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研究生:莊麗瑾
研究生(外文):Li-Jin Chuang
論文名稱:Atg1在果蠅發育上的調控基因篩選分析
論文名稱(外文):A Screen for modifiers of Atg1-mediated signaling in Drosophila development
指導教授:陳光超陳光超引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:62
中文關鍵詞:細胞自噬細胞凋亡果蠅
外文關鍵詞:AutophagyApoptosisDrosophilaAtg1
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細胞自噬是具有高度保留性的過程,涉及囊泡分泌及細胞質內蛋白質和胞器的降解。Atg1是一種絲氨酸/酥氨酸激酶,並且已經知道受到Tor訊號傳遞途徑的調控。研究發現在酵母菌中,Atg1的激酶活性是CVT訊息傳遞途徑以及細胞自噬所必須。因此Atg1可能是一個可以調控細胞自噬很多步驟的一個調節點。
我利用果蠅為實驗材料,研究結果顯示大量表現Atg1,在果蠅複眼的發育上會造成細胞死亡和眼睛表面粗糙的表型。儘管目前的研究已經發現一些會與Atg1有所關聯的蛋白質,然而要鑑定出其他在細胞自噬過程中可能與Atg1有交互作用的受質仍是一個困難的任務。因此為了尋找參與在Atg1所調控細胞自噬過程中未知且新穎的基因,我利用表現Atg1 所造成粗糙眼睛的表型做了果蠅眼睛發育上的調控基因篩選分析。利用能代表果蠅百分之七十基因組的連續染色體缺陷,在測試了277株染色體缺陷的果蠅中,我發現了26個品系的染色體缺陷果蠅能有效抑制Atg1所造成的訊息傳遞途徑。我利用一系列的遺傳分析縮小候選基因的位置,並且找到了三個可能與Atg1有關聯且參與在細胞死亡訊息傳遞途徑的基因。
這些能夠與Atg1作用的調控蛋白將為細胞死亡與細胞自噬的研究帶來新的發展。我目前正在調查這些調控蛋白是否能與Atg1有物理上的交互作用,並且在細胞死亡的過程中扮演什麼樣的生物功能。


Abstracts
Autophagy is a highly conserved cellular process that involves vesicle-mediated sequestration and degradation of cytoplasmic proteins and organelles. Atg1 is a Ser/Thr kinase that is regulated by TOR-dependent signaling. In yeast, studies have found the requirement for Atg1 kinase activity in both CVT and autophagy. Thus, Atg1 is representing a nodal point for controlling multiple steps in autophagic process in response to various stresses. I have examined that overexpression of Drosophila Atg1 in the developing compound eye triggers cell death and results in eye roughness. Although a number of proteins have been found to associate with Atg1, the identification of Atg1 substrates important for autophagy remains a difficult task. To identify novel genes involved in the Atg1-mediated pathway, I carried out a dominant modifier screen of the Atg1-induced rough eye phenotype using contiguous chromosomal deficiencies that represent more than 70% of the Drosophila genome. Of the 277 deficiencies tested, 26 were identified as suppressors of Atg1 signaling. I characterize a subset of autosomal regions that strongly interact with Atg1. Three novel genes will likely identify Atg1 regulators and should shed some light on how cells are regulated by the balance between cell survival and cell death. I am currently investigating whether these regulators have physical interaction with Atg1 and what is the biological function in cell death.


Table of contents
Abstract in Chinese…………………………………………………….....1
Abstract……………………………………………………………………2
Introduction……………………………..…………………………… 3~20
The cell biology of autophagy……………………………………….3
The molecular machinery of autophagy…………………………..4~9
Atg1 plays a critical role in autophagy…………………………..9~12
Cellular functions of autophagy………………………………..12~20
Materials and Methods .…………………....…………………….…21~24
Results………………………………………………………………..25~33
Induction of apoptosis by Atg1 disrupts the ommatidial structure in a kinase-dependent manner……………………………….……...25~27
Genetic screen of the dAtg1-induced rough eye phenotype…...27~29
Candidates of genetic screen inhibits Atg1-induced phenotypes and cell death.………………………………………………………29~32
Modifiers inhibit starvation-induced auto- phagosome formation…………………………………..…………………...32~33
Discussion……………………………………………………………34~39
The ability of dAtg1 to induce cell death…………………… ...34~35
Relationship between autophagy and dAtg1-induced apoptosis.35~39
Reference……………………………………………………………..40~46
Figures………………………………………………………………..47~58
Figure 1.Overexpression of Atg1 induces apoptosis in a kinase-dependent manner………………………………………......47
Figure 2. Induction of apoptosis by Atg1 disrupts the ommatidial structure in kinase- dependent manner……………………………48
Figure 3. Genetic screen for dominant modifier in Drosophila based on a dAtg1-dependent phenotype…………………………………49
Figure 4. Coexpression of dAtg1 and dificiency line can rescue the rough eye phenotype……………………………………………...50
Figure 5. Coexpression of the dificiency lines can rescue dAtg1-induced apoptosis………………………………………….51
Figure 6. Narrow down the candidate region with its corresponding complementary deficiency lines……………………………….52-53
Figure 7. The alignment of the novel protein………...…...………54
Figure 8. The candidate gene can rescue dAtg1-induced eye phenotype and apoptosis……………………………….…………55
Figure 9. The candidate gene can rescue ptc>dAtg1-induced wing phenotype…………………………………………………………56
Figure 10. Deletion of the novel protein activity can inhibit starvation-induced autophagy…………………………..…………57
Figure 11. A proposed model for the dAtg1-mediated cell death...58
Table...………………………………………………………………..59~62
Table 1. The dificiency lines which have genetic interaction with dAtg1 in Drosophila chromosome……………………………59~60
Table 2. The dificiency lines which have genetic interaction with dAtg-cat in Drosophila chromosome…………………………61~62

Akdemir, F., Farkas, R., Chen, P., Juhasz, G., Medved''ova, L., Sass, M., Wang, L., Wang, X., Chittaranjan, S., Gorski, S.M., et al. (2006). Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death. Development 133, 1457-1465.

Bangs, P., and White, K. (2000). Regulation and execution of apoptosis during Drosophila development. Dev Dyn 218, 68-79.

Betin, V.M., and Lane, J.D. (2009). Atg4D at the interface between autophagy and apoptosis. Autophagy 5, 1057-1059.

Boya, P., Gonzalez-Polo, R.A., Casares, N., Perfettini, J.L., Dessen, P., Larochette, N., Metivier, D., Meley, D., Souquere, S., Yoshimori, T., et al. (2005). Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 25, 1025-1040.

Brachmann, C.B., and Cagan, R.L. (2003). Patterning the fly eye: the role of apoptosis. Trends Genet 19, 91-96.

Bursch, W., Ellinger, A., Gerner, C., Frohwein, U., and Schulte-Hermann, R. (2000). Programmed cell death (PCD). Apoptosis, autophagic PCD, or others? Ann N Y Acad Sci 926, 1-12.

Chan, E.Y., and Tooze, S.A. (2009). Evolution of Atg1 function and regulation. Autophagy 5, 758-765.

Cheong, H., Nair, U., Geng, J., and Klionsky, D.J. (2008). The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae. Mol Biol Cell 19, 668-681.

Codogno, P., and Meijer, A.J. (2005). Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 12 Suppl 2, 1509-1518.

Edinger, A.L., and Thompson, C.B. (2004). Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 16, 663-669.

Fingar, D.C., and Blenis, J. (2004). Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23, 3151-3171.

Fukunaga, E., Inoue, Y., Komiya, S., Horiguchi, K., Goto, K., Saitoh, M., Miyazawa, K., Koinuma, D., Hanyu, A., and Imamura, T. (2008). Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells. J Biol Chem 283, 35660-35667.

Gao, S., Alarcon, C., Sapkota, G., Rahman, S., Chen, P.Y., Goerner, N., Macias, M.J., Erdjument-Bromage, H., Tempst, P., and Massague, J. (2009). Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling. Mol Cell 36, 457-468.

Gozuacik, D., and Kimchi, A. (2004). Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891-2906.

Gozuacik, D., and Kimchi, A. (2006). DAPk protein family and cancer. Autophagy 2, 74-79.

Gu, Z., Gilbert, D.J., Valentine, V.A., Jenkins, N.A., Copeland, N.G., and Zambetti, G.P. (2000). The p53-inducible gene EI24/PIG8 localizes to human chromosome 11q23 and the proximal region of mouse chromosome 9. Cytogenet Cell Genet 89, 230-233.

Hamasaki, M., and Yoshimori, T. (2010). Where do they come from? Insights into autophagosome formation. FEBS Lett 584, 1296-1301.

Hara, T., and Mizushima, N. (2009). Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17? Autophagy 5, 85-87.

Hara, T., Takamura, A., Kishi, C., Iemura, S., Natsume, T., Guan, J.L., and Mizushima, N. (2008). FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J Cell Biol 181, 497-510.

He, B., Lu, N., and Zhou, Z. (2009). Cellular and nuclear degradation during apoptosis. Curr Opin Cell Biol 21, 900-912.

He, C., and Klionsky, D.J. (2007). Atg9 trafficking in autophagy-related pathways. Autophagy 3, 271-274.

Heymann, D. (2006). Autophagy: A protective mechanism in response to stress and inflammation. Curr Opin Investig Drugs 7, 443-450.

Hosokawa, N., Sasaki, T., Iemura, S., Natsume, T., Hara, T., and Mizushima, N. (2009). Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5, 973-979.

Inoue, Y., and Imamura, T. (2008). Regulation of TGF-beta family signaling by E3 ubiquitin ligases. Cancer Sci 99, 2107-2112.

Izzi, L., and Attisano, L. (2004). Regulation of the TGFbeta signalling pathway by ubiquitin-mediated degradation. Oncogene 23, 2071-2078.

Jung, C.H., Jun, C.B., Ro, S.H., Kim, Y.M., Otto, N.M., Cao, J., Kundu, M., and Kim, D.H. (2009). ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell 20, 1992-2003.

Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19, 5720-5728.

Kalkan, T., Iwasaki, Y., Park, C.Y., and Thomsen, G.H. (2009). Tumor necrosis factor-receptor-associated factor-4 is a positive regulator of transforming growth factor-beta signaling that affects neural crest formation. Mol Biol Cell 20, 3436-3450.

Kamada, Y., Yoshino, K., Kondo, C., Kawamata, T., Oshiro, N., Yonezawa, K., and Ohsumi, Y. (2010). Tor directly controls the Atg1 kinase complex to regulate autophagy. Mol Cell Biol 30, 1049-1058.

King, M.A., Hands, S., Hafiz, F., Mizushima, N., Tolkovsky, A.M., and Wyttenbach, A. (2008). Rapamycin inhibits polyglutamine aggregation independently of autophagy by reducing protein synthesis. Mol Pharmacol 73, 1052-1063.

Lee, J.-Y., Koga, H., Kawaguchi, Y., Tang, W., Wong, E., Gao, Y.-S., Pandey, U.B., Kaushik, S., Tresse, E., Lu, J., et al. (2010). HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy. EMBO J 29, 969-980.
Lee, K.S., Iijima-Ando, K., Iijima, K., Lee, W.J., Lee, J.H., Yu, K., and Lee, D.S. (2009). JNK/FOXO-mediated neuronal expression of fly homologue of peroxiredoxin II reduces oxidative stress and extends life span. J Biol Chem 284, 29454-29461.

Lehar, S.M., Nacht, M., Jacks, T., Vater, C.A., Chittenden, T., and Guild, B.C. (1996). Identification and cloning of EI24, a gene induced by p53 in etoposide-treated cells. Oncogene 12, 1181-1187.

Levine, B., and Klionsky, D.J. (2004). Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6, 463-477.

Liang, Y.Y., Lin, X., Liang, M., Brunicardi, F.C., ten Dijke, P., Chen, Z., Choi, K.W., and Feng, X.H. (2003). dSmurf selectively degrades decapentaplegic-activated MAD, and its overexpression disrupts imaginal disc development. J Biol Chem 278, 26307-26310.

Lum, J.J., DeBerardinis, R.J., and Thompson, C.B. (2005). Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol 6, 439-448.

Martin, D.N., and Baehrecke, E.H. (2004). Caspases function in autophagic programmed cell death in Drosophila. Development 131, 275-284.

Melendez, A., and Neufeld, T.P. (2008). The cell biology of autophagy in metazoans: a developing story. Development 135, 2347-2360.

Melendez, A., Talloczy, Z., Seaman, M., Eskelinen, E.L., Hall, D.H., and Levine, B. (2003). Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301, 1387-1391.

Mork, C.N., Faller, D.V., and Spanjaard, R.A. (2007). Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide. FEBS Lett 581, 5440-5444.

Nakatogawa, H., Ichimura, Y., and Ohsumi, Y. (2007). Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165-178.

Neufeld, T.P., and Baehrecke, E.H. (2008). Eating on the fly: function and regulation of autophagy during cell growth, survival and death in Drosophila. Autophagy 4, 557-562.
Nishida, Y., Arakawa, S., Fujitani, K., Yamaguchi, H., Mizuta, T., Kanaseki, T., Komatsu, M., Otsu, K., Tsujimoto, Y., and Shimizu, S. (2009). Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461, 654-658.

Podos, S.D., Hanson, K.K., Wang, Y.C., and Ferguson, E.L. (2001). The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev Cell 1, 567-578.

Polager, S., Ofir, M., and Ginsberg, D. (2008). E2F1 regulates autophagy and the transcription of autophagy genes. Oncogene 27, 4860-4864.

Ravikumar, B., Duden, R., and Rubinsztein, D.C. (2002). Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet 11, 1107-1117.

Ravikumar, B., Vacher, C., Berger, Z., Davies, J.E., Luo, S., Oroz, L.G., Scaravilli, F., Easton, D.F., Duden, R., O''Kane, C.J., et al. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet 36, 585-595.

Reggiori, F., Tucker, K.A., Stromhaug, P.E., and Klionsky, D.J. (2004). The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure. Dev Cell 6, 79-90.

Reme, C.E., Wolfrum, U., Imsand, C., Hafezi, F., and Williams, T.P. (1999). Photoreceptor autophagy: effects of light history on number and opsin content of degradative vacuoles. Invest Ophthalmol Vis Sci 40, 2398-2404.

Richie, D.L., and Askew, D.S. (2008). Autophagy: a role in metal ion homeostasis? Autophagy 4, 115-117.

Richie, D.L., Fuller, K.K., Fortwendel, J., Miley, M.D., McCarthy, J.W., Feldmesser, M., Rhodes, J.C., and Askew, D.S. (2007). Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus. Eukaryot Cell 6, 2437-2447.

Rowland, A.M., Richmond, J.E., Olsen, J.G., Hall, D.H., and Bamber, B.A. (2006). Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans. J Neurosci 26, 1711-1720.
Sarkar, S., Ravikumar, B., Floto, R.A., and Rubinsztein, D.C. (2009). Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell Death Differ 16, 46-56.

Scott, R.C., Juhasz, G., and Neufeld, T.P. (2007). Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death. Curr Biol 17, 1-11.

Shaw, R.J., and Cantley, L.C. (2006). Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441, 424-430.

Shen, W., and Ganetzky, B. (2009). Autophagy promotes synapse development in Drosophila. J Cell Biol 187, 71-79.

Siegrist, S.E., Haque, N.S., Chen, C.H., Hay, B.A., and Hariharan, I.K. (2010). Inactivation of both foxo and reaper promotes long-term adult neurogenesis in Drosophila. Curr Biol 20, 643-648.

Stephan, J.S., Yeh, Y.Y., Ramachandran, V., Deminoff, S.J., and Herman, P.K. (2009). The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy. Proc Natl Acad Sci U S A 106, 17049-17054.

Suzuki, K., and Ohsumi, Y. (2007). Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae. FEBS Lett 581, 2156-2161.

Toda, H., Mochizuki, H., Flores, R., 3rd, Josowitz, R., Krasieva, T.B., Lamorte, V.J., Suzuki, E., Gindhart, J.G., Furukubo-Tokunaga, K., and Tomoda, T. (2008). UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly. Genes Dev 22, 3292-3307.

Tourneur, L., and Chiocchia, G. (2010). FADD: a regulator of life and death. Trends Immunol.

Tsujimoto, Y., and Shimizu, S. (2005). Another way to die: autophagic programmed cell death. Cell Death Differ 12 Suppl 2, 1528-1534.

Vellai, T., Bicsak, B., Toth, M.L., Takacs-Vellai, K., and Kovacs, A.L. (2008). Regulation of cell growth by autophagy. Autophagy 4, 507-509.

Wang, C.W., and Klionsky, D.J. (2003). The molecular mechanism of autophagy. Mol Med 9, 65-76.
Wang, C.W., Stromhaug, P.E., Kauffman, E.J., Weisman, L.S., and Klionsky, D.J. (2003). Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage. J Cell Biol 163, 973-985.

Xie, Z., and Klionsky, D.J. (2007). Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9, 1102-1109.

Yorimitsu, T., and Klionsky, D.J. (2005). Autophagy: molecular machinery for self-eating. Cell Death Differ 12 Suppl 2, 1542-1552.

Yorimitsu, T., and Klionsky, D.J. (2007). Endoplasmic reticulum stress: a new pathway to induce autophagy. Autophagy 3, 160-162.

Yorimitsu, T., Nair, U., Yang, Z., and Klionsky, D.J. (2006). Endoplasmic reticulum stress triggers autophagy. J Biol Chem 281, 30299-30304.

Yoshimori, T. (2004). Autophagy: a regulated bulk degradation process inside cells. Biochem Biophys Res Commun 313, 453-458.

Young, A.R., Chan, E.Y., Hu, X.W., Kochl, R., Crawshaw, S.G., High, S., Hailey, D.W., Lippincott-Schwartz, J., and Tooze, S.A. (2006). Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes. J Cell Sci 119, 3888-3900.

Yue, Z., Jin, S., Yang, C., Levine, A.J., and Heintz, N. (2003). Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A 100, 15077-15082.

Zhao, X., Ayer, R.E., Davis, S.L., Ames, S.J., Florence, B., Torchinsky, C., Liou, J.S., Shen, L., and Spanjaard, R.A. (2005). Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness. Cancer Res 65, 2125-2129.

T., Tani, T., Wooten, M.W., and Wang, F. (2007). Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons. Proc Natl Acad Sci U S A 104, 5842-5847.

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