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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 劉雅雯(Ya-Wen Liu) | |
| dc.contributor.author | Tsung-Lin Hsieh | en |
| dc.contributor.author | 謝宗霖 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:47:24Z | - |
| dc.date.copyright | 2020-08-26 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-04 | |
| dc.identifier.citation | Abram, C.L., Seals, D.F., Pass, I., Salinsky, D., Maurer, L., Roth, T.M., and Courtneidge, S.A. (2003). The adaptor protein fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells. J Biol Chem 278, 16844-16851. Ahn, S., Kim, J., Lucaveche, C.L., Reedy, M.C., Luttrell, L.M., Lefkowitz, R.J., and Daaka, Y. (2002). Src-dependent Tyrosine Phosphorylation Regulates Dynamin Self-assembly and Ligand-induced Endocytosis of the Epidermal Growth Factor Receptor. Journal of Biological Chemistry 277, 26642-26651. Ahn, S., Maudsley, S., Luttrell, L.M., Lefkowitz, R.J., and Daaka, Y. (1999). Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling. J Biol Chem 274, 1185-1188. Baron, V., Alengrin, F.o., and Van Obberghen, E. (1998). 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(2016b). Actin bundling by dynamin 2 and cortactin is implicated in cell migration by stabilizing filopodia in human non-small cell lung carcinoma cells. Int J Oncol 49, 877-886. Zhu, J., Zhou, K., Hao, J.J., Liu, J., Smith, N., and Zhan, X. (2005). Regulation of cortactin/dynamin interaction by actin polymerization during the fission of clathrin-coated pits. J Cell Sci 118, 807-817 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15521 | - |
| dc.description.abstract | 侵襲體是一種受到Src 磷酸酶所調控、並且由肌動蛋白所組成的結構，這樣的結構對於細胞的貼附、移動、侵襲是非常重要的。另一方面，一個能夠調控膜組織的三磷酸鳥苷水解酶 dynamin-2 (Dyn2) 被發現會聚集到侵襲體的附近去幫助細胞貼附結構的胞吞作用，以及調控束狀的肌動蛋白進而影響侵襲體的軟硬程度。然而，目前還不清楚是什麼機制來調控Dyn2的活性以及如何促使他聚集到侵襲體附近。有鑑於酪胺酸磷酸酶Src 已經被發現是一個很重要的調控者來調控侵襲體的形成，同時又能夠去磷酸化Dyn2，因此我們猜測Src對於Dyn2的磷酸化也許是一個很重要的機制來去調控他在侵襲體上的活性。為了驗證以上的推測，我們針對Dyn2上會被Src磷酸酶磷酸化的位點進行突變，並且利用細胞實驗及生化實驗進行觀察。我們發現仿磷酸化(Y597E)及不能被磷酸化(Y597F)兩種Dyn2的突變蛋白都對於Dyn2本身的活性以及他和肌動蛋白之間的交互作用有很大的影響，甚至進一步的影響侵襲體的外觀形狀及功能。除此之外，仿磷酸化(Y597E) Dyn2突變會增加和肌動蛋白接合的能力，但會降低和脂質的親和力。總而言之，我們發現酪胺酸磷酸酶Src 對於Dyn2的磷酸化是一個很重要的機制來調控Dyn2在侵襲體附近的活性。 | zh_TW |
| dc.description.abstract | Podosomes are Src kinase regulated, actin-rich structures critical for cell adhesion, migration and invasion. While the membrane remodeling GTPase dynamin-2 (Dyn2) has been found to be enriched at podosomes to mediate adhesion molecule endocytosis as well as to regulate the bundling of actin and the stiffness of podosome, the mechanism orchestrating the activity and recruitment of Dyn2 to podosome is still unclear. Given that tyrosine kinase Src is the critical regulator of podosome assembly and Dyn2 is a substrate of Src kinase, we hypothesize that Src kinase-mediated phosphorylation of Dyn2 may play an important role for its activity at podosome. We thus investigate the effect of Dyn2 mutants on the Src phosphorylation sites with cell biological and biochemical approaches. We found that both the phospho-mimetic (Y597E) and phospho-deficient (Y597F) mutants of Dyn2 have significant impacts on its activity as well as Dyn2-actin association that further alter podosome morphology and function. Strikingly, the phospho-mimetic Dyn2 displays higher actin bundling activity, but a decreased lipid binding ability. Together, our findings demonstrate that Src kinase phosphorylation serve as a critical regulation to dictate Dyn2 activity at podosome structures. | en |
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| dc.description.tableofcontents | 論文審定書............................................................. i Acknowledgement........................................................ii 中文摘要 ...............................................................iii ABSTRACT...............................................................iv Table of Contents......................................................v Chapter 1 Introduction...............................................1 1.1 The structure and functions of podosome..........................1 1.2 The function of dynamin-2 at podosome............................4 1.3 Dyn2 is a substrate of Src kinase ...............................7 Chapter 2 Materials and Methods......................................10 2.1 Cell culture, transfection......................................10 2.2 Matrix degradation assay .......................................11 2.3 Immunofluorescent staining and imaging.............................12 2.4 Fix image quantification .......................................13 2.5 Transmission EM ...............................................13 2.6 Protein purification and GST pulldown assay.....................13 2.7 Liposome preparation............................................14 2.8 F-actin bundle/ liposome binding sedimentation assay............15 2.9 GTPase activity assay...........................................16 2.10 Transferrin uptake assay .......................................16 2.11 Statistical analysis............................................17 Chapter 3 Results....................................................18 3.1 Phospho-deficient dynamin-2 mutant (Dyn2Y597F) is unable to target to podosome .......................................................................18 3.2 Phospho-mimetic Dynamin-2 mutant (Dyn2Y597E) enriches at actin puncta in C2C12 myoblasts..............................................................19 3.3 The phosphorylation on Dyn2Y231 results in abnormal aggregation in C2C12 myoblasts..............................................................19 3.4 The Dyn2Y597E perfectly enrich to podosome rosette in c-Src transformed NIH3T3 cells. ...............................................................20 3.5 The expression of Dyn2Y597F inhibits ECM degradation function of c-Src transformed NIH3T3 cells...............................................21 3.6 The Dyn2Y597E increase podosome lifetime .......................22 3.7 Dyn2Y597E displays increased actin binding ability..............23 3.8 Dyn2Y597E has enhanced basal GTPase activity....................24 3.9 Dyn2Y597E has increased self-assembly ability...................24 3.10 Dyn2Y597E shows decreased lipid binding ability. ...............26 3.11 Dyn2Y597E disturbs transferrin uptake of c-Src transformed NIH3T3 cells. 26 3.12 Both Dyn2Y597E and Y597F disturb transferrin uptake internalization in HeLa cells. ...............................................................27 3.13 The Dyn2 biochemical activity mutant affect podosome degradation function. .......................................................................28 Chapter 4 Discussion ...............................................30 4.1 The Y597 phosphorylation switch Dyn2 affinity between actin and membrane 30 4.2 The Y597 phosphorylation regulate Dyn2 activity at podosome 32 4.3 The regulation of dynamin activity between phosphorylation/ dephosphorylation ........................................................................33 4.4 The Dyn2Y597E phosphorylation inhibit endocytosis................34 Chapter 5 Figures.....................................................36 Figure 1. Current understandings of podosome and dynamic................36 Figure 2. The phospho-deficient Dyn2Y597F mutant shows decreased enrichment to podosome in C2C12 myotube...............................................38 Figure 3. Dyn2Y597E mutant enriches with actin puncta in C2C12 myoblast.40 Figure 4. The mutant of Dyn2Y231 tyrosine site results in abnormal aggregation. 42 Figure 5. Dyn2Y597E strongly enriches to the podosome rosette in c-Src transformed NIH3T3 cells. ........................................................44 Figure 6. Dyn2Y597F affects podosome formation and ECM degradation function.46 Figure 7. The Dyn2Y597E increases the size of podosome. ................48 Figure 8. Dyn2Y597E increases podosome lifetime.........................50 Figure 9. Dyn2Y597E has higher F-actin bundling activity with GTP. 52 Figure 10. Dyn2Y597E shows enhanced actin bundling ability under TEM. 54 Figure 11. Dyn2 Y597E shows enhanced basal GTPase activity..............56 Figure 12. The self-assembly activity of Dyn2 mutants...................58 Figure 13. Dyn2Y597E shows decreased lipid binding ability..............60 Figure 14. Dyn2Y597E shows disturbed transferrin internalization in c-Src transformed NIH3T3 cells. ........................................................62 Figure 15. Dyn2Y597E disturbs transferrin uptake in HeLa cells. ........64 Figure 16. Dyn2Y597F is enriched at microtubule.........................66 Figure 17. The ECM degradation ability of different Dyn2 mutants........68 Figure 18. The schematic diagram of Src-mediated Dyn2 activity at podosome structure. ........................................................................70 Chapter 6 Reference ................................................72 | |
| dc.language.iso | en | |
| dc.title | 探討Src磷酸化調控之dynamin-2在侵襲體的活性及功能 | zh_TW |
| dc.title | Src-mediated Dynamin-2 Phosphorylation Regulates Its Activity at Podosome | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李芳仁(Fang-Jen Lee),陳炳宏(Ping-Hung Chen) | |
| dc.subject.keyword | 侵襲體,dynamin-2,Src 磷酸酶磷酸化, | zh_TW |
| dc.subject.keyword | Podosome,dynamin-2,Src kinase phosphorylation, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202002359 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-05 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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