The Drosophila Tumor Suppressor Vps25 Prevents Nonautonomous Overproliferation by Regulating Notch Trafficking

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Developmental Cell, Vol. 9, 687–698, November, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.devcel.2005.09.019 The Drosophila Tumor Suppressor vps25 Prevents Nonautonomous Overproliferation by Regulating Notch Trafficking

Thomas Vaccari and David Bilder* face, which will determine the cell’s sensitivity to ligand Department of Molecular and Cell Biology (Sorkin and Von Zastrow, 2002). University of California, Berkeley While the stimuli for internalization vary among re- Berkeley, California 94720 ceptors, ubiquitination of endocytosed receptors is an emerging common theme (Hicke and Dunn, 2003). Ubiquitination may play a role in promoting receptor internalization per se, as well as the eventual fate of the Summary internalized receptor. Internalized receptors generally traffic along one of two itineraries: either they are re- Cell-cell signaling coordinates proliferation of meta- turned to the cell surface via a recycling endosome, or zoan tissues during development, and its alteration they are transported to the lysosome for degradation. can induce malignant transformation. Endocytosis Ubiquitination often promotes the latter route. Many regulates signaling by controlling the levels and ac- ubiquitinated receptors reach a prelysosomal endocytic tivity of transmembrane receptors, both prior to and compartment called the multivesicular body (MVB) (Gruen- following ligand engagement. Here, we identify Vps25, berg and Stenmark, 2004; Katzmann et al., 2002). In- a component of the ESCRT machinery that regulates ward budding of the limiting membrane then carries endocytic sorting of signaling receptors, as an un- these receptors into internal vesicles of the MVB lumen, conventional type of Drosophila tumor suppressor. which permits the entire receptor to be degraded upon vps25 mutant cells undergo autonomous neoplastic- subsequent fusion with the lysosome. like transformation, but they also stimulate nonauton- The cellular machinery that sorts ubiquitinated pro- omous cell proliferation. Endocytic trafficking defects teins for lysosomal degradation has been identified in vps25 cells cause endosomal accumulation of the through yeast genetic screening. Mutations in a group signaling receptor Notch and enhanced Notch signal- of genes called “class E vps” (vesicular protein sorting) ing. Increased Notch activity leads to ectopic produc- genes give rise to an expanded endosomal compart- tion of the mitogenic JAK-STAT pathway ligand Un- ment that accumulates ubiquitinated transmembrane paired, which is secreted from mutant cells to induce proteins destined for degradation (Raymond et al., 1992). overproliferation of the surrounding epithelium. Our Eleven of the identified class E vps proteins form four data show that defects in endocytic sorting can both complexes, termed Hrs/Stam and ESCRT (endosomal transform cells and, through heterotypic signaling, al- sorting complex required for transport) -I, -II, and -III ter the behavior of neighboring wild-type tissue. (Babst et al., 2002a, 2002b; Bache et al., 2003; Katz- mann et al., 2001). Extensive biochemical and cell bio- Introduction logical experiments (Babst, 2005) have led to a model in which Hrs binds ubiquitinated cargo in the early en- Metazoan animals must coordinate the proliferation of dosome and then recruits the ESCRT-I complex from tissues to permit normal development of individual or- the cytoplasm. Endosomal recruitment brings ESCRT-I gans and the organism as a whole. Coordination of tis- in proximity to and activates ESCRT-II, which receives sue growth occurs through signal transduction net- the ubiquitinated cargo. ESCRT-II then induces the as- works that regulate the underlying cell cycle machinery. sembly of ESCRT-III, to which ESCRT-II passes its These signal transduction networks rely on surface re- cargo in kind. The net result of this process is to con- ceptors to communicate with neighboring cells. Proper centrate lysosome-destined cargo into a membrane regulation of this network is essential, since aberrant subdomain that will invaginate inward, isolating trans- cell-cell communication arising from alterations in re- membrane receptors from the general cellular cyto- ceptor number or function can lead to uncoordinated plasm. Since receptor signaling can occur from endo- proliferation and the formation of benign and malig- somal environments as well as from the cell surface nant tumors. (Gonzalez-Gaitan and Stenmark, 2003; Miaczynska et While the importance of spatiotemporally controlled al., 2004), sorting by ESCRT proteins may modulate the ligand and receptor synthesis in controlling cell-cell amplitude and kinetics of various signal transduction signaling has long been known, a recently appreciated pathways. mode of signal regulation involves endocytosis. Endo- Here, we show that the ESCRT-II complex member cytosis of ligand-bound, activated receptors can turn Vps25 acts as an unconventional type of Drosophila tu- off signaling by removing the receptor from the cell sur- mor suppressor. In addition to cell-autonomous loss of face, or it can promote signaling by bringing an acti- cell polarity and proliferation control, mutations in vated receptor in proximity to internally localized signal vps25 cause ectopic mitogenic signaling. In mutant tis- transduction components (Gonzalez-Gaitan, 2003; Le sue, Notch is trapped in an endosomal compartment, Roy and Wrana, 2005; Seto et al., 2002). In addition to and inappropriate Notch activation results. This ectopic these mechanisms, endocytosis can control the steady- Notch activity induces production of the secreted sig- state level of nonactivated receptors on the cell sur- nal Unpaired (Upd), which stimulates excess cell division in neighboring wild-type cells. A mutation in a sin- *Correspondence: [email protected] gle gene thus causes both persistent proliferation of Developmental Cell 688

Figure 1. The A3 Mutation Causes Cell-Autonomous Epithelial Disorganization and Overproliferation (A–C) Epithelial disorganization in A3 mutant tissue revealed by phalloidin staining (red). Follicle cells homozygous for A3 in a stage-7 egg chamber (GFP positive in [A]) are misshapen and multilayered, in contrast to cuboidal wild-type (WT) follicle cells. Wing disc cells homozygous for A3 (marked by the absence of GFP in [B] and [C]) show a similar cell-autonomous disruption of epithelial organization. (D–F) Polarity defects in A3 imaginal disc cells (marked by the absence of GFP). aPKC (blue in [D]) is exclusively apical in WT (GFP positive) cells, but it is distributed throughout the membrane in mutant cells, while Dlg (magenta in [E]) and Ecad (red in [F]) lose their WT junctional localization and show reduced levels. The arrowheads in (D)–(F) point to the (D) apical, (E) lateral, and (F) junctional staining observed in WT cells. (G) Size comparison of staged WT and A3 eye discs stained with phalloidin shows that A3 discs are initially smaller than WT, but that they continue to proliferate after wild-type discs have ceased growth prior to pupation. (H) Size comparison of resultant WT and A3 third instar larvae. The latter become giant during their extended larval life. The scale bars correspond to 10 ␮m in (A), (C), and (D)–(F), to 100 ␮m in (B) and (G), and to 1 mm in (H). mutant cells and also induction of overproliferation in respectively (Tepass et al., 2001). As opposed to its po- surrounding tissue. larized and restricted localization in wild-type cells, aPKC in A3 cells displays a redistribution throughout the entire cell cortex (Figure 1D). By contrast, levels of Results Dlg (Figure 1E) and Ecad (Figure 1F) are reduced when The A3 Mutation Causes Epithelial Disorganization compared to the surrounding wild-type cells. A3 cells and Cell-Autonomous Overproliferation thus lose apicobasal polarity and show an expansion In a mitotic recombination-based screen for genes that of the apical plasma membrane in particular. control epithelial organization, we identified a mutation Loss of apicobasal polarity in A3 cells is cell autono- called A3 that causes disruption of cell shapes in both mous and is seen both in mosaic clones as well as follicular and imaginal epithelia. Follicle cells homozy- when eye imaginal discs consisting predominantly of gous for A3 lose their cuboidal morphology and pile mutant cells are generated by using the eyFLP/cell le- upon each other, particularly at the poles of egg cham- thal system (Newsome et al., 2000). Strikingly, in the bers (Figure 1A). Similarly, in genetically mosaic imagi- latter case, in which almost all of the wild-type cells are nal discs, A3 cells are round and are arranged in multilay- removed from the disc, enormous overproliferation of ered masses of cells (Figure 1B). These masses consist of mutant tissue, in addition to epithelial defects, is seen. mutant cells immediately adjacent to the pseudostratified Analyses of staged larvae demonstrate that A3 mutant columnar monolayer of surrounding wild-type epithelial eye discs do not grow more rapidly than wild-type eye cells (Figure 1C), demonstrating that loss of epithelial discs, but that they continue to proliferate during a lar- character is strictly cell autonomous. val phase that is extended for more than 3 days after Proper epithelial organization requires polarization of wild-type animals have pupated (Figure 1G). Larvae the plasma membrane into apical and basolateral mem- containing entirely mutant A3 eye discs are giant and brane domains separated by adherens junctions. We eventually die; they show only initial signs of pupation assayed polarity in A3 mutant cells by staining with an- (Figure 1H). The epithelial disorganization and overpro- tibodies against the atypical protein kinase C (aPKC), liferation of A3 mutant eye discs resemble that caused Discs-large (Dlg), and E-cadherin (Ecad), which are by mutations in the Drosophila neoplastic tumor sup- markers for the apical, lateral, and junctional domains, pressor genes scribble, discs-large, lethal giant larvae Proliferation Control by Drosophila vps25 689

Figure 2. A3 Disrupts Drosophila vps25 (A) Diagram of the genomic region surrounding CG14750/vps25. (B) Drosophila vps25 coding sequence with locations of mutant alleles. (C) Vps25 protein. Homology to the Winged Helix A and B domains (WHA, WHB) of human Vps25 is indicated. The transposon insertion K08904 interrupts the coding region in WHA, while the EMS-induced mutation A3 deletes 9 amino acids in WHB. (D and E) (D) Disruption of epithelial organization in A3 mutant clones (lacking GFP, with phalloidin staining in red) of eye imaginal discs is restored by transgenic provision of (E) Vps25. (F and G) A3 clones also accumulate ubiquitinated proteins (magenta in [F] and [G]), but ubiquitin clearance is restored to mutant cells that express (G) Vps25. The scale bar corresponds to 10 ␮m.

(Bilder, 2004), and, in particular, avalanche (avl), whose complex that mediates protein sorting within the endo- elimination from eye discs causes a nearly identical somal pathway (Babst et al., 2002b). The crystal struc- phenotype (Lu and Bilder, 2005). These phenotypes ture of Vps25p shows that it contains two “winged- demonstrate that A3 mutates an unidentified Drosoph- helix” (WHA/B) domains (Teo et al., 2004). The K08904 ila tumor suppressor gene. transposon insertion is predicted to cause termination at residue 32, in the midst of the WHA domain, while The A3 Mutation Disrupts Drosophila vps25 the A3 deletion removes residues 109–118 at the begin- We used deficiency mapping to localize A3 to the ning of the WHB domain (Figure 2C). WHA mediates 44D4-5 region on chromosome 2R. A3 fails to comple- associations with Vps22p and Vps36p to form a func- ment Df(2R)Exel8047, limiting the A3 region to seven tional ESCRT-II complex, while WHB interacts with the candidate genes (Figure 2A). Complementation tests ESCRT-III complex protein Vps20p (Hierro et al., 2004; within this region established that A3 also fails to com- Teo et al., 2004). Since, in yeast, ESCRT-II regulates plement the lethal transposon insertion K08904 (Bellen ESCRT-III formation (Babst et al., 2002b), both WHA et al., 2004), which is inserted in the coding sequence and WHB domains are likely to be required for Vps25 of the predicted gene CG14750 (Figure 2B). Sequencing function. Lethal phase analysis of Drosophila trans-het- of A3 homozygous genomic DNA identified a 27 base erozygous between A3, K08904, and Df(2R)Exel8047 pair deletion in the second exon of CG14750 (Figure reveals that all combinations die as sluggish L1 larvae, 2B), suggesting that the A3 epithelial phenotype is suggesting that both alleles confer null phenotypes. caused by loss of CG14750 function. To confirm this, Yeast Vps25p and other ESCRT proteins are required we recombined K08904 onto an FRT chromosome and for sorting of ubiquitinated membrane proteins; in mu- found that the K08904 mosaic phenotype is indistinguish- tant yeast, ubiquitinated cargo is not transported to the able from that of A3 (data not shown). Furthermore, provi- vacuole (Katzmann et al., 2002). To test whether CG14750 sion of transgenic CG14750 from an inducible heat shock plays a Vps25p-like role in Drosophila, we stained A3 promoter restores wild-type morphology to A3 mosaic mosaic tissue with an antibody that recognizes ubiq- clones (Figures 2D and 2E). These data establish that the uitinated proteins. In wild-type tissue, the anti-ubiquitin defects in A3 cells arise from disruption of CG14750. signal is present only at low levels (Jekely and Rorth, Although the predicted CG14750 protein contains no 2003). However, in A3 mutant tissue, the anti-ubiquitin obvious motifs, BLAST searches reveal that it is homol- signal accumulates to high levels in intracellular puncta ogous throughout its 174 amino acid length to the yeast that stain with endosomal markers (Figure 2F; data not protein Vps25p. Vps25p is a component of the ESCRT- shown). This phenotype is consistent with a failure to II (endosomal sorting complex required for transport-II) route ubiquitinated cargo for degradation, and provi- Developmental Cell 690

sion of transgenic CG14750 restores ubiquitinated protein clearance to the mutant tissue (Figure 2G). These results indicate that CG14750 is both structurally and functionally homologous to yeast Vps25p. We will therefore refer to CG14750 as Vps25.

Nonautonomous Induction of Proliferation by vps25 Mutant Tissue To study the impact of vps25 loss on the development of epithelial-derived organs in the adult, we examined genetically mosaic eyes 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 (Tapon et al., 2001). The eyes resulting from vps25 mosaic animals consist solely of pigmented cells, indicating that vps25 mutant tissue fails to contribute to the adult eye (Figures 3A and 3C). Surpris- ingly, despite the absence of mutant tissue, these eyes are dramatically overgrown and bulging (Figures 3B and 3D). This phenotype is reminiscent of that caused by mutations in certain Drosophila hyperplastic tumor suppressor genes such as hippo, salvador, and warts/ lats (Jia et al., 2003; Tapon et al., 2002; Udan et al., 2003; Wu et al., 2003; Xu et al., 1995). 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 (Brumby and Figure 3. Nonautonomous Induction of Proliferation by vps25 Mu- tant Tissue Richardson, 2003). Thus, vps25 mutants cause excess growth of adult tissue in a manner that is distinct from (A–D) Adult eyes deriving from larvae genetically mosaic for a WT or vps25 mutant chromosome. Homozygous WT or vps25 cells are other previously described Drosophila tumor suppres- marked by the absence of red pigment in bright field images, dem- sor genes. We focused subsequent work on under- onstrating the absence of vps25 cells in mosaic eyes ([C], compare standing this difference. WT in [A]). Scanning electron micrographs show the tissue over- The absence of mutant cells but overproliferation of growth of genotypically WT tissue in vps25 mosaics ([D], compare wild-type tissue in vps25 mosaic animals suggests that WT in [B]). $ the mutant cells are eliminated but stimulate increased (E–I ) Histochemistry of mosaic eye imaginal discs; homozygous cells are marked by the absence of GFP. (E and F) Both WT and cell proliferation in a nonautonomous fashion. We vps25 mutant cells are present in mosaic discs; note the tissue tested this hypothesis by analyzing vps25 mosaic eye overgrowth of vps25 discs. The differentiation marker Elav (red) is imaginal discs with markers for differentiation, prolifer- lost in (G) vps25 cells, and (H) apoptotic caspases (blue) are acti- ation, and cell death. vps25 mutant cells do not form vated cell autonomously. BrdU incorporation (red in [I]) reveals non- preommatidial clusters and fail to express the neuronal autonomous elevation of mitotic activity in genotypically WT cells # $ marker Elav, demonstrating that they do not undergo (arrowhead in [I ]) bordering vps25 mutant clones (outlined in [I ]). The scale bars correspond to 100 ␮m in (A)–(F) and to 10 ␮min terminal differentiation (Figure 3G). Moreover, caspases (G)–(I). are activated in this tissue, indicating that mutant cells are undergoing apoptosis (Figure 3H). Lack of terminal differentiation and induction of apoptosis can account seen in wing discs (Figure 1B and data not shown), indi- for the absence of mutant tissue in the adult eye. Nev- cating that vps25 mutant tissue stimulates proliferation ertheless, vps25 mosaic eye discs overall are signifi- of surrounding wild-type cells in multiple epithelia. cantly larger than wild-type eye discs (Figures 3E and 3F). The increased size of the eye disc, like that of the Trafficking Defects Lead to Endosomal Notch adult eye, is rescued by provision of transgenic Vps25 Accumulation in vps25 Cells (data not shown). To identify the source of increased The involvement of Vps25p in endocytosis in yeast sug- disc size, we carried out BrdU incorporation assays to gested that proliferation defects in vps25 mosaic Dro- mark proliferating cells. These assays reveal a strong sophila tissue might arise from defects in trafficking of increase in DNA synthesis in many wild-type cells sur- membrane proteins. To explore this possibility, we ana- rounding large mutant clones (Figure 3I). This increase lyzed vps25 mosaic eye discs by using immunohisto- is evident over more than ten cell diameters, and it is chemistry against the transmembrane receptor Notch, seen only in cells anterior to the morphogenetic furrow. which is highly endocytic. In mutant tissue, we found Tissue size and BrdU incorporation increases are also alterations in subcellular localization of Notch (Figure Proliferation Control by Drosophila vps25 691

4A). In wild-type cells, Notch is found at the apical surface as well as in internal puncta (Figure 4B). By contrast, in vps25 cells, Notch is depleted from the cell surface, and there is a strong accumulation of Notch in intracellular puncta (Figure 4C) that are larger than their wild-type counterparts. This accumulation is seen with antibodies directed against both N- and C-terminal epi- topes (Figure 4D), indicating that both the extracellular and intracellular domains of Notch are trapped in puncta. 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, we performed a trafficking assay by labeling cell surface Notch in live eye imaginal discs (see Experimental Procedures). In wild-type tissue at 4°C, a restrictive temperature for endocytosis, labeling is predominantly seen on the apical plasma membrane (Fig- ure 4E).A1hrshift to a permissive temperature results in the relocalization of most of the surface labeling into intracellular puncta (Figure 4F). After 6 hr, no labeling is apparent, indicating that most endocytosed Notch has been degraded, due to lysosomal delivery (Figure 4G). 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 (Fig- ures 4H–4J). Moreover, vesicular accumulation of Notch in mutant cells persists at late time points when Notch has disappeared from surrounding wild-type cells (Fig- ures 4G and 4J). Wild-type tissue treated with chloroquine and other inhibitors of lysosomal degradation also displays persistent Notch accumulation (Figures 4K–4M and data not shown). 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.

Notch Accumulates with Ubiquitinated Proteins in a Malformed Early Endocytic Compartment To characterize the compartment in which Notch accumulates, we costained mutant tissue with markers for endocytic structures. In wild-type cells, most Notch puncta colocalize with late endosomal markers such as Figure 4. Trafficking Defects Lead to Endosomal Notch Accumula- Rab7GFP (Hori et al., 2004; Weber et al., 2003; Wilkin tion in vps25 Cells et al., 2004). Only occasional colocalization with Avl, (A–D) vps25 mosaic eye imaginal discs stained with anti-Notch which encodes a syntaxin localized to early endosomes (A–C) ECD or (D) ICD in blue; GFP marks WT tissue, and red phalloi- (Lu and Bilder, 2005) and Hrs, which is required for MVB din reveals the cell cortex. (A) Aberrant Notch localization is seen formation (Lloyd et al., 2002), is seen (Figures 5A and in vps25 mutant clones. (B) In contrast to WT, (C) Notch is depleted 5D), suggesting that Notch transits through early endo- from the apical surface and accumulates in intracellular puncta. (D) somes and MVBs before taking up residence in the late Intracellular accumulation of the Notch ICD epitope is seen as well. endosome prior to degradation. In tissues mutant for Arrowheads in (B) and (D) point to the apical staining observed in WT cells. hrs, which in yeast acts upstream of ESCRT-mediated (E–M) (E–G) Endocytosis assay with anti-Notch ECD to label Notch sorting (Katzmann et al., 2003), Notch accumulates pri- at the surface of live imaginal discs. In WT tissue, surface bound marily in the early endosome, as shown by extensive anti-Notch ECD is internalized into endosomes after 1 hr and is colocalization with Avl (Figure 5C) and 2XFYVE-GFP degraded after 6 hr. (H–J) In vps25 tissue, anti-Notch ECD is inter- (Jekely and Rorth, 2003). These Avl-positive early endo- nalized but is not degraded, persisting 6 hr after labeling. (K–M) A ␮ somes are enlarged in hrs tissue, but they retain a similar phenotype is observed in WT tissue treated with 200 M chloroquine to inhibit lysosomal degradation. spherical morphology resembling that of wild-type en- The scale bars correspond to 100 ␮m in (A) and to 10 ␮m in (B)–(M). dosomes. By contrast, in vps25 cells, Notch accumulates in an extensive intracellular compartment that displays highly irregular morphology. This compartment is Developmental Cell 692

activity, and we tested this hypothesis by assaying the expression of E(spl)mb-lacZ, which is transcribed in response to Notch signaling (Nellesen et al., 1999). In clones of vps25 mutant cells, we observed higher levels of E(spl)mb-lacZ expression than in the neighboring wild-type cells (Figures 6A and 6B). 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 (Bailey and Posakony, 1995) (data not shown). Interestingly, Notch reporters are not induced in cells mutant for avl and hrs despite the increased levels of Notch protein in these cells (Lu and Bilder, 2005). Ectopic Notch activity thus correlates with the ability of each endocytic mutant to promote nonautonomous tissue overgrowth. 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 (Harrison et al., 1998). 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 (Chao et al., 2004). Upd can also be induced by ectopic Notch and can cause nonautonomous proliferation of eye tissue (Bach et al., 2003; Tsai and Sun, 2004). In wild-type eye discs, Upd expression Figure 5. Notch Accumulates in an Aberrant Early Endocytic Com- is largely lost by the third instar (Figure 6F). However, partment in vps25 mutant clones in third instar discs, Upd is (A–C$)WTandvps25 mutant eye disc tissue stained for Notch (red) found at high levels in a cell-autonomous fashion (Fig- and Avl (blue). (A) In WT cells, Notch is distinct from Avl-positive ures 6C and 6G). Induction of an Upd-lacZ transgene early endosomes. (B) In vps25 cells, endosomal structures are en- (Sun et al., 1995) is seen in many vps25 mutant clones, larged, irregular, and accumulate excess Notch, but Notch and Avl suggesting that Upd expression is predominantly due remain largely separate. (C) hrs mutant cells also accumulate ex- to increased transcription (Figure 6D). To assay whether cess Notch, but Notch shows a high degree of colocalization with Upd was indeed involved in the excess proliferation of Avl, and the endosomal signal remains regular. (D and E$) WT and mutant eye disc tissue stained for Notch (red) vps25 mosaic eyes, we tested the effects of reducing and Hrs (green). (D) In WT cells, Notch and Hrs puncta often abut, levels of STAT92E, which transduces the extracellular and colocalization is uncommon. (E) In vps25 cells, Notch overlaps Upd signal to the nucleus of receiving cells (Hou et al., strongly with Hrs in the irregular endosomal compartment. 1996). Heterozygosity for STAT92E does not reduce the T scale bar corresponds to 10 ␮m. frequency of vps25 clones, nor the presence of Upd in these clones (Figures 6E and 6H). However, it partially suppresses the tissue overgrowth of both vps25 mo- enriched for both Avl and, particularly, for Hrs (Figures saic imaginal discs as well as adult eyes (Figures 6H 5B and 5E). Avl and Hrs show largely complementary and 6K), implicating the Upd-activated JAK-STAT path- localization within the vps25 mutant compartment, and way in the vps25 phenotype. only a fraction of Avl and Notch overlap, as in wild- To test whether Notch is in fact responsible for the type cells (Figures 5A and 5B). However, by contrast, ectopic Upd expression observed in vps25 mutant substantial amounts of Notch colocalize with Hrs in clones, we reduced the levels of Notch in vps25 clones vps25 mutant tissue (Figure 5E). Loss of vps25 thus al- by means of a Notch RNA interference construct (UAS- ters the endocytic distribution of Notch as well as the N-IR)(Presente et al., 2002). In vps25 mutant cells morphology of the endosomal compartment. expressing UAS-N-IR, Notch does not accumulate in- tracellularly, and its level is reduced compared to sur- Notch-Induced Upd Promotes Nonautonomous rounding wild-type cells (Figure 6M). Moreover, in striking Overproliferation contrast to the ectopic Upd expression observed in The endosomal accumulation of Notch in vps25 cells is clones of vps25 mutant cells (Figure 6L#), vps25 mutant provocative, since Notch overexpression itself is capa- cells expressing N-IR display no Upd accumulation ble of promoting nonautonomous tissue growth in imagi- (Figure 6M#). Taken together, the data indicate that ex- nal discs (Artavanis-Tsakonas et al., 1999). We hypothe- cess Notch activity in vps25 cells induces Upd to in- sized that vps25 cells might contain ectopic Notch crease proliferation in wild-type cells. Proliferation Control by Drosophila vps25 693

Figure 6. Notch-Induced Upd Promotes Non- autonomous Overproliferation (A–K) Upregulation of Notch targets in vps25 cells (marked by the absence of GFP). The mb-lacZ reporter (blue in [B]), Upd (red in [C], [E], and [F]–[H]), and upd-lacZ (magenta in [D]) are cell-autonomously increased in vps25 tissue as compared to WT tissue. (F–K) Heterozygosity for STAT92E does not reduce the production of Upd within vps25 mutant cells (mutant clones are outlined in [G] and [H]), but it does suppress the nonautonomous tissue overgrowth of adult eyes ([K], compare with [J]). (L–M#) Reducing Notch levels by RNAi in vps25 mutant cells prevents ectopic Upd accumulation. vps25 mutant cells (outlined in [L]) and vps25 mutant cells expressing Notch-IR (outlined in [M]) are stained for Notch and Upd. Note the absence of Notch and Upd expression in vps25 mutant cells expressing Notch-IR. The scale bars correspond to 100 ␮m in (A), (B), and (F)–(K) and to 10 ␮m in (C)–(E) and (L) and (M).

Discussion teractions observed during malignant transformation of mammalian tissues. Genetic screens in Drosophila have lent insight into the molecules and mechanisms that control tissue growth ESCRT Mutant Phenotype in a Metazoan and cell proliferation and therefore prevent tumor for- Class E vps proteins have been studied in cultured ver- mation. Here, we show that the ESCRT-II complex pro- tebrate cells (Babst, 2005), but the early lethality of mu- tein Vps25 is a member of an unconventional class of tant mice and cell cycle arrest seen in tissue-specific Drosophila tumor suppressors, whose loss causes both inactivation have hampered functional analyses in mam- cell-autonomous overproliferation and nonautonomous mals (Ruland et al., 2001; Wagner et al., 2003). Here, overproliferation of surrounding tissue. we describe the null phenotype of an ESCRT complex A model for tissue transformation in vps25 mosaic member in a metazoan tissue. As in yeast and mam- epithelia is shown in Figure 7. In wild-type epithelial mals, loss of ESCRT-II function in flies causes accumu- cells, Notch is endocytosed and degraded via MVB lation of ubiquitinated proteins in an enlarged endoso- sorting in endosomes (Figure 7A). In vps25 mutant mal structure, indicating that the cell biological role of cells, Notch is endocytosed but fails to be degraded ESCRT-II is conserved across phylogeny. Trapping of due to impaired MVB sorting; thus, it accumulates in Notch in an early endosomal compartment in hrs mu- enlarged endosomes. vps25 mutant cells also fail to tants, and in an Hrs-positive compartment in vps25 mu- polarize, to exit the cell cycle and to differentiate; they tants, is also consistent with the ordering of class E are later eliminated by apoptosis (Figure 7B). Due to protein functions in yeast. ESCRT-II physically interacts ectopic Notch activation, vps25 mutant cells produce with both ESCRT-I and ESCRT-III, and identical pheno- and secrete Upd. Via the JAK-STAT pathway, the ec- types on endosomal organization and sorting are seen topic Upd promotes extra growth of the neighboring in yeast mutant for any ESCRT complex member. Thus, wild-type epithelium (Figure 7C). This heterotypic sig- the phenotype described here probably represents that naling process echoes aspects of the tumor-host in- of general ESCRT absence in flies. Developmental Cell 694

gle hrs allele (Lloyd et al., 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.

Autonomous and Nonautonomous Promotion of Cell Division in vps25 Mutants 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 (Bilder, 2004; Lu and Bilder, 2005). 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 (Lu and Bilder, 2005). However, they have quite different affects on Notch localization and Notch pathway activity. 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.B., unpublished data). While we have not directly tested a requirement for Notch in the cell- autonomous overproliferation of vps25 mutant tissue, the avl phenotype suggests that altered activities of other mistrafficked membrane proteins, perhaps including the apical membrane determinant Crumbs (Lu and Bilder, 2005; Tepass et al., 1990), are responsible for the persistent proliferation of mutant cells. By contrast, the nonautonomous tissue growth induced by vps25, but not avl, cells is spurred by Notch- Figure 7. A Model of Tissue Transformation in vps25 Mosaic Epi- mediated production of the extracellular ligand Upd. thelia Overexpression of Upd, either alone or in response to (A) WT cells internalize Notch and degrade it via endosomal MVB ectopic Notch expression, can cause hyperproliferation sorting. of cells anterior to the morphogenetic furrow resem- (B) Due to impaired MVB sorting, vps25 mutant cells fail to degrade bling the phenotype of vps25 mosaic clones. Moreover, Notch, which accumulates in enlarged endosomes. vps25 mutant cells also lose epithelial polarity and fail to arrest proliferation. Upd overexpression phenotypes, like vps25 mutant (C) Notch activation in vps25 mutant cells triggers ectopic Upd ex- phenotypes, are suppressed by STAT92E heterozygos- pression, which, in turn, promotes overproliferation of the sur- ity (Bach et al., 2003; Chao et al., 2004). Importantly, rounding WT cells via the JAK-STAT pathway. 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 appro- We were surprised to find strong differences between priate cellular response in an inappropriate develop- the Drosophila mutant phenotypes of vps25 and hrs. mental context that leads to tumorous tissue growth. In yeast, deletion of the hrs homolog vps27 causes an In mammalian immune cells, ectopic Notch pathway endosomal and cargo accumulation phenotype that is activity can lead to lymphomas (Weijzen et al., 2002; indistinguishable from deletion of ESCRT complex mem- Zweidler-McKay and Pear, 2004), while, during normal bers (Piper et al., 1995). However, the immunoreactivity development, Notch activation induces production of to Notch, ubiquitin, and the early endosomal marker interleukin-4 and related molecules (Amsen et al., 2004; Avl, which is largely regular in hrs cells (Jekely and Maillard et al., 2005), which, like Upd, signal through Rorth, 2003), is highly irregular in vps25 cells. Loss of the JAK-STAT pathway. Intriguingly, a recent report de- vps25, which acts downstream of hrs in the endosomal scribes a critical role for Interleukin-8 expression in sorting pathway in yeast (Babst et al., 2002b) thus neovascularization of Ras-transformed human cells causes a more severe disruption in endosomal organi- (Sparmann and Bar-Sagi, 2004). While a number of zation than hrs. It remains possible that other Drosoph- cytokines are amongst the secreted factors that can ila gene products can partially substitute for Hrs or that be produced in tumors (Coussens and Werb, 2002), we the N-terminally truncated protein produced by the sin- know of no examples in which mammalian Upd or- Proliferation Control by Drosophila vps25 695

thologs specifically have been shown to modify the tu- which differs between avl, hrs, and vps25 mutant cells. mor environment of Notch-induced malignancies, for Possible mechanisms for inappropriate activation in- instance by promoting angiogenesis or recruiting stroma. clude coaccumulation of Notch and its ligands, pro- Nevertheless, we speculate that heterotypic signaling longed exposure to γ-secretase, or eventual dissoci- by Upd-like factors might alter the proliferation rates of ation of the heterodimer in the endosomal environment. untransformed cells relative to tumor cells, ultimately These possibilities are not mutually exclusive, and the favoring tumor expansion. Further investigations will be altered organization of the vps25 endosome in addition required to establish whether the vps25 mutant pheno- to the absence of flux through the compartment is likely type represents a novel inductive mechanism relevant to contribute. Future studies will discriminate among to mammalian tumor-host interactions. these possible mechanisms of ectopic Notch activation. Endosomal Sorting and Notch Activation Notch and its ligands are distributed widely through- Complexity, Endocytosis, and Cancer out development, yet Notch activity is highly localized While we have concentrated on the Notch pathway in to specific times and places. Many posttranslational this work, it is clear that many molecules are trapped mechanisms are involved in restricting Notch activity, in vps25 endosomes and that vps25 mutations are phe- including ligand presentation, modification of the re- notypically pleiotropic due to alterations in a number of ceptor by sugars, and proteolytic processing to create signaling pathways. For instance, STAT92E suppression active receptor forms (Schweisguth, 2004). Each of of vps25 phenotypes is less complete than STAT92E sup- these processes both provides a potential point of reg- pression of overproliferation mediated by Upd alone ulation and ensures that inappropriate activation does (Bach et al., 2003), suggesting that additional factors not occur, which is critical due to the potent effects contribute to vps25-induced tissue overgrowth. One of Notch-mediated signals. The vps25 phenotype de- candidate that merits exploration is the MAPK signaling scribed here highlights another mechanism that pre- cascade, since vps25 mutants enhance gain-of-func- vents inappropriate activation: endocytic sorting of tion alleles of EGFR (Elp) and MAPK (Sem) (data not receptor that is being cleared from the cell surface. In shown). The latter evidence is consistent with the per- wild-type cells, Notch is continuously internalized and sistent MAPK signaling described in several class E degraded in the lysosome to maintain steady-state mutant tissues, including hrs in flies and TSG101 in levels of surface expression and therefore receptor mammals (Babst et al., 2000; Lloyd et al., 2002). availability. For this process to accurately control sig- Thus, the complexity of the vps25 mutant phenotype naling, the cell must ensure that the unliganded Notch emphasizes that endosomal sorting is a point of con- is not activated during internalization (Kanwar and For- tact between diverse signaling pathways, and a likely tini, 2004). Our results indicate that prevention of Notch regulatory nexus for normal development and for path- activation requires MVB sorting, and they suggest that ology. Since human tumors benefit from the coordi- rapid transit through the endosomal environment is re- nated disruption of multiple signaling pathways, sub- quired to prevent this inappropriate activation. version of endosomal sorting may be one susceptible How could the endosomal accumulation of Notch in route toward malignant transformation. Increasing evi- vps25 cells lead to ectopic Notch signaling? The ter- dence implicates defects in trafficking of specific re- minal proteolytic cleavage in Notch activation is medi- ceptors in the ontogeny of mammalian tumors (Bache ated by the membrane bound γ-secretase activity pro- et al., 2004; Polo et al., 2004). Moreover, the ESCRT-I vided by Presenillin and its associated proteins (Fortini, complex member TSG101 was originally isolated for a 2002). The site of γ-secretase activity is controversial, tumor suppressive function in cultured cells, although with some evidence pointing toward the cell surface such a role in vivo has not been established (Li and and other evidence pointing to an endosome (Paster- Cohen, 1996). The accessibility of Drosophila tissues, nak et al., 2004). Interestingly, ubiquitination of Notch along with the availability of mutations that block spe- has recently been linked to both its internalization and cific steps of endocytic traffic, will help to elucidate its activation. A partially processed form of mammalian how endocytosis affects metazoan signaling and the Notch requires ubiquitination for efficient γ-secretase consequent effects on cell proliferation during develop- cleavage and activation (Gupta-Rossi et al., 2004), ment as well as tumorigenesis. while several Drosophila ubiquitin ligases seem to influ- ence an endosomal sorting decision specifying degra- Experimental Procedures dation rather than activation of unliganded Notch (Hori et al., 2004; Sakata et al., 2004). In this regard, it is nota- Drosophila Genetics A3 ble that although hrs and vps25 mutant cells both The FRT42D chromosome was used to generate the vps25 mutation and to recombine the P element K08904; animals referred to contain elevated levels of ubiquitinated proteins, only in the text are the null allele vps25A3. Other alleles used include vps25 mutants show ectopic Notch signaling. The dif- avl1,(Lu and Bilder, 2005), hrsD28 (Lloyd et al., 2002), and ferences in Notch signaling activity could in theory arise PZ(STAT92E)(Hou et al., 1996). Notch target experiments used from differences in the amount of Notch trapped in the E(spl)mb-LacZ (Nellesen et al., 1999) and Upd-LacZ (Sun et al., different endocytic mutants. However, very high amounts 1995). Mutant follicle cell clones were generated by using the heat of Notch are present in avl mutant cells, which do not shock-MARCM system (Lee and Luo, 2001), and mutant wing disc clones were generated by using Ubxflp (Hong et al., 2003). Mosaic show ectopic Notch signaling (Lu and Bilder, 2005). eyes and eye imaginal discs were generated by using eyFLP as Therefore, we favor the possibility that ectopic Notch described in Tapon et al. (2001), while completely mutant eye discs activity may be due to the locus of endocytic trapping, (referred to in the text as vps25 or A3 eye discs) were generated Developmental Cell 696

by using eyFLP and a recessive cell lethal chromosome as de- by different notch ligands on antigen-presenting cells. Cell 117, scribed in Newsome et al. (2000). 515–526. Artavanis-Tsakonas, S., Rand, M.D., and Lake, R.J. (1999). Notch Molecular Biology signaling: cell fate control and signal integration in development. A3 The mutation in vps25 alleles was identified by sequencing PCR Science 284, 770–776. products amplified from genomic DNA; at least two independent reactions were sequenced for each allele. cDNA encoding the Babst, M. (2005). A protein’s final ESCRT. Traffic 6, 2–9. Vps25 coding sequence was cloned into pCasperhs for transgenic Babst, M., Odorizzi, G., Estepa, E.J., and Emr, S.D. (2000). Mamma- rescue experiments. Primer sequences are available upon request. lian tumor susceptibility gene 101 (TSG101) and the yeast homo- logue, Vps23p, both function in late endosomal trafficking. Traffic Immunostainings and Microscopy 1, 248–258. For immunostainings and adult eye pictures, genotypes were as Babst, M., Katzmann, D.J., Estepa-Sabal, E.J., Meerloo, T., and A3 follows: FRT42D eyFLP/+; FRT42D vps25 /FRT42D P (mini-w, ubi- Emr, S.D. (2002a). Escrt-III: an endosome-associated heterooligo- A3 GFP) ubxFLP/+; FRT42D vps25 /FRT42D P(mini-w, ubi-GFP) hsFLP, meric protein complex required for mvb sorting. Dev. Cell 3, 271– A3 P (UAS CD8-GFP)/+; FRT42D vps25 /FRT42D P(Tubulin-GAL80); 282. P(Tubulin-GAL4)/+ eyFLP/+; FRT42D vps25A3/FRT42D P (mini-w, Babst, M., Katzmann, D.J., Snyder, W.B., Wendland, B., and Emr, arm-lac-Z) FRT42D vps25A3/FRT42D P(mini-w, cl); eyGAL4, P(UAS- FLP)/+ eyFLP/+; FRT42D vps25A3/FRT42D P (mini-w, ubi-GFP); S.D. (2002b). Endosome-associated complex, ESCRT-II, recruits P(hsVps25)/+ eyFLP/+; FRT42D vps25A3/FRT42D P (mini-w, ubi- transport machinery for protein sorting at the multivesicular body. GFP); P(M4-lac-Z)/+ eyFLP/+; FRT42D vps25A3/FRT42D P (mini-w, Dev. Cell 3, 283–289. ubi-GFP); P(upd-lac-Z)/+ eyFLP/+; FRT40 hrsD22/FRT40 P (mini-w, Bach, E.A., Vincent, S., Zeidler, M.P., and Perrimon, N. (2003). A ubi-GFP) eyFLP/+; FRT80 avl1/FRT80 M(3))/+ eyFLP/+; FRT42D sensitized genetic screen to identify novel regulators and compo- vps25A3/FRT42D P (mini-w, ubi-GFP); PZ(STAT92E)/+ hsFLP, P nents of the Drosophila janus kinase/signal transducer and activa- (UAS CD8-GFP)/ P (mini-w, UAST-Ni); FRT42D vps25A3/FRT42D tor of transcription pathway. Genetics 165, 1149–1166. P(Tubulin-GAL80); P(Tubulin-GAL4)/+. Bache, K.G., Raiborg, C., Mehlum, A., and Stenmark, H. (2003). Ovaries and imaginal disc tissues were fixed and stained as de- STAM and Hrs are subunits of a multivalent ubiquitin-binding com- scribed (Tanentzapf et al., 2000; Zeitler et al., 2004) with rhodamine- plex on early endosomes. J. Biol. Chem. 278, 12513–12521. phalloidin (Molecular Probes) and primary antibodies against the following antigens: Avl (Lu and Bilder, 2005); aPKC (Santa Cruz Bio- Bache, K.G., Slagsvold, T., and Stenmark, H. (2004). Defective technology); Hrs (Lloyd et al., 2002); Upd (Harrison et al., 1998); downregulation of receptor tyrosine kinases in cancer. EMBO J. 23, Notch ECD, Notch ICD, Ecad, Elav, Dlg (all from Developmental 2707–2712. Studies Hybridoma Bank); Ubiquitin FK2 (Biomol); activated Cas- Bailey, A.M., and Posakony, J.W. (1995). Suppressor of hairless di- pase-3 (Signal Transduction Technologies); and BrdU (BD Biosci- rectly activates transcription of enhancer of split complex genes in ences). Secondary antibodies were from Molecular Probes. All response to Notch receptor activity. Genes Dev. 9, 2609–2622. images are single confocal sections taken with a TCS microscope Bellen, H.J., Levis, R.W., Liao, G., He, Y., Carlson, J.W., Tsang, G., (Leica) by using 16×/NA 0.5 or 63×/NA 1.4 oil lenses. Scanning Evans-Holm, M., Hiesinger, P.R., Schulze, K.L., Rubin, G.M., et al. electron micrographs of adult eyes prepared by following standard (2004). The BDGP gene disruption project: single transposon inser- protocols (Kimmel et al., 1990) were taken with an XL-30 ESEM tions associated with 40% of Drosophila genes. Genetics 167, microscope (Phillips). Images were edited with Adobe Photoshop 761–781. 7.0 and were assembled with Adobe Illustrator 10. Bilder, D. (2004). Epithelial polarity and growth control: links from Staging Experiments, BrdU Incorporation, the Drosophila neoplastic tumor suppressors. Genes Dev. 18, and Trafficking Assay 1909–1925. Wild-type and eyflp vps25/cl larvae were staged at L2 as described Brumby, A.M., and Richardson, H.E. (2003). scribble mutants coop- in Sullivan and Thummel (2003). BrdU was incorporated in imaginal erate with oncogenic Ras or Notch to cause neoplastic overgrowth discs by using a 90 min pulse to reveal ectopic S phases. For Notch in Drosophila. EMBO J. 22, 5769–5779. A3 endocytosis assays, vps25 mosaic eye discs were dissected and Chao, J.L., Tsai, Y.C., Chiu, S.J., and Sun, Y.H. (2004). Localized cultured in the presence of anti-Notch ECD by following the proto- Notch signal acts through eyg and upd to promote global growth ␮ col of Le Borgne and Schweisguth (2003). A total of 200 M chlo- in Drosophila eye. Development 131, 3839–3847. roquine (Sigma) was added to the culture medium to inhibit lysosomal degradation (Incardona et al., 2002). Similar results were Coussens, L.M., and Werb, Z. (2002). Inflammation and cancer. Na- obtained by using Leupeptin or Ammonium Chloride, two other ture 420, 860–867. known inhibitors of lysosomal degradation. Fortini, M.E. 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