142 POSTERS: Cell Division and Growth Control
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142 POSTERS: Cell Division and Growth Control 157A An expression-based approach to identifying factors that mediate cell competition. Claire de la Cova, Laura A. Johnston. Genetics and Development, Columbia University, New York, NY. Cell competition is a process that results in elimination of “losing” cells and survival of “winning” cells within growing organs of both vertebrates and Drosophila melanogaster. In a mosaic Drosophila wing imaginal disc, cells expressing high levels of dMyc compete against and eliminate their neighbors with lower dMyc levels. “Losing” cells die frequently and their competitive elimination requires the pro-apoptotic gene hid. Because little is known about the signal that kills “losing” cells, and no biological markers specific to cell competition exist, we sought to better characterize “winning” and “losing” cells using a gene expression approach. We have generated and isolated competing cell populations from the wing imaginal disc and used gene expression microarrays identify mRNA expression changes in “winning” dMyc cells and wildtype “losing” cells. A large number of significant expression changes occur in dMyc-expressing cells (750 genes at ≥1.5-fold). On the other hand, when growing near dMyc-expressing cells, wildtype “losing” cells display a comparably small number of significant expression changes (58 genes at ≥1.5-fold). We have selected several candidate genes whose expression levels are altered in “losing” cells and used a simple clonal assay of cell competition in the wing imaginal disc to explore their roles in the competitive process. We find that some of our candidate genes contribute to the cell death or to the growth disadvantage of “losing” cells, while others are required for the survival of dMyc- expressing “winning” cells. This data raises the possibility that both “winning” and “losing” cells produce signals necessary for cell competition. 158B Characterization of a Mutation that Produces Cell Competition. Yassi Hafezi, Iswar Hariharan. Molecular and Cell Biology, Univ. of California, Berkeley, Berkeley, CA. The goal of this study is to address the mechanistic basis of a phenomenon known as cell competition. In Drosophila melanogaster, viable yet slow-growing clones of cells can be eliminated from a tissue when they are adjacent to certain faster-growing cells. The basis of competitive ability and the mechanism by which cells eliminate other cells are unclear. Our laboratory has previously identified mutants in over twenty loci in an extensive screen for genes that negatively regulate growth in the developing Drosophila eye. In this study we characterize one viable mutation from this screen and show that it is involved in cell competition. We are currently trying to map the mutation to identify the gene responsible for this phenotype. We are also testing the effects of this mutation on molecules previously implicated in cell competition, dMyc and Decapentaplegic (Dpp), to determine whether it competes in the same way or through a novel mechanism. Ultimately, we hope to better understand this interesting cell behavior. 159C A functional analysis of cell competition using Drosophila cell culture. Nanami Senoo-Matsuda, Laura A. Johnston. Department of Genetics & Development, College of Physicians & Surgeons, Columbia University, New York, NY 10032. Our studies have revealed that developing wing cells in Drosophila melanogaster that differ in expression levels of the growth regulator dMyc can compete, leading to the apoptotic death of the cells with less dMyc (“losers”) and over-representation of cells with more dMyc (“winners”) in the wing (de la Cova et al., 2004). This phenomenon, called cell competition, seems to play a crucial role in the control of organ size. To identify the genetic logic underlying cell competition, we have developed an S2 cell-culture based assay for cell competition to use in a genome-wide, functional RNAi screen. We have made stable cell lines that inducibly express either dMyc or the PI3K, Dp110, or constitutively express GFP, and established an in vitro model of cell competition. Using a variety of co-culture assays we find that cell competition is induced in a dMyc-concentration and time-dependent manner. Our results indicate that cell competition does not require direct cell-to-cell contact and is the result of factors secreted from both the “winner” and “loser” cell population. We will discuss these results and our efforts to identify genes required for competition to occur. POSTERS: Cell Division and Growth Control 143 160A Studies on the regulation of dMyc expression by Insulin and Nutrients signaling. Rajendra Chilukuri1, Federica Parisi2, Daniela Grifoni2, Paola Bellosta1. 1) City College-CUNY,New York, NY; 2) University of Bologna, Italy. myc is a gene whose deregulation is prominent in cancer, and is a critical regulator of growth in flies and mammals. Genetic studies in vertebrates and invertebrates suggest that signals from the conserved patterns organizing morphogens such as Insulin, BMP/Dpp/TGF-b Wnt/Wingless, and Hedgehog contribute to this program, but it is unclear how they monitor and regulate growth. Our and others microarray analysis revealed that the majority of Myc target genes play a role in ribosome biogenesis, protein synthesis and metabolism, consistent with dMyc’s role in cellular growth. We recently demonstrated in vitro using S2 cells, that stimulation of cells with Insulin increases Myc protein levels and this event is dependent on Tor signaling. Our data are consistent with a role of dMyc on Insulin and Nutrients signaling. Most recent data will be presented. 161B Drosophila TCTP is a new component of the TSC pathway. Ya-Chieh Hsu1, Kwang-Wook Choi1,2. 1) Program in Developmental Biology, Baylor College Med, Houston, TX; 2) Molec & Cell Biol, Baylor College Med, Houston, TX. Cellular growth and proliferation are properly coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous Sclerosis (TSC) is a benign tumor syndrome caused by mutations in either TSC1 or TSC2. Recent studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. TSC2 has been shown to be the GTPase activating protein (GAP) for Rheb, but a guanine nucleotide exchange factor (GEF) that facilitates the GDP/GTP exchange on Rheb has not been identified. We have found genetic and biochemical evidence which suggests that the highly conserved protein Translationally Controlled Tumor Protein (TCTP) is a new component of the TSC pathway. Reducing dTCTP levels affects cell size, cell number and organ size, which mimics the Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to TSC1 and dRheb, but acts upstream of ds6k, a downstream target of dRheb. In addition, dTCTP directly associates with dRheb and displays GEF activity to it both in vivo and in vitro. Expression of the human TCTP (hTCTP) can rescue dTCTP mutant phenotypes. Since hTCTP is also able to interact with human Rheb (hRheb) and stimulates its GTP/GDP exchange, the function of TCTP in the TSC pathway is likely to be conserved throughout evolution. Currently we are identifying critical amino acids mediating the function of dTCTP and dRheb. 162C The role of CUL4-DDB1 in the control of growth and CDT1/DUP levels during Drosophila development. Hyun O Lee, Sima Zacharek. GMB, University of North Carolina, Chapel Hill, NC. CDT1/DUP is an essential replication licensing factor that is degraded at the onset of S phase via ubiquitin-mediated proteolysis to ensure that the genome is replicated only once per cell cycle. The CUL4DDB1 E3 ubiquitin ligase is necessary for the regulated proteolysis of CDT1/DUP after DNA damage, but whether it plays an essential role in the destruction of CDT1/DUP at the beginning of S phase is unclear. In order to examine this issue and to determine the in vivo function of CUL4DDB1 we isolated and characterized mutations in the essential Drosophila Cul4 and Ddb1 genes. Cul4 and Ddb1 null mutants develop until the 1st or 2nd larval instar stage, and then display phenotypes consistent with a growth defect: The mutant animals can survive for up to 10 days without developing further and fail to incorporate BrdU in most cells. Clones of Ddb1 null mutant cells generated by mitotic recombination in larval imaginal discs are reduced in size relative to control clones. Similarly, Cul4 mutant cells grow slowly and are eventually eliminated from the imaginal epithelia most likely via competition with phenotypically normal neighboring cells. Depletion of either CUL4 or DDB1 in homozygous mutant larvae or by RNAi in cultured S2 or HeLa cells results in mild hyper-accumulation of CDT1/ DUP. DDB1 and CDT1/DUP were detected in CUL4 immunocomplexes. Thus, we were surprised to find that clones of either Ddb1 or Cul4 mutant imaginal cells demonstrated normal CDT1/DUP degradation at the G1-S transition, suggesting that CUL4DDB1 is not necessary for cell cycle regulated CDT1/DUP degradation and that the observed hyper-accumulation may be due to growth or cell cycle arrest. Recent results in vertebrate systems suggest redundancy between CUL4 and CUL1 E3 ligases in the control of CDT1/ DUP degradation during the cell cycle. However, cells in Cul1 or Cul1 Cul4 double mutant clones also fail to hyper-accumulate CDT1/DUP. 144 POSTERS: Cell Division and Growth Control 163A Identification and characterisation of novel regulators of insulin signalling. Shivanthy M Visvalingam, Deborah C.I Goberdhan, Clive Wilson. Department of Physiology, Anatomy & Genetics, Le Gros Clark Building, Oxford University, Oxford, GB. The Insulin/Insulin-like growth factor signalling (IIS) cascade is a highly conserved pathway which regulates growth and metabolism in response to the availability of nutrients. Investigating this pathway has immense importance in understanding tumorigenesis, a process which is frequently upregulated, and type 2 diabetes where IIS is reduced.