Background The Drosophila gene erupted (ept) encodes the fly homolog of human Tumor Susceptibility Gene-101 (TSG101), which functions as part of the conserved ESCRT-1 complex to facilitate the movement of cargoes through the endolysosomal pathway. Loss of ept or other genes that encode components of the endocytic machinery (e.g. synatxin7/avalanche, rab5, and vps25) produces disorganized overgrowth of imaginal disc tissue. Excess cell division is postulated to be a primary cause of these ‘neoplastic’ phenotypes, but the autonomous effect of these mutations on cell cycle control has not been examined. Principal Findings Here we show that disc cells lacking ept function display an altered cell cycle profile indicative of deregulated progression through the G1-to-S phase transition and express reduced levels of the tumor suppressor ortholog and G1/S inhibitor Rbf1. Genetic reductions of the Drosophila aPKC kinase (DaPKC), which has been shown to promote tumor growth in other fly tumor models, prevent both the ept neoplastic phenotype and the reduction in Rbf1 levels that otherwise occurs in clones of ept mutant cells; this effect is coincident with changes in localization of Notch and Crumbs, two proteins whose sorting is altered in ept mutant cells. The effect on Rbf1 can also be blocked by removal of the γ-secretase component presenilin, suggesting that cleavage of a γ-secretase target influences Rbf1 levels in ept mutant cells. Expression of exogenous rbf1 completely ablates ept mutant eye tissues but only mildly affects the development of discs composed of cells with wild type ept. Conclusions Together, these data show that loss of ept alters nuclear cell cycle control in developing imaginal discs and identify the DaPKC, presenilin, and rbf1 genes as modifiers of molecular and cellular phenotypes that result from loss of ept.
References
[1]
Hariharan IK, Bilder D (2006) Regulation of imaginal disc growth by tumor-suppressor genes in Drosophila. Annu Rev Genet 40: 335–361.
[2]
Bilder D (2004) Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors. Genes Dev 18: 1909–1925.
[3]
Vaccari T, Bilder D (2005) The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking. Dev Cell 9: 687–698.
[4]
Lu H, Bilder D (2005) Endocytic control of epithelial polarity and proliferation in Drosophila. Nat Cell Biol 7: 1132–1139.
[5]
Thompson BJ, Mathieu J, Sung HH, Loeser E, Rorth P, et al. (2005) Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev Cell 9: 711–720.
[6]
Moberg KH, Schelble S, Burdick SK, Hariharan IK (2005) Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev Cell 9: 699–710.
[7]
Childress JL, Acar M, Tao C, Halder G (2006) Lethal giant discs, a novel C2-domain protein, restricts notch activation during endocytosis. Curr Biol 16: 2228–2233.
[8]
Herz HM, Chen Z, Scherr H, Lackey M, Bolduc C, et al. (2006) vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis. Development 133: 1871–1880.
[9]
Bishop N, Woodman P (2001) TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes. J Biol Chem 276: 11735–11742.
[10]
Katzmann DJ, Babst M, Emr SD (2001) Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106: 145–155.
[11]
Baonza A, Freeman M (2005) Control of cell proliferation in the Drosophila eye by Notch signaling. Dev Cell 8: 529–539.
[12]
Firth LC, Baker NE (2005) Extracellular signals responsible for spatially regulated proliferation in the differentiating Drosophila eye. Dev Cell 8: 541–551.
[13]
Schaeffer V, Althauser C, Shcherbata HR, Deng WM, Ruohola-Baker H (2004) Notch-dependent Fizzy-related/Hec1/Cdh1 expression is required for the mitotic-to-endocycle transition in Drosophila follicle cells. Curr Biol 14: 630–636.
[14]
Shcherbata HR, Althauser C, Findley SD, Ruohola-Baker H (2004) The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions. Development 131: 3169–3181.
[15]
Ferres-Marco D, Gutierrez-Garcia I, Vallejo DM, Bolivar J, Gutierrez-Avino FJ, et al. (2006) Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing. Nature 439: 430–436.
[16]
Herranz H, Stamataki E, Feiguin F, Milán M (2006) Self-refinement of Notch activity through the transmembrane protein Crumbs: modulation of gamma-secretase activity. EMBO 7: 297–302.
[17]
Jekely G, Rorth P (2003) Hrs mediates downregulation of multiple signalling receptors in Drosophila. EMBO Rep 4: 1163–1168.
[18]
Balklava Z, Pant S, Fares H, Grant BD (2007) Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic. Nat Cell Biol 9: 1066–1073.
[19]
Assemat E, Bazellieres E, Pallesi-Pocachard E, Le Bivic A, Massey-Harroche D (2008) Polarity complex proteins. Biochim Biophys Acta 1778: 614–630.
[20]
Suzuki A, Ohno S (2006) The PAR-aPKC system: lessons in polarity. J Cell Sci 119: 979–987.
[21]
Henrique D, Schweisguth F (2003) Cell polarity: the ups and downs of the Par6/aPKC complex. Curr Opin Genet Dev 13: 341–350.
[22]
Yan R, Small S, Desplan C, Dearolf CR, Darnell JE Jr (1996) Identification of a Stat gene that functions in Drosophila development. Cell 84: 421–430.
[23]
Hou XS, Melnick MB, Perrimon N (1996) Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 84: 411–419.
[24]
Sotillos S, Diaz-Meco MT, Caminero E, Moscat J, Campuzano S (2004) DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila. J Cell Biol 166: 549–557.
[25]
Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, et al. (1999) The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153: 135–177.
[26]
Wolff T, Ready DF (1991) The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave. Development 113: 841–850.
[27]
White K, Grether ME, Abrams JM, Young L, Farrell K, et al. (1994) Genetic control of programmed cell death in Drosophila. Science 264: 677–683.
[28]
Ryoo HD, Bergmann A, Gonen H, Ciechanover A, Steller H (2002) Regulation of Drosophila IAP1 degradation and apoptosis by reaper and ubcD1. Nat Cell Biol 20: 20.
[29]
Holley CL, Olson MR, Colon-Ramos DA, Kornbluth S (2002) Reaper eliminates IAP proteins through stimulated IAP degradation and generalized translational inhibition. Nat Cell Biol 14: 14.
[30]
Yoo SJ, Huh JR, Muro I, Yu H, Wang L, et al. (2002) Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms. Nat Cell Biol 4: 416–424.
[31]
Lisi S, Mazzon I, White K (2000) Diverse domains of THREAD/DIAP1 are required to inhibit apoptosis induced by REAPER and HID in Drosophila. Genetics 154: 669–678.
[32]
Goyal L, McCall K, Agapite J, Hartwieg E, Steller H (2000) Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function. Embo J 19: 589–597.
[33]
Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA (1999) The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID. Cell 98: 453–463.
[34]
Du W, Vidal M, Xie JE, Dyson N (1996) RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila. Genes Dev 10: 1206–1218.
[35]
Lee T, Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24: 251–254.
[36]
Wang H, Somers GW, Bashirullah A, Heberlein U, Yu F, et al. (2006) Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts. Genes Dev 20: 3453–3463.
[37]
Mahoney MB, Parks AL, Ruddy DA, Tiong SY, Esengil H, et al. (2006) Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers. Genetics 172: 2309–2324.
[38]
Struhl G, Adachi A (2000) Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins. Mol Cell 6: 625–636.
[39]
Struhl G, Greenwald I (2001) Presenilin-mediated transmembrane cleavage is required for Notch signal transduction in Drosophila. Proc Natl Acad Sci U S A 98: 229–234.
[40]
Vaccari T, Lu H, Kanwar R, Fortini ME, Bilder D (2008) Endosomal entry regulates Notch receptor activation in Drosophila melanogaster. J Cell Biol 180: 755–762.
[41]
Dyson N (1998) The regulation of E2F by pRB-family proteins. Genes Dev 12: 2245–2262.
[42]
Pignoni F, Zipursky SL (1997) Induction of Drosophila eye development by decapentaplegic. Development 124: 271–278.
[43]
Lee CY, Robinson KJ, Doe CQ (2006) Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation. Nature 439: 594–598.
[44]
Lee CY, Andersen RO, Cabernard C, Manning L, Tran KD, et al. (2006) Drosophila Aurora-A kinase inhibits neuroblast self-renewal by regulating aPKC/Numb cortical polarity and spindle orientation. Genes Dev 20: 3464–3474.
[45]
Rolls MM, Albertson R, Shih HP, Lee CY, Doe CQ (2003) Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia. J Cell Biol 163: 1089–1098.
[46]
Smith CA, Lau KM, Rahmani Z, Dho SE, Brothers G, et al. (2007) aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb. Embo J 26: 468–480.
[47]
Lai EC (2002) Protein degradation: four E3s for the notch pathway. Curr Biol 12: R74–78.
[48]
van den Heuvel S, Dyson NJ (2008) Conserved functions of the pRB and E2F families. Nat Rev Mol Cell Biol 9: 713–724.
[49]
Lloyd TE, Atkinson R, Wu MN, Zhou Y, Pennetta G, et al. (2002) Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila. Cell 108: 261–269.
[50]
Moberg KH, Bell DW, Wahrer DC, Haber DA, Hariharan IK (2001) Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413: 311–316.
[51]
Pellock BJ, Buff E, White K, Hariharan IK (2007) The Drosophila tumor suppressors Expanded and Merlin differentially regulate cell cycle exit, apoptosis, and Wingless signaling. Dev Biol 304: 102–115.
