請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7665
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊維元 | |
dc.contributor.author | Chun-Wei Chen | en |
dc.contributor.author | 陳君瑋 | zh_TW |
dc.date.accessioned | 2021-05-19T17:49:29Z | - |
dc.date.available | 2021-08-21 | |
dc.date.available | 2021-05-19T17:49:29Z | - |
dc.date.copyright | 2017-08-28 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-18 | |
dc.identifier.citation | Adler, J., and Parmryd, I. (2010). Quantifying Colocalization by Correlation: The Pearson correlation Coefficient is Superior to the Mander’s Overlap Coefficient. Cytometry Part A 77A, 733-742.
Anding, A.L., and Baehrecke, E.H. (2017). Cleaning House: Selective Autophagy of Organelles. Dev Cell 41, 10-22. Baens, M., Noels, H., Broeckx, V., Hagens, S., Fevery, S., Billiau, A.D., Vankelecom, H., and Marynen, P. (2006). The Dark Side of EGFP: Defective Polyubiquitination. PLOS ONE 1, e54. Bingol, B., Tea, J.S., Phu, L., Reichelt, M., Bakalarski, C.E., Song, Q., Foreman, O., Kirkpatrick, D.S., and Sheng, M. (2014). The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy. Nature 510, 370-375. Bottone, M.G., Santin, G., Aredia, F., Bernocchi, G., Pellicciari, C., and Scovassi, A.I. (2013). Morphological Features of Organelles during Apoptosis: An Overview. Cells 2, 294-305. Chernousova, S., and Epple, M. (2017). Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent. Gene Ther 24, 282-289. Chu, Y.P., Hung, Y.H., Chang, H.Y., and Yang, W.Y. (2017). Chapter Fourteen - Assays to Monitor Lysophagy. In Methods in Enzymology, J.M.B.-S.P. Lorenzo Galluzzi, and K. Guido, eds. (Academic Press), pp. 231-244. Clague, M.J., Coulson, J.M., and Urbe, S. (2012). Cellular functions of the DUBs. J Cell Sci 125, 277-286. Colosimo, A., Goncz, K.K., Holmes, A.R., Kunzelmann, K., Novelli, G., Malone, R.W., Bennett, M.J., and Gruenert, D.C. (2000). Transfer and expression of foreign genes in mammalian cells. Biotechniques 29, 314-318, 320-312, 324 passim. Cornelissen, T., Haddad, D., Wauters, F., Van Humbeeck, C., Mandemakers, W., Koentjoro, B., Sue, C., Gevaert, K., De Strooper, B., Verstreken, P., et al. (2014). The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy. Human Molecular Genetics 23, 5227-5242. Dikic, I., and Bremm, A. (2014). DUBs counteract parkin for efficient mitophagy. The EMBO Journal 33, 2442-2443. Durcan, T.M., Tang, M.Y., Pérusse, J.R., Dashti, E.A., Aguileta, M.A., McLelland, G.L., Gros, P., Shaler, T.A., Faubert, D., Coulombe, B., et al. (2014). USP8 regulates mitophagy by removing K6‐linked ubiquitin conjugates from parkin. The EMBO Journal 33, 2473-2491. Fabris, C., Valduga, G., Miotto, G., Borsetto, L., Jori, G., Garbisa, S., and Reddi, E. (2001). Photosensitization with Zinc (II) Phthalocyanine as a Switch in the Decision between Apoptosis and Necrosis. Cancer Research 61, 7495-7500. Galluzzi, L., Baehrecke, E.H., Ballabio, A., Boya, P., Bravo-San Pedro, J.M., Cecconi, F., Choi, A.M., Chu, C.T., Codogno, P., Colombo, M.I., et al. (2017). Molecular definitions of autophagy and related processes. EMBO J, 1-26. Hsieh, C.W., Chu, C.H., Lee, H.M., and Yuan Yang, W. (2015). Triggering mitophagy with far-red fluorescent photosensitizers. Sci Rep 5, 10376. Hung, Y.-H., Chen, L.M.-W., Yang, J.-Y., and Yuan Yang, W. (2013). Spatiotemporally controlled induction of autophagy-mediated lysosome turnover. Nat Commun 4. Hunt, M.A., Currie, M.J., Robinson, B.A., and Dachs, G.U. (2010). Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents. Journal of Biomolecular Techniques : JBT 21, 66-72. 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. The EMBO Journal 19, 5720-5728. Karbowski, M., and Youle, R.J. (2003). Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ 10, 870-880. Keusekotten, K., Elliott, Paul R., Glockner, L., Fiil, Berthe K., Damgaard, Rune B., Kulathu, Y., Wauer, T., Hospenthal, Manuela K., Gyrd-Hansen, M., Krappmann, D., et al. (2013). OTULIN Antagonizes LUBAC Signaling by Specifically Hydrolyzing Met1-Linked Polyubiquitin. Cell 153, 1312-1326. Khaminets, A., Behl, C., and Dikic, I. (2015). Ubiquitin-Dependent And Independent Signals In Selective Autophagy. Trends in Cell Biology 26, 6-16. Kirkin, V., McEwan, D.G., Novak, I., and Dikic, I. (2009). A Role for Ubiquitin in Selective Autophagy. Molecular Cell 34, 259-269. Komander, D., Clague, M.J., and Urbe, S. (2009). Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10, 550-563. Kondapalli, C., Kazlauskaite, A., Zhang, N., Woodroof, H.I., Campbell, D.G., Gourlay, R., Burchell, L., Walden, H., Macartney, T.J., Deak, M., et al. (2012). PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol 2, 120080. Koyano, F., Okatsu, K., Kosako, H., Tamura, Y., Go, E., Kimura, M., Kimura, Y., Tsuchiya, H., Yoshihara, H., Hirokawa, T., et al. (2014). Ubiquitin is phosphorylated by PINK1 to activate parkin. Nature 510, 162-166. Kraft, C., Peter, M., and Hofmann, K. (2010). Selective autophagy: ubiquitin-mediated recognition and beyond. Nat Cell Biol 12, 836-841. Lemasters, J.J. (2005). Selective Mitochondrial Autophagy, or Mitophagy, as a Targeted Defense Against Oxidative Stress, Mitochondrial Dysfunction, and Aging. Rejuvenation Research 8, 3-5. Li, L., Hailey, D.W., Soetandyo, N., Li, W., Lippincott-Schwartz, J., Shu, H.-b., and Ye, Y. (2008). Localization of A20 to a lysosome-associated compartment and its role in NFκB signaling. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1783, 1140-1149. Liu, H.S., Jan, M.S., Chou, C.K., Chen, P.H., and Ke, N.J. (1999). Is green fluorescent protein toxic to the living cells? Biochemical and biophysical research communications 260, 712-717. Magraoui, F.E., Reidick, C., Meyer, H.E., and Platta, H.W. (2015). Autophagy-Related Deubiquitinating Enzymes Involved in Health and Disease. Cells 4, 596-621. Maurisse, R., De Semir, D., Emamekhoo, H., Bedayat, B., Abdolmohammadi, A., Parsi, H., and Gruenert, D.C. (2010). Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnology 10, 9. Mei, Y., Hahn, A.A., Hu, S., and Yang, X. (2011). The USP19 Deubiquitinase Regulates the Stability of c-IAP1 and c-IAP2. Journal of Biological Chemistry 286, 35380-35387. Mevissen, Tycho E.T., Hospenthal, Manuela K., Geurink, Paul P., Elliott, Paul R., Akutsu, M., Arnaudo, N., Ekkebus, R., Kulathu, Y., Wauer, T., El Oualid, F., et al. (2013). OTU Deubiquitinases Reveal Mechanisms of Linkage Specificity and Enable Ubiquitin Chain Restriction Analysis. Cell 154, 169-184. Narendra, D., Tanaka, A., Suen, D.F., and Youle, R.J. (2008). Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 183, 795-803. Nath, S., Dancourt, J., Shteyn, V., Puente, G., Fong, W.M., Nag, S., Bewersdorf, J., Yamamoto, A., Antonny, B., and Melia, T.J. (2014). Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat Cell Biol 16, 415-424. Nunnari, J., and Suomalainen, A. (2012). Mitochondria: In Sickness and in Health. Cell 148, 1145-1159. Okamoto, K. (2014). Organellophagy: Eliminating cellular building blocks via selective autophagy. The Journal of Cell Biology 205, 435-445. Okatsu, K., Uno, M., Koyano, F., Go, E., Kimura, M., Oka, T., Tanaka, K., and Matsuda, N. (2013). A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment. Journal of Biological Chemistry 288, 36372-36384. Reyes-Turcu, F.E., Ventii, K.H., and Wilkinson, K.D. (2009). Regulation and Cellular Roles of Ubiquitin-Specific Deubiquitinating Enzymes. Annual Review of Biochemistry 78, 363-397. Satoo, K., Noda, N.N., Kumeta, H., Fujioka, Y., Mizushima, N., Ohsumi, Y., and Inagaki, F. (2009). The structure of Atg4B–LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy. The EMBO Journal 28, 1341-1350. Shaid, S., Brandts, C.H., Serve, H., and Dikic, I. (2013). Ubiquitination and selective autophagy. Cell Death Differ 20, 21-30. Sowa, M.E., Bennett, E.J., Gygi, S.P., and Harper, J.W. (2009). Defining the Human Deubiquitinating Enzyme Interaction Landscape. Cell 138, 389-403. Taghizadeh, R.R., and Sherley, J.L. (2008). CFP and YFP, but not GFP, provide stable fluorescent marking of rat hepatic adult stem cells. J Biomed Biotechnol 2008, 453590. Tanida, I., Ueno, T., and Kominami, E. (2004). LC3 conjugation system in mammalian autophagy. The International Journal of Biochemistry & Cell Biology 36, 2503-2518. Tu, Z., He, G., Li, K.X., Chen, M.J., Chang, J., Chen, L., Yao, Q., Liu, D.P., Ye, H., Shi, J., et al. (2005). An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains. Electronic Journal of Biotechnology 8, 113-120. Urbé, S., Liu, H., Hayes, S.D., Heride, C., Rigden, D.J., and Clague, M.J. (2012). Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions. Molecular Biology of the Cell 23, 1095-1103. Valente, E.M., Abou-Sleiman, P.M., Caputo, V., Muqit, M.M.K., Harvey, K., Gispert, S., Ali, Z., Del Turco, D., Bentivoglio, A.R., Healy, D.G., et al. (2004). Hereditary Early-Onset Parkinson's Disease Caused by Mutations in PINK1. Science 304, 1158-1160. Wang, Y., Serricchio, M., Jauregui, M., Shanbhag, R., Stoltz, T., Di Paolo, C.T., Kim, P.K., and McQuibban, G.A. (2015). Deubiquitinating enzymes regulate PARK2-mediated mitophagy. Autophagy 11, 595-606. Xie, Z., and Klionsky, D.J. (2007). Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9, 1102-1109. Yang, J.H., Gross, R.L., Basinger, S.F., and Wu, S.M. (2001). Apoptotic cell death of cultured salamander photoreceptors induced by cccp: CsA-insensitive mitochondrial permeability transition. J Cell Sci 114, 1655-1664. Yang, J.Y., and Yang, W.Y. (2011). Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells. Autophagy 7, 1230-1238. Ziegler, U., and Groscurth, P. (2004). Morphological Features of Cell Death. Physiology 19, 124-128. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7665 | - |
dc.description.abstract | 細胞自噬 (autophagy) 是一種細胞內的自我分解機制,調控細胞中物質的去與留,分解後並加以循環利用。需要被清除的物質會被自噬小體 (autophagosome)包覆後與溶酶體 (lysosome)結合,進而被溶酶體中的水解酵素分解。在分類上,細胞自噬可以由所吞噬的物質之特定性,而分成非特定性細胞自噬與特定性細胞自噬(nonselective and selective autophagy)。在特定性細胞自噬的過程中,特定的胞中物質,例如:受傷的胞器,會被泛素(ubiquitin)標定,進而被特定性細胞自噬受器(selective autophagy receptors) 辨識而形成自噬小體進行吞噬與分解。泛素在這個過程扮演了重要的角色;因此,去泛素化酶(deubiquitinases, DUBs) 在調控這個過程中也是不可或缺。在其中一種特定性細胞自噬,胞器自噬(organellophagy) 中,有部分參與其中的去泛素化酶已經被發現,他們去除標定在將被清除的胞器上的泛素,而抑制了胞器自噬的產生,但也尚有許多參與胞器自噬的去泛素化酶尚未被發掘。先前的研究有利用影像結果找尋去泛素化酶,但其方法並非使用大量、系統化的地毯式搜尋,難免會有漏網之魚。因此我們想要發展出一套以影像為主的搜索策略,在人體近一百種的去泛素化酶中,搜尋在胞器自噬中擔任負調控角色的去泛素化酶。
這套策略假設當目標去泛素化酶過度表現時,標定在受傷胞器上的泛素將會被去泛素化酶切除進而抑制胞器自噬的進行。為了使這套策略能夠系統化搜尋目標,我們首先建立了接上增強型綠色螢光蛋白(EGFP)的去泛素化酶質體庫,並將各質體一一表現於海拉細胞(HeLa cells) 中,並以光破壞的方式誘導特定的胞器自噬產生。接著利用免疫螢光染色(immunofluorescence) 標定出泛素與特定胞器的位置,並利用ScanR影像分析軟體計算每個細胞中的去泛素化酶的表現量與泛素標定在特定胞器上的面積,此二參數應該呈現負相關;也就是當目標去泛素化酶的表現量越高,泛素標定在特定胞器上的面積就越低。這個搜索策略可以只計算在特定胞器上的泛素(而非整個細胞中的泛素);而且由於基因轉染的效率(transfection efficiency)因各個細胞而異,因此以單一細胞為單位,可以在一盤細胞中得到不同的去泛素化酶的表現量,便可由量化分析比較不同去泛素化酶表現量下,泛素標定在特定胞器上的面積。 我們所設計的影像式搜索方法首先由目前了解較詳細的粒線體自噬(mitophagy) 來測試其可行性。我們將已知會抑制粒線體自噬去泛素化酶USP30 表現於海拉細胞中進行測試,確定這個策略是可行的,不過仍有一些問題存在於控制組的實驗結果中,需要進一步的改正。同時,我們也準備了材料,欲將此策略應用在其他種胞器自噬中。質體庫目前已經建立了27個去泛素化酶質體,並且在目前了解不多的溶酶體自噬中測試了10個去泛素化酶,其中兩個有可能是目標,需要進一步的檢測才能確定。總而言之,我們所建立的影像搜索方法經過改正ㄧ些問題後,相信可以應用在鑑定抑制胞器自噬的去泛素化酶過程中。 | zh_TW |
dc.description.abstract | Autophagy is an intracellular digestion mechanism, termed as “self-eating” processes in quality control of cellular components. Autophagy can be categorized as nonselective and selective autophagy by the specificity of the targeted cargo, which is engulfed by autophagosomes and degraded by fusion of lysosomes. In selective autophagy, specific cellular components, such as organelles, are targeted by ubiquitin (Ub), providing a recognition by selective autophagy receptors for autophagosome formation. Since ubiquitination plays an important role in the targeting of substrates for selective autophagy, especially organelle-autophagy, the deubiquitinases (DUBs), which oppose ubiquitination, can be a key factor for suppressing organelle-autophagy. However, which DUBs are involved in organellophagy remains partially unidentified. In addition, previous studies utilize images in aid of identifying DUBs in selective autophagy, but not in a systematic and statistical ways. Therefore, the goal of the study is to establish an image-based screening strategy to robustly identify which DUBs regulate autophagic organelles turnover from over a hundred DUBs within human genome.
The assumption underlying this strategy is that ubiquitination of damaged organelle will be suppressed by the overexpression of the DUBs that regulate organelle autophagy. To enable the strategy systematically, DUBs were first cloned into EGFP vector and overexpressed within HeLa cells. To clearly observe the organelle autophagy in cells, the dye-labeled organelles were specifically damaged by light induction. By quantifying the Ub signal on the damaged organelles with immunofluorescence in a cellular scale by ScanR analysis software, the correlation between EGFP-DUB signal intensity and Ub signal area should be negative with the overexpression of the DUB candidates. This strategy can not only quantify the ubiquitination specifically on the damaged organelles cell by cell, but also acquire different DUB expression level in each cell due to the transfection efficiency. This image-based screening strategy was first examined through parkin-mediated mitophagy, and one of the known DUBs for natively regulating parkin-mediated mitophagy, USP30, was tested in the assays. However, the problem in the EGFP control groups remained to be revised. Meanwhile, we prepared for applying the assay to identify the DUBs that regulate autophagic turnover of other types of organelles. So far, 27 DUBs were prepared, with 9 of them testing in lysophgy, and one DUB in Golgiohagy. There were 2 candidates showed in the lysophagy testing, and further screening need to be done. To sum up, the image-based screening strategy can become a powerful method after validation. | en |
dc.description.provenance | Made available in DSpace on 2021-05-19T17:49:29Z (GMT). No. of bitstreams: 1 ntu-106-R04b46010-1.pdf: 4970322 bytes, checksum: a99e3ff90e77eb4f23c11082dffb78b2 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | Introduction 1
Materials and Methods 10 Materials 10 Equipment 13 Methods 15 Plasmids 15 Cloning 15 Cell culture and seeding 17 Transfection 17 Lysophagy assay 18 Mitophagy assay 19 Golgiphagy assay 19 Immunofluorescence 20 Fluorescence-activated cell sorting (FACs) 20 Cellular imaging and analysis 21 Statistics 25 Results 27 Shading correction is required for image processing. 27 ScanR assay testing by expressing EGFP-USP30 in mitophagy 27 The ScanR analysis assay is applicable on single cell measurement 29 The increase of cell number sorting stable cell line by FACs were suggested when utilizing the ScanR analysis assay. 30 The ScanR assay was tested by illuminated Mitotracker induced mitophagy. 32 Test the ScanR assay by changing the transfection methods 32 Test the ScanR assay by different fixation conditions. 33 The preceding work for applying screening assay 34 The progress of setting up the DUB library 34 The conditions in damaging lysosomes 35 The DUBs tested in lysophagy: OTUD6A and USP10 may be the candidates in reversing lysophagy 36 The Flag-tagged and myc-tagged vectors showed no significant impact on Ub recruitment to damaged lysosomes. 37 The DUB tested in Golgiphagy: VCPIP1 may not be the DUB for opposing Golgiphagy. 38 Discussion 39 The key points in ScanR analysis assay 39 The negative correlation of EGFP groups 39 Optional ways for screening 41 Figures and Tables 43 References 90 | |
dc.language.iso | en | |
dc.title | 建立影像式搜索策略鑑別參與特定性細胞自噬的去泛素化酶 | zh_TW |
dc.title | An image-based screening strategy to identify deubiquitinases in selective autophagy | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳光超,黃馥 | |
dc.subject.keyword | Selective autophagy,organellophagy,deubiquitinases,ubiquitin,ScanR, | zh_TW |
dc.relation.page | 97 | |
dc.identifier.doi | 10.6342/NTU201704022 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2017-08-19 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-106-1.pdf | 4.85 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。