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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許家維 | zh_TW |
| dc.contributor.advisor | Jia-Wei Hsu | en |
| dc.contributor.author | 賴冠妘 | zh_TW |
| dc.contributor.author | Kuan-Yun Lai | en |
| dc.date.accessioned | 2024-09-12T16:21:28Z | - |
| dc.date.available | 2024-09-13 | - |
| dc.date.copyright | 2024-09-12 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-09 | - |
| dc.identifier.citation | Bairstow, S.F., K. Ling, X.J. Su, A.J. Firestone, C. Carbonara, and R.A. Anderson. 2006. Type Iγ661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis. J Biol Chem. 281:20632-20642.
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Cockcroft. 2020. Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane. Essays Biochem. 64:513-531. Mills, I.G., S. Urbe, and M.J. Clague. 2001. Relationships between EEA1 binding partners and their role in endosome fusion. J Cell Sci. 114:1959-1965. Navaroli, D.M., K.D. Bellve, C. Standley, L.M. Lifshitz, J. Cardia, D. Lambright, D. Leonard, K.E. Fogarty, and S. Corvera. 2012. Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis. Proc. Natl. Acad. Sci. U. S. A. 109:E471-480. Nielsen, E., S. Christoforidis, S. Uttenweiler-Joseph, M. Miaczynska, F. Dewitte, M. Wilm, B. Hoflack, and M. Zerial. 2000a. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J Cell Biol. 151:601-612. Nielsen, E., S. Christoforidis, S. Uttenweiler-Joseph, M. Miaczynska, F. Dewitte, M. Wilm, B. Hoflack, and M. Zerial. 2000b. Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain. J Cell Biol. 151:601-612. O'Sullivan, M.J., and A.J. Lindsay. 2020. The Endosomal Recycling Pathway-At the Crossroads of the Cell. Int J Mol Sci. 21. Padrón, D., Y.J. Wang, M. Yamamoto, H. Yin, and M.G. Roth. 2003. Phosphatidylinositol phosphate 5-kinase Iβ recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis. J Cell Biol. 162:693-701. Pearse, B.M., and M.S. Robinson. 1990. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 6:151-171. Qi, Y., Z. Liang, Z. Wang, G. Lu, and G. Li. 2015. Determination of Rab5 activity in the cell by effector pull-down assay. Methods Mol Biol. 1298:259-270. Rink, J., E. Ghigo, Y. Kalaidzidis, and M. Zerial. 2005. Rab conversion as a mechanism of progression from early to late endosomes. Cell. 122:735-749. Rong, Y., M. Liu, L. Ma, W. Du, H. Zhang, Y. Tian, Z. Cao, Y. Li, H. Ren, C. Zhang, L. Li, S. Chen, J. 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An unexpected role for PI4,5P 2 in EGF receptor endosomal trafficking. Cell Cycle. 12:1991-1992. Tan, X.J., N. Thapa, S. Choi, and R.A. Anderson. 2015. Emerging roles of PtdIns(4,5) - beyond the plasma membrane. J Cell Sci. 128:4047-4056. Tsai, M.T., N. Katagiri, N. Ohbayashi, K. Iwasaki, N. Ohkohchi, S.T. Ding, Y. Kanaho, and Y. Funakoshi. 2017. Regulation of HGF-induced hepatocyte proliferation by the small GTPase Arf6 through the PIP(2)-producing enzyme PIP5K1A. Sci Rep. 7:9438. Wallroth, A., and V. Haucke. 2018. Phosphoinositide conversion in endocytosis and the endolysosomal system. J Biol Chem. 293:1526-1535. Xie, Z., S.M. Chang, S.D. Pennypacker, E.Y. Liao, and D.D. Bikle. 2009. Phosphatidylinositol-4-phosphate 5-kinase 1alpha mediates extracellular calcium-induced keratinocyte differentiation. Mol Biol Cell. 20:1695-1704. Yang, C., and X. Wang. 2021. Lysosome biogenesis: Regulation and functions. J Cell Biol. 220. Yuan, W., and C. Song. 2020. The Emerging Role of Rab5 in Membrane Receptor Trafficking and Signaling Pathways. Biochem Res Int. 2020:4186308. Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2:107-117. Zhang, Z., T. Zhang, S. Wang, Z. Gong, C. Tang, J. Chen, and J. Ding. 2014. Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5. Elife. 3. Zoncu, R., R.M. Perera, D.M. Balkin, M. Pirruccello, D. Toomre, and P. De Camilli. 2009. A phosphoinositide switch controls the maturation and signaling properties of APPL endosomes. Cell. 136:1110-1121. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95618 | - |
| dc.description.abstract | 磷脂醯肌醇-4,5-二磷酸（PI(4,5)P2）是一種重要的脂質類的信號分子，在細胞中有許多種功能，其中包括訊號傳遞、分子分泌以及囊泡運輸。磷脂醯肌醇-4,5-二磷酸是由磷脂酰肌醇-5-磷酸酶（PIP5K）磷酸化磷脂醯肌醇4-磷酸（PI(4)P）而成。而磷脂酰肌醇-5-磷酸酶可進一步細分為磷脂酰肌醇-5-磷酸酶1A（PIP5K1A）、磷脂酰肌醇-5-磷酸酶1B（PIP5K1B）和磷脂酰肌醇-5-磷酸酶1C（PIP5K1C）。在先前的研究發現磷脂醯肌醇-4,5-二磷酸可以調節載鐵蛋白受體（TfR）和表皮生長因子受體（EGFR）的內吞運輸。本研究中，我們發現細胞缺失磷脂酰肌醇-5-磷酸酶1A（PIP5K1A）的會抑制內吞作用以及分子運輸到晚期胞內體（Late endosome）或溶酶體（Lysosome）的運輸。此外，缺失磷脂酰肌醇-5-磷酸酶1A（PIP5K1A）的情況下也抑制了載鐵蛋白受體（TfR）回收到細胞膜表面，使其累積在胞內體。除此之外，我們還發現到在缺失磷脂酰肌醇-5-磷酸酶1A（PIP5K1A）的情況下，會使得胞內體成熟的途徑也受到抑制，另外也造成了胞內體重要調節蛋白Rab5活性顯著下調。總括而言，我們的結果表明脂酰肌醇-5-磷酸酶1A（PIP5K1A）不僅在調節內吞運輸途徑中發揮特定作用，還在胞內體的成熟以及空間分佈中有扮演其角色。 | zh_TW |
| dc.description.abstract | Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is a key lipid signaling molecule that plays several cellular functions in cells, including signal transduction, secretion, and vesicular trafficking. PI(4,5)P2 is generated from PI(4)P by class 1 PIP5K (Phosphatidylinositol-4-phosphate 5-kinase). Class 1 PIP5K, generating PI(4,5)P2 from PI(4)P, can be further classified as PIP5K1A, PIP5K1B and PIP5K1C. Previous studies demonstrated that PI(4,5)P2 regulates the endocytic transport of transferrin receptor (TfR) and epidermal growth factor receptor (EGFR). Yet, the mechanism that distinguishes PIP5K isoform-dependent endocytic transport has remained elusive. In this study, we examined several intracellular transport pathways for the role of PIP5K during endocytic transport, including clathrin-mediated endocytosis, transferrin recycling pathway, and lysosomal transport. We found that loss of PIP5K1A specifically inhibits cargo internalization to the early endosome, and EGFR transport to the late endosome/lysosome. In addition, knockdown of PIP5K1A inhibited TfR recycling back to the plasma membrane, leading to the accumulation of TfR within the recycling endosome. Moreover, colocalization of the late endosome or recycling endosome with early endosome was dramatically increased, and the early endosome was clustered at the perinuclear region in siPIP5K1A-treated cells, indicating the disorder of the endosomal membrane system. Furthermore, the activity of Rab5, a master early endosome regulator, was downregulated under the PIP5K1A knockdown cells. Together, our results identify the specific role of PIP5K1A not only participates in regulating endocytic transport pathways but also organizing the spatial distribution of endosomes. These advances our understanding of how endosomal membrane systems and transport are tightly interconnected. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-09-12T16:21:28Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-09-12T16:21:28Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 論文口試委員審定書 i
致謝 ii 摘要 iii Abstract iv Table of contents vi Chapter 1 Introduction 1 1.1 Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a crucial lipid, which participate in several cellular processes. 1 1.2 PI(4,5)P2 is generated from phosphatidylinositol-4-phosphate 5-kinase (PIP5K) class I family. 2 1.3 Three isoforms of PIP5K1 play different role in cellular processes 2 1.4 Early endosome is an essential compartment for cargo sorting. 4 1.5 Rab5 plays a pivotal role in modulating the endosomal system, influencing both the sorting processes and the trafficking. 4 1.6 Study background 5 Chapter 2 Material and Method 7 2.1 Cell line and Cell culture 7 2.2 siRNA transfection (knockdown) 7 2.3 Plasmid transfection 7 2.4 Plasmids construct 8 2.5 In vivo endocytic protein transport assay 8 2.5.1 Transferrin endocytosis 8 2.5.2 Transferrin (Tf) recycling 9 2.5.3 EGFR endocytosis 10 2.6 Immunofluorescence 10 2.7 Western Blotting 11 2.7.1 Sample preparation 11 2.7.2 SDS-PAGE and Immunoblotting 11 2.8 Rab5 activity assay 12 2.8.1 Expression of GST-R5BD Fusion Proteins 12 2.8.2 Purification of GST-R5BD Fusion Proteins 12 2.8.3 Preparation of Cell Lysates 13 2.8.4 GST-R5BD Pull-Down Assay 14 2.9 Confocal microscopy. 14 2.11 Analysis 14 2.12 Chemical, antibodies 15 2.12.1 Antibody 15 2.12.2 Reagent 17 2.12.3 siRNA sequence 18 2.12.4 RT-PCR 19 2.12.5 PCR 19 Chapter 3 Results 21 3.1 PIP5K1A is primarily responsible for producing PI(4,5)P2 on early endosomes. 21 3.2 PIP5K1A promotes EGFR internalization and EGFR degradative pathway. 22 3.3 Internalization and recycling of transferrin receptors are inhibited in siPIP5K1A-treated cells. 23 3.4 The kinase activity of PIP5K1A promotes EGFR endocytosis, transferrin endocytosis and TfR recycling. 24 3.5 PIP5K1A plays an crucial role in remodeling the endosomal membrane and cargo trafficking within the endosomal system. 25 3.6 PIP5K1A kinase ability increases Rab5 activity in vivo. 27 3.7 PIP5K1A determines the spatial and structural distribution of early endosomes through kinase-independent manner. 29 3.8 Endosomal localization of PIP5K1A is necessary for early endosome distribution and cargo trafficking. 30 Chapter 4 Conclusion 32 Chapter 5 Discussion 32 5.1 The role of PIP5K1A in transport pathways 32 5.2 Role of PI(4,5)P2 in endosomal maturation 34 5.2.1 Lipid conversion failure impairs endosomal maturation 34 5.2.2 Decreased Rab5 activity impairs endosomal maturation. 35 5.3 Changes in phosphoinositide composition can alter Rab5 functions. 36 Chapter 6 Figures 38 Figure 1. PIP5K1A, PIP5K1B and PIP5K1C gene expression levels after knocking down. 38 Figure 2. PIP5K1A poses a PIP5K1A-specific effect on the distribution of PI(4,5)P2 on early endosomes. 40 Figure 3. Knocking down PIP5K1A decreases GFP-C1-PLCẟ-PH colocalized with early endosomes. 41 Figure 4. PIP5K1A is found on early endosomes. 42 Figure 5. PIP5K1A protein expression level is significantly downregulated after RNA interference. 43 Figure 6. PIP5K1A promotes EGFR internalization. 44 Figure 7. PIP5K1A promotes EGFR transporting to lysosomes. 45 Figure 8. Internalization of transferrin receptors is inhibited in siPIP5K1A-treated cells. 46 Figure 9. PIP5K1A depletion causes transferrin receptors recycling inhibited. 47 Figure 10. Loss of PIP5K1A inhibits transferrin recycling. 48 Figure 11. The kinase activity of PIP5K1A promotes EGFR endocytosis. 49 Figure 12. The kinase activity of PIP5K1A for generating PI(4,5)P2 promotes TfR endocytosis. 50 Figure 13. PI(4,5)P2 produced by PIP5K1A can promote transferrin receptor recycling transport. 51 Figure 14. PIP5K1A depletion causes early endosome clustering and inhibit early endosome mature into late endosome. 52 Figure 15. Knocking down PIP5K1A inhibits early endosome maturing into recycling endosome. 53 Figure 16. EGFR sorting to the late endosome is delayed in siPIP5K1A-treated cells. 54 Figure 17. Transferrin sorting to recycling endosomes is impaired in siPIP5K1A-treated cells. 55 Figure 18. PI(4,5)P2 generating by PIP5K1A participates in maintaining endosomal maturation. 56 Figure 19. Using Rab5 binding domain (R5BD) of Rab5 effector can effectively pull down active form Rab5 (GTP-bound Rab5). 57 Figure 20. All three isotypes in PIP5K1 family promote Rab5 activity. 58 Figure 21. PIP5K1A depletion decreases Rab5-GTP levels. 59 Figure 22. Rab5 activity is PIP5K1A kinase-dependent. 60 Figure 23. Early endosome clustering can be reversed by both wild-type and kinase-dead mutant PIP5K1A. 61 Figure 24. V5-PIP5K1A-Fis1 can significantly redirect PIP5K1A toward mitochondria outer membrane. 62 Figure 25. Endosomal localization of PIP5K1A for generating PI(4,5)P2 determines transferrin endocytosis. 64 Reference 65 | - |
| dc.language.iso | en | - |
| dc.subject | 磷脂酰肌醇-5-磷酸酶- 1A | zh_TW |
| dc.subject | 早期胞內體分佈 | zh_TW |
| dc.subject | 內膜系統 | zh_TW |
| dc.subject | 囊泡運輸 | zh_TW |
| dc.subject | 5-二磷酸 | zh_TW |
| dc.subject | 磷脂醯肌醇-4 | zh_TW |
| dc.subject | endosomal maturation | en |
| dc.subject | endocytic transport | en |
| dc.subject | 5)P2 | en |
| dc.subject | PI(4 | en |
| dc.subject | Rab5 activity | en |
| dc.subject | organelle cluster | en |
| dc.title | 脂質磷酸酶PIP5K1A調節內吞蛋白運輸以及維持內膜系統完整性 | zh_TW |
| dc.title | Lipid Kinase PIP5K1A Regulates Endocytic Protein Transport and Remodels Endosomal Membrane System | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 劉雅雯;李家瑋 | zh_TW |
| dc.contributor.oralexamcommittee | Ya-Wen Liu;Chia-Wei Lee | en |
| dc.subject.keyword | 磷脂酰肌醇-5-磷酸酶- 1A,磷脂醯肌醇-4,5-二磷酸,囊泡運輸,內膜系統,早期胞內體分佈, | zh_TW |
| dc.subject.keyword | PI(4,5)P2,endocytic transport,endosomal maturation,organelle cluster,Rab5 activity, | en |
| dc.relation.page | 71 | - |
| dc.identifier.doi | 10.6342/NTU202403588 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-08-12 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2029-08-13 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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