues. In tissues composed of arrested cells, vps25 mutant cells survive well and remain epithelial. These effects can be explained by two well-known phenomena. (1) The process of cell proliferation presents a significant challenge to epithelial integrity, with cells often rounding up within the epithelium during cell division. Consequently, mutants that interfere with a cell's epithelial organization are more likely to manifest in proliferating tissues. (2) Proliferating tissues exhibit cell competition, in which competing cells attempt to induce apoptosis of their neighbors. Apoptosis of vps25 mutant cells is caused by cell competition, because it can be rescued by reducing the competitiveness of neighboring cells. Note, however, that alleviation of cell competition does not rescue the defects in epithelial organization in vps25 mutant cells. The results indicate that loss of epithelial organization causes cells to become sensitive to cell competition, a conclusion supported by analysis of other mutations that affect epithelial integrity (Thompson, 2005).
Why should loss of epithelial organization render cells sensitive to competition? This effect could be viewed as an example of 'antagonistic pleiotropy', in which mutations that cause a potentially malignant effect also trigger a safety mechanism, such as sensitivity to apoptosis (the c-Myc oncogene being a well characterized example. Indeed, single genes whose mutation is sufficient to cause immediate and complete malignant transformation of cells may turn out to be very rare. Consequently, multiple mutations are necessary for tumorigenesis, and this analysis of vps25 provides an illustrative example (Thompson, 2005).
As far as apoptosis is concerned, the process disguises the oncogenic potential of the vps25 mutation, which is revealed upon blockade of apoptosis by a second genetic change. Apoptosis can be prevented either by alleviating competition from neighbors, or by expression of the caspase inhibitor, p35. In both cases, blockade of apoptosis allows the mutant clone to grow to form a large, amorphous mass. The complete lack of epithelial organization is clearly visible within these clones and contrasts with adjacent wild-type tissue, which overproliferates but remains epithelial. Thus, imaginal discs containing such clones are a complex mixture of mutant and wild-type cells that can grow to form a large, tumor-like mass that prevents the onset of proliferation arrest and differentiation at metamorphosis. Finally, when such discs are transplanted into an adult host, the mutant cells are capable of growth and metastasis to a second site within the host's body (Thompson, 2005).
The malignant transformations observed occur because the mutant clone possesses three key properties: it produces signals that drive cell proliferation; apoptosis is prevented; and cells lose their epithelial organization, progressing to metastasis. Note that, in imaginal discs, simply driving proliferation (for example, with activated Notch signaling) and blocking apoptosis causes tissue overgrowth, but epithelial architecture remains intact, and proliferation arrest and differentiation occur normally at metamorphosis. This work supports the view that, at least in Drosophila, loss of epithelial integrity is a critical event that synergizes with aberrant survival and proliferation to generate malignancies. The potential role of genes regulating epithelial organization in human cancer remains to be fully explored, but early results are interesting (Thompson, 2005).
The best-studied ESCRT component in mammals is Tsg101/Vps27. Drosophila Tsg101 has recently been found to have a loss-of-function phenotype similar to vps25 (Moberg, 2005). Tsg101 was first identified by antisense methods as having potential tumor suppressor activity in NIH 3T3 cells, but knockouts of Tsg101 were later shown to cause apoptosis in mouse cell lines and in mouse tissues. The consequence of blocking apoptosis in mouse cells mutant for Tsg101 has not been examined, but the results in Drosophila suggest that this may be a worthwhile line of inquiry, particularly in those tissues in which activated Notch signaling has been implicated in tumor formation (Thompson, 2005).
These findings underscore the importance of cooperative interactions between multiple genetic changes in a Drosophila model of tumorigenesis. In addition, they demonstrate that Drosophila Vps25 possesses several properties of a tumor suppressor. The basis for these properties lies in the requirement for Vps25 in ESCRT-mediated sorting of transmembrane proteins into MVBs -- a function that is conserved throughout the animal kingdom. These findings, therefore, raise the possibility that mutations in human ESCRT components could cooperate with other antiapoptotic mutations in cancer (Thompson, 2005).
In a mitotic recombination-based screen for genes that control epithelial organization, a mutation called A3 was identifed that causes disruption of cell shapes in both follicular and imaginal epithelia. Follicle cells homozygous for A3 lose their cuboidal morphology and pile upon each other, particularly at the poles of egg chambers. Similarly, in genetically mosaic imaginal discs, A3 cells are round and are arranged in multilayered masses of cells. These masses consist of mutant cells immediately adjacent to the pseudostratified columnar monolayer of surrounding wild-type epithelial cells, demonstrating that loss of epithelial character is strictly cell autonomous (Vaccari, 2005).
Proper epithelial organization requires polarization of the plasma membrane into apical and basolateral membrane domains separated by adherens junctions. Polarity in A3 mutant cells was assayed by staining with antibodies against the atypical protein kinase C (aPKC), Discs-large (Dlg), and E-cadherin (Ecad), which are markers, respectively, for the apical, lateral, and junctional domains. As opposed to its polarized and restricted localization in wild-type cells, aPKC in A3 cells displays a redistribution throughout the entire cell cortex. By contrast, levels of Dlg and Ecad are reduced when compared to the surrounding wild-type cells. A3 cells thus lose apicobasal polarity and show an expansion of the apical plasma membrane in particular (Vaccari, 2005).
Loss of apicobasal polarity in A3 cells is cell autonomous and is seen both in mosaic clones as well as when eye imaginal discs consisting predominantly of mutant cells are generated by using the eyFLP/cell lethal system. Strikingly, in the latter case, in which almost all of the wild-type cells are removed from the disc, enormous overproliferation of mutant tissue, in addition to epithelial defects, is seen. Analyses of staged larvae demonstrate that A3 mutant eye discs do not grow more rapidly than wild-type eye discs, but that they continue to proliferate during a larval phase that is extended for more than 3 days after wild-type animals have pupated. Larvae containing entirely mutant A3 eye discs are giant and eventually die; they show only initial signs of pupation. The epithelial disorganization and overproliferation of A3 mutant eye discs resemble that caused by mutations in the Drosophila neoplastic tumor suppressor genes scribble, discs-large, lethal giant larvae, and, in particular, avalanche (avl), whose elimination from eye discs causes a nearly identical phenotype (Lu, 2005). These phenotypes demonstrate that A3 mutates an unidentified Drosophila tumor suppressor gene (Vaccari, 2005).
Deficiency mapping was used to localize A3 to the 44D4-5 region on chromosome 2R. A3 fails to complement Df(2R)Exel8047, limiting the A3 region to seven candidate genes. Complementation tests within this region have established that A3 also fails to complement the lethal transposon insertion K08904, which is inserted in the coding sequence of the predicted gene CG14750. Sequencing of A3 homozygous genomic DNA identified a 27 base pair deletion in the second exon of CG14750, suggesting that the A3 epithelial phenotype is caused by loss of CG14750 function. To confirm this, K08904 was recombined onto an FRT chromosome, and it was found that the K08904 mosaic phenotype is indistinguishable from that of A3. Furthermore, provision of transgenic CG14750 from an inducible heat shock promoter restores wild-type morphology to A3 mosaic clones. These data establish that the defects in A3 cells arise from disruption of CG14750 (Vaccari, 2005).
Although the predicted CG14750 protein contains no obvious motifs, BLAST searches reveal that it is homologous throughout its 174 amino acid length to the yeast protein Vps25p. Vps25p is a component of the ESCRT-II (endosomal sorting complex required for transport-II) complex that mediates protein sorting within the endosomal pathway (Babst, 2002b). The crystal structure of Vps25p shows that it contains two 'winged-helix' (WHA/B) domains (Teo, 2004). The K08904 transposon insertion is predicted to cause termination at residue 32, in the midst of the WHA domain, while the A3 deletion removes residues 109-118 at the beginning of the WHB domain. WHA mediates associations with Vps22p and Vps36p to form a functional ESCRT-II complex, while WHB interacts with the ESCRT-III complex protein Vps20p (Hierro, 2004 and Teo, 2004). Since, in yeast, ESCRT-II regulates ESCRT-III formation (Babst, 2002b), both WHA and WHB domains are likely to be required for Vps25 function. Lethal phase analysis of Drosophila trans-heterozygous between A3, K08904, and Df(2R)Exel8047 reveals that all combinations die as sluggish L1 larvae, suggesting that both alleles confer null phenotypes (Vaccari, 2005).
Yeast Vps25p and other ESCRT proteins are required for sorting of ubiquitinated membrane proteins; in mutant yeast, ubiquitinated cargo is not transported to the vacuole. To test whether CG14750 plays a Vps25p-like role in Drosophila, A3 mosaic tissues were stained with an antibody that recognizes ubiquitinated proteins. In wild-type tissue, the anti-ubiquitin signal is present only at low levels. However, in A3 mutant tissue, the anti-ubiquitin signal accumulates to high levels in intracellular puncta that stain with endosomal markers. This phenotype is consistent with a failure to route ubiquitinated cargo for degradation, and provision of transgenic CG14750 restores ubiquitinated protein clearance to the mutant tissue. These results indicate that CG14750 is both structurally and functionally homologous to yeast Vps25p. CG14750 will therefore be referred to as Vps25 (Vaccari, 2005).
To study the impact of vps25 loss on the development of epithelial-derived organs in the adult, genetically mosaic eyes were examined in which mutant clones were produced alongside wild-type cells during larval stages. Cells containing wild-type chromosomes were marked with a pigment-producing transgene such that, following recombination, only vps25 homozygous cells will lack pigment. The eyes resulting from vps25 mosaic animals consist solely of pigmented cells, indicating that vps25 mutant tissue fails to contribute to the adult eye. Surprisingly, despite the absence of mutant tissue, these eyes are dramatically overgrown and bulging. This phenotype is reminiscent of that caused by mutations in certain Drosophila hyperplastic tumor suppressor genes such as hippo, salvador, and warts/lats. However, in these latter cases, it is the mutant tissue that shows extra growth, whereas, in vps25 mosaic eyes, it is genotypically wild-type tissue that is overgrown. By further contrast, in genetically mosaic eye disc clones other neoplastic tumor suppressor genes do not affect the proliferation of neighboring wild-type cells. Thus, vps25 mutants cause excess growth of adult tissue in a manner that is distinct from other previously described Drosophila tumor suppressor genes. Subsequent work has focused on understanding this difference (Vaccari, 2005).
The absence of mutant cells but overproliferation of wild-type tissue in vps25 mosaic animals suggests that the mutant cells are eliminated but stimulate increased cell proliferation in a nonautonomous fashion. This hypothesis was tested by analyzing vps25 mosaic eye imaginal discs with markers for differentiation, proliferation, and cell death. vps25 mutant cells do not form preommatidial clusters and fail to express the neuronal marker Elav, demonstrating that they do not undergo terminal differentiation. Moreover, caspases are activated in this tissue, indicating that mutant cells are undergoing apoptosis. Lack of terminal differentiation and induction of apoptosis can account for the absence of mutant tissue in the adult eye. Nevertheless, vps25 mosaic eye discs overall are significantly larger than wild-type eye discs. The increased size of the eye disc, like that of the adult eye, is rescued by provision of transgenic Vps25. To identify the source of increased disc size, BrdU incorporation assays were carried out to mark proliferating cells. These assays reveal a strong increase in DNA synthesis in many wild-type cells surrounding large mutant clones. This increase is evident over more than ten cell diameters, and it is seen only in cells anterior to the morphogenetic furrow. Tissue size and BrdU incorporation increases are also seen in wing discs, indicating that vps25 mutant tissue stimulates proliferation of surrounding wild-type cells in multiple epithelia (Vaccari, 2005).
The involvement of Vps25p in endocytosis in yeast suggested that proliferation defects in vps25 mosaic Drosophila tissue might arise from defects in trafficking of membrane proteins. To explore this possibility, vps25 mosaic eye discs were analyzed by using immunohistochemistry against the transmembrane receptor Notch, which is highly endocytic. In mutant tissue, alterations were found in subcellular localization of Notch. In wild-type cells, Notch is found at the apical surface as well as in internal puncta. By contrast, in vps25 cells, Notch is depleted from the cell surface, and there is a strong accumulation of Notch in intracellular puncta that are larger than their wild-type counterparts. This accumulation is seen with antibodies directed against both N- and C-terminal epitopes, indicating that both the extracellular and intracellular domains of Notch are trapped in puncta (Vaccari, 2005).
The intracellular accumulation of Notch in vps25 cells could, in theory, result from a defect in either exocytic or endocytic transport. To distinguish between these possibilities, a trafficking assay was performedby labeling cell surface Notch in live eye imaginal discs. In wild-type tissue at 4°C, a restrictive temperature for endocytosis, labeling is predominantly seen on the apical plasma membrane. A 1 hr shift to a permissive temperature results in the relocalization of most of the surface labeling into intracellular puncta. After 6 hr, no labeling is apparent, indicating that most endocytosed Notch has been degraded, due to lysosomal delivery. In vps25 mutant tissue, surface-labeled Notch is removed from the plasma membrane, but it progressively accumulates in large vesicles of endocytic origin resembling the puncta seen in fixed tissue samples. Moreover, vesicular accumulation of Notch in mutant cells persists at late time points when Notch has disappeared from surrounding wild-type cells. Wild-type tissue treated with chloroquine and other inhibitors of lysosomal degradation also display persistent Notch accumulation. These trafficking experiments establish that vps25 cells can internalize Notch, but that a block in subsequent trafficking causes persistent accumulation in an endocytic compartment rather than degradation (Vaccari, 2005).
To characterize the compartment in which Notch accumulates, mutant tissue was costained with markers for endocytic structures. In wild-type cells, most Notch puncta colocalize with late endosomal markers such as Rab7GFP. Only occasional colocalization with Avl, which encodes a syntaxin localized to early endosomes (Lu, 2005) and Hrs, which is required for MVB formation, is seen, suggesting that Notch transits through early endosomes and MVBs before taking up residence in the late endosome prior to degradation. In tissues mutant for hrs, which in yeast acts upstream of ESCRT-mediated sorting, Notch accumulates primarily in the early endosome, as shown by extensive colocalization with Avl and 2XFYVE-GFP. These Avl-positive early endosomes are enlarged in hrs tissue, but they retain a spherical morphology resembling that of wild-type endosomes. By contrast, in vps25 cells, Notch accumulates in an extensive intracellular compartment that displays highly irregular morphology. This compartment is enriched for both Avl and, particularly, for Hrs. Avl and Hrs show largely complementary localization within the vps25 mutant compartment, and only a fraction of Avl and Notch overlap, as in wild-type cells. However, by contrast, substantial amounts of Notch colocalize with Hrs in vps25 mutant tissue. Loss of vps25 thus alters the endocytic distribution of Notch as well as the morphology of the endosomal compartment (Vaccari, 2005).
The endosomal accumulation of Notch in vps25 cells is provocative, since Notch overexpression itself is capable of promoting nonautonomous tissue growth in imaginal discs. It was hypothesized that vps25 cells might contain ectopic Notch activity; this was tested by assaying the expression of E(spl)mβ-lacZ, which is transcribed in response to Notch signaling. In clones of vps25 mutant cells, higher levels of E(spl)mβ-lacZ expression were observed than in the neighboring wild-type cells. This increase does not occur outside of mutant cells, suggesting that it results from effects on the Notch receptor itself, rather than on ligands on neighboring cells. Similar results were obtained by assessing the expression of E(spl)m4-lacZ, another reporter of Notch activity. Interestingly, Notch reporters are not induced in cells mutant for avl and hrs despite the increased levels of Notch protein in these cells (Lu, 2005). Ectopic Notch activity thus correlates with the ability of each endocytic mutant to promote nonautonomous tissue overgrowth (Vaccari, 2005).
The observation that vps25 mutant tissue, like Notch-overexpressing tissue, can increase proliferation in surrounding cells over a distance suggests that vps25 mutant cells emit a mitogenic signal. A good candidate for such a signal is Unpaired (Upd), a secreted cytokine-like molecule that activates the JAK-STAT pathway in flies. In the second larval instar, Upd is transcribed at the posterior margin of the eye disc in response to Notch signaling; this localized production is thought to promote the proliferation of the entire eye disc. Upd can also be induced by ectopic Notch and can cause nonautonomous proliferation of eye tissue. In wild-type eye discs, Upd expression is largely lost by the third instar. However, in vps25 mutant clones in third instar discs, Upd is found at high levels in a cell-autonomous fashion. Induction of an Upd-lacZ transgene is seen in many vps25 mutant clones, suggesting that Upd expression is predominantly due to increased transcription. To assay whether Upd is indeed involved in the excess proliferation of vps25 mosaic eyes, the effects were tested of reducing levels of STAT92E, which transduces the extracellular Upd signal to the nucleus of receiving cells. Heterozygosity for STAT92E does not reduce the frequency of vps25 clones, nor the presence of Upd in these clones. However, it partially suppresses the tissue overgrowth of both vps25 mosaic imaginal discs as well as adult eyes, implicating the Upd-activated JAK-STAT pathway in the vps25 phenotype (Vaccari, 2005).
To test whether Notch is in fact responsible for the ectopic Upd expression observed in vps25 mutant clones, the levels of Notch in vps25 clones were reduced by means of a Notch RNA interference construct (UAS-N-IR). In vps25 mutant cells expressing UAS-N-IR, Notch does not accumulate intracellularly, and its level is reduced compared to surrounding wild-type cells. Moreover, in striking contrast to the ectopic Upd expression observed in clones of vps25 mutant cells, vps25 mutant cells expressing N-IR display no Upd accumulation. Taken together, the data indicate that excess Notch activity in vps25 cells induces Upd to increase proliferation in wild-type cells (Vaccari, 2005).
Reference names in red indicate recommended papers.
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date revised: 5 April 2006
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