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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李芳仁(Fang-Jen Lee) | |
dc.contributor.author | Tai-Chen Tsai | en |
dc.contributor.author | 蔡岱蓁 | zh_TW |
dc.date.accessioned | 2021-06-17T04:36:45Z | - |
dc.date.available | 2023-10-03 | |
dc.date.copyright | 2018-10-03 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-08 | |
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Yeh, T.C., et al., Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. J Biol Chem, 1996. 271(6): p. 2921-8. 21. Okamura-Oho, Y., T. Miyashita, and M. Yamada, Distinctive tissue distribution and phosphorylation of IRSp53 isoforms. Biochem Biophys Res Commun, 2001. 289(5): p. 957-60. 22. Oda, K., et al., Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1. Cytogenet Cell Genet, 1999. 84(1-2): p. 75-82. 23. Miyahara, A., et al., Genomic structure and alternative splicing of the insulin receptor tyrosine kinase substrate of 53-kDa protein. J Hum Genet, 2003. 48(8): p. 410-4. 24. Kang, J., H. Park, and E. Kim, IRSp53/BAIAP2 in dendritic spine development, NMDA receptor regulation, and psychiatric disorders. Neuropharmacology, 2016. 100: p. 27-39. 25. Scita, G., et al., IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions. Trends Cell Biol, 2008. 18(2): p. 52-60. 26. Ahmed, S., W.I. Goh, and W. Bu, I-BAR domains, IRSp53 and filopodium formation. Semin Cell Dev Biol, 2010. 21(4): p. 350-6. 27. Zhao, H., A. Pykalainen, and P. Lappalainen, I-BAR domain proteins: linking actin and plasma membrane dynamics. Curr Opin Cell Biol, 2011. 23(1): p. 14-21. 28. Yamagishi, A., et al., A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein. J Biol Chem, 2004. 279(15): p. 14929-36. 29. Millard, T.H., et al., Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53. EMBO J, 2005. 24(2): p. 240-50. 30. Mattila, P.K., et al., Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J Cell Biol, 2007. 176(7): p. 953-64. 31. Govind, S., et al., Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin. J Cell Biol, 2001. 152(3): p. 579-94. 32. Choi, J., et al., Regulation of dendritic spine morphogenesis by insulin receptor substrate 53, a downstream effector of Rac1 and Cdc42 small GTPases. J Neurosci, 2005. 25(4): p. 869-79. 33. Disanza, A., et al., CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP. EMBO J, 2013. 32(20): p. 2735-50. 34. Oikawa, T., et al., IRSp53 mediates podosome formation via VASP in NIH-Src cells. PLoS One, 2013. 8(3): p. e60528. 35. Sathe, M., et al., Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis. Nat Commun, 2018. 9(1): p. 1835. 36. Kast, D.J., et al., Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors. Nat Struct Mol Biol, 2014. 21(4): p. 413-22. 37. Krugmann, S., et al., Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex. Curr Biol, 2001. 11(21): p. 1645-55. 38. Miki, H., et al., IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature, 2000. 408(6813): p. 732-5. 39. Miki, H. and T. Takenawa, WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac. Biochem Biophys Res Commun, 2002. 293(1): p. 93-9. 40. Connolly, B.A., et al., Tiam1-IRSp53 complex formation directs specificity of rac-mediated actin cytoskeleton regulation. Mol Cell Biol, 2005. 25(11): p. 4602-14. 41. Suetsugu, S., et al., Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP(3), and Rac. J Cell Biol, 2006. 173(4): p. 571-85. 42. Abou-Kheir, W., et al., Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2. J Cell Sci, 2008. 121(Pt 3): p. 379-90. 43. Lim, K.B., et al., The Cdc42 effector IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics. J Biol Chem, 2008. 283(29): p. 20454-72. 44. Heung, M.Y., et al., Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53. Eur J Cell Biol, 2008. 87(8-9): p. 699-708. 45. Robens, J.M., et al., Regulation of IRSp53-dependent filopodial dynamics by antagonism between 14-3-3 binding and SH3-mediated localization. Mol Cell Biol, 2010. 30(3): p. 829-44. 46. Disanza, A., et al., Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex. Nat Cell Biol, 2006. 8(12): p. 1337-47. 47. Millard, T.H., J. Dawson, and L.M. Machesky, Characterisation of IRTKS, a novel IRSp53/MIM family actin regulator with distinct filament bundling properties. J Cell Sci, 2007. 120(Pt 9): p. 1663-72. 48. Suetsugu, S., et al., The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation. J Biol Chem, 2006. 281(46): p. 35347-58. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70739 | - |
dc.description.abstract | 二磷酸線苷核醣基化因子 (Arfs) 是一群調控膜運輸、胞器完整性及細胞骨架動態性的小分子鳥嘌呤核苷酸結合蛋白 (Small GTP binding protein),其中與其結構相似的腺嘌呤核苷二磷酸核醣化相似因子4 (Arl4s) 家族包含了三個成員,Arl4A,Arl4C及Arl4D。在這篇論文中,我們發現一個新的Arl4D的作用蛋白-IRSp53。IRSp53受到兩個小分子鳥嘌呤核苷酸結合蛋白,Cdc42及Rac1的調控,分別決定其促進絲狀偽足 (filopodia) 或是板狀偽足 (lamellipodia) 的形成。透過酵母菌雙雜交系統 (yeast two hybrid) 的檢測,只有Arl4D會專一和IRSp53結合,Arl4A及Arl4C不會。進一步由試管結合實驗 (in vitro binding assay) 及免疫共沉降 (co-immunoprecipitation) 實驗驗證,我們發現持續活化及不活化的Arl4D都能和IRSp53結合。由酵母菌雙雜交系統及試管結合實驗,我們發現IRSp53 N端的229個胺基酸殘基對於兩者的結合是重要的,且和過去被報導的Rac1結合位相同。靜默IRSp53不會改變Arl4D在膜上的分布,相反地,剔除Arl4D會讓IRSp53誘使的突出數目減少,此外,只有持續活化的Arl4D會降低IRSp53誘使的絲狀偽足的長度。透過免疫共沉降實驗,我們發現Arl4D能夠和Cdc42及IRSp53存在於同一個複合體當中。總而言之,我們發現一個新的小分子鳥嘌呤核苷酸結合蛋白Arl4D能夠和IRSp53交互作用,Arl4D可能透過未知的調控方式參與在絲狀偽足的形成當中。 | zh_TW |
dc.description.abstract | ADP-ribosylation factors (Arfs) are small GTP binding proteins involved in membrane transport, the maintenance of organelle integrity and cytoskeletal dynamics. The Arf-like 4 (Arl4) family is composed of three isoforms, Arl4A, Arl4C and Arl4D. Here, we identified a novel interacting protein of Arl4D, called insulin substrate receptor p53 (IRSp53). IRSp53 is a main actin modulator that regulates filopodia and lamellipodia formation, mediated by two small GTPases, Cdc42 and Rac1, respectively. Through yeast two hybrid, we found that only Arl4D, but not Arl4A or Arl4C, interacted with IRSp53. Confirmed by in vitro binding assay and co-immunoprecipitation, we demonstrated that both constitutively active and inactive mutants of Arl4D interacted with IRSp53. Revealed from yeast two hybrid and in vitro binding assay, the shortest region important for their interaction resided in the N terminal 229 residues of IRSp53, which is the same as reported Rac1 binding region. Knockdown of IRSp53 did not affect the membrane localization of Arl4D. By contrast, knockout of Arl4D decreased the number of IRSp53-induced filopodium-like protrusions. In addition, coexpression of WT and constitutively active but not inactive mutant of Arl4D reduced the average length of IRSp53-induced protrusions. Last but not least, Arl4D was found within the same complex with Cdc42 and IRSp53 from co-immunoprecipitation. In conclusion, we found that Arl4D is a novel small GTPase that interacts with IRSp53, which might regulate filopodia formation through unknown mechanism via IRSp53. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T04:36:45Z (GMT). No. of bitstreams: 1 ntu-107-R05448005-1.pdf: 3467151 bytes, checksum: 83e2333417d786681668b08e5cd3ca5b (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iii Abstract iv Abbreviations vi Contents vii 1. Introduction 1 1-1. Small GTP binding protein 1 1-2. ADP-ribosylation factor (Arf family) 1 1-3. ADP-ribosylation factor-like 4 (Arl4) family 2 1-4. Small G protein interplay in actin cytoskeleton remodeling 3 1-5. Overview of IRSp53/BAIAP2 4 1-5-1. Isoforms of IRSp53/BAIAP2 4 1-5-2. I-BAR domain/IMD and membrane curvature 4 1-5-3. Physiological function of IRSp53 5 1-5-4. Lamellipodia and filopodia formation mediated by Rac1 and Cdc42, respectively 6 1-5-5. Role of phosphorylation 8 2. Materials and methods 9 2-1. Antibodies 9 2-2. Cell culture and transfection 9 2-3. Expression Plasmids 9 2-4. Lentivirus delivered shRNA knockdown system 10 2-5. pSUPER RNAi system for Arl4D 11 2-6. Yeast two hybrid 11 2-7. Recombinant proteins production and purification 12 2-8. In vitro binding assay 14 2-9. Co-immunoprecipitation 15 2-10. Immunofluorescence (IF) 15 2-11. Proximity ligation assay 16 2-12. Transwell migration assay 17 3. Results 18 3-1. Identification of IRSp53 as a novel interacting protein of Arl4 family. 18 3-2. IRSp53 specifically interacts with Arl4D. 19 3-3. Confirm interaction by proximity ligation assay. 20 3-4. Binding of Arl4D to IRSp53 is nucleotide-independent. 21 3-5. Overexpression of Arl4D reduces the length of protrusions induced by IRSp53. 22 3-6. IRSp53 N terminal 229 residues are sufficient for its interaction with Arl4D. 23 3-7. Knockdown of IRSp53/BAIAP2 does not affect the plasma membrane localization of Arl4D. 24 3-8. Knockdown of Arl4D slightly reduces IRSp53-induced protrusions. 25 3-9. Arl4D forms a complex with Cdc42 and IRSp53. 26 3-10. Relationship of Arl4D and Rac1 with IRSp53 27 3-11. Examination of whether Arl4D induces migration through IRSp53. 28 4. Discussion 30 5. Tables 34 Table 1. Antibodies used in the thesis 34 Table 2. Oligonucleotides used in the thesis 35 6. Figures 36 Figure 1. Arl4D WT specifically interacts with IRSp53 in yeast two hybrid. 36 Figure 2. Arl4D specifically interacts with IRSp53 both in vitro and in vivo. 37 Figure 3. WT, QL and TN mutants of Arl4D co-immunoprecipitate with IRSp53. 39 Figure 4. WT, QL and TN mutants of Arl4D directly interact with IRSp53 in vitro. 40 Figure 5. Arl4D, and also Arl4A, colocalize on IRSp53-induced protrusions. 42 Figure 6. WT and constitutively active Arl4D reduce the average length of IRSp53-induced protrusions. 44 Figure 7. IRSp53 1-229 residues are important for Arl4D interaction. 46 Figure 8. Deletion mutants of IRSp53 lose interaction with Ar4D, however, they form severe aggregations when expressed in COS 7 cells. 49 Figure 9. Knockdown of IRSp53/BAIAP2 does not affect the membrane localization of Arl4D. 50 Figure 10. Knockdown of Arl4D does not affect IRSp53-induced protrusions. 51 Figure 11. Knockout of Arl4D reduces the number of IRSp53-induced protrusions. 52 Figure 12. Arl4D exists in the same complex with Cdc42 and IRSp53. 53 Figure 13. Both Arl4D and IRSp53 promote cell migration. 54 Figure 14. Proposed working model. 56 7. Supplementary figures 57 Figure S1. Arl4A* Q79L interacts with IRSp53 1-340 much stronger than Arl4A Q79L. 57 Figure S2. Arl4D interacts with IRSp53 revealed by proximity ligation assay. 59 Figure S3. The second and third experimental repeat of figure 3. 60 Figure S4. The second and third experimental repeat of figure 4. 60 Figure S5. The second and third experimental repeat of figure 8A. 61 Figure S6. The second and third experimental repeat of figure 8B. 62 Figure S7. Direct interaction of Rac1 and IRSp53 is not detected in in vitro binding assay. 64 8. References 65 | |
dc.language.iso | en | |
dc.title | 人類腺嘌呤核苷二磷酸核醣化相似因子四與其結合蛋白IRSp53/BAIAP2特性之探討 | zh_TW |
dc.title | Functional characterization of human Arl4D and its interacting protein, IRSp53/BAIAP2 | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周祖述(Tzuu-Shuh Jou),陳瑞華(Ruey-Hwa Chen),黃佩欣(Pei-Hsin Huang),劉雅雯(Ya-Wen Liu) | |
dc.subject.keyword | 腺嘌呤核?二磷酸核醣化相似因子4D,IRSp53/BAIAP2,Cdc42,肌動蛋白細胞骨架重塑,絲狀偽足,板狀偽足, | zh_TW |
dc.subject.keyword | Arl4D,IRSp53/BAIAP2,Cdc42,actin cytoskeleton remodeling,filopodia,lamellipodia, | en |
dc.relation.page | 71 | |
dc.identifier.doi | 10.6342/NTU201802758 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-08 | |
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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