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

CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector 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 divi- sion 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

Load more