請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90227
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡丰喬 | zh_TW |
dc.contributor.advisor | Feng-Chiao Tsai | en |
dc.contributor.author | 曾宇岑 | zh_TW |
dc.contributor.author | Yu-Chen Tseng | en |
dc.date.accessioned | 2023-09-24T16:06:07Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-09-23 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-08-09 | - |
dc.identifier.citation | 1. Hsueh, Y.-P. The role of the MAGUK protein CASK in neural development and synaptic function. Curr. Med. Chem. 13, 1915–1927 (2006).
2. Atasoy, D., Schoch, S., Ho, A., et al. Deletion of CASK in mice is lethal and impairs synaptic function. Proc Natl Acad Sci USA. 7, 2525-2530 (2007). 3. Becker, M., Mastropasqua, F., Reising, J. P., Maier, S., Ho, M. L., Rabkina, I., Li, D., Neufeld, J., Ballenberger, L., Myers, L., Moritz, V., Kele, M., Wincent, J., Willfors, C., Sitnikov, R., Herlenius, E., Anderlid, B. M., Falk, A., Bölte, S., & Tammimies, K. Presynaptic dysfunction in CASK-related neurodevelopmental disorders. Transl. Psychiatry 10, 312. (2020). 4. Hsueh YP. The role of the MAGUK protein CASK in neural development and synaptic function. Curr Med Chem 13, 1915–1927( 2006). 5. Lehtonen S, Ryan JJ, Kudlicka K, Iino N, Zhou H, Farquhar MG. Cell junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and alpha-actinin are components of the nephrin multiprotein complex. Proc Natl Acad Sci U S A. 102(28): 9814-9819. (2005). 6. Malik, B. R. & Hodge, J. J. L. CASK and CaMKII function in Drosophila memory. Front Neurosci 8, 178 (2014). 7. Chao, H.-W., Hong, C.-J., Huang, T.-N., Lin, Y.-L. & Hsueh, Y.-P. SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis. J. Cell Biol. 182, 141–155 (2008). 8. Mustroph, J. et al. Loss of CASK Accelerates Heart Failure Development. Circ Res 128, 1139–1155 9. Ojeh, N., Pekovic, V., Jahoda, C. & Määttä, A. The MAGUK-family protein CASK is targeted to nuclei of the basal epidermis and controls keratinocyte proliferation. J. Cell. Sci. 121, 2705–2717 (2008). 10. Qu, J., Zhou, Y., Li, Y., Yu, J. & Wang, W. CASK regulates Notch pathway and functions as a tumor promoter in pancreatic cancer. Arch Biochem Biophys 701, 108789 (2021). 11. Kaech, S. M., Whitfield, C. W. & Kim, S. K. The LIN-2/LIN-7/LIN-10 Complex Mediates Basolateral Membrane Localization of the C. elegans EGF Receptor LET-23 in Vulval Epithelial Cells. Cell 94, 761–771 (1998). 12. Aravindan, R. G. et al. CASK interacts with PMCA4b and JAM-A on the mouse sperm flagellum to regulate Ca2+ homeostasis and motility. J. Cell. Physiol. 227, 3138–3150 (2012). 13. Martinez-Estrada OM, Villa A, Breviario F, Orsenigo F, Dejana E, Bazzoni G. Association of junctional adhesion molecule with calcium/calmodulin-dependent serine protein kinase (CASK/LIN-2) in human epithelial caco-2 cells. J Biol Chem. 276(12):9291-9296. (2001) 14. Ohno H, Hirabayashi S, Kansaku A, et al. Carom: a novel membrane-associated guanylate kinase-interacting protein with two SH3 domains. Oncogene. 22, 8422-8431. 15. Mukherjee K, Sharma M, Urlaub H, et al. CASK Functions as a Mg2+-independent neurexin kinase. Cell. 133(2):328-339. (2008). 16. Alday-Parejo B, Ghimire K, Coquoz O, et al. MAGI1 localizes to mature focal adhesion and modulates endothelial cell adhesion, migration and angiogenesis. Cell Adh Migr. 15(1):126-139. (2021). 17. Rios-Doria J, Day KC, Kuefer R, et al. The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells. J Biol Chem. 278(2):1372-1379. (2003) 18. Liu S, Xiong X, Zhao X, Yang X, Wang H. F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases. J Hematol Oncol. 8:47. (2015). 19. Burridge K. Focal adhesions: a personal perspective on a half century of progress. FEBS J. 284(20):3355-3361. (2017). 20. Mori T, Kasem EA, Suzuki-Kouyama E, et al. Deficiency of calcium/calmodulin-dependent serine protein kinase disrupts the excitatory-inhibitory balance of synapses by down-regulating GluN2B. Mol Psychiatry. 24(7):1079-1092. (2019) 21. Almeida-Souza L, Frank RAW, García-Nafría J, et al. A Flat BAR Protein Promotes Actin Polymerization at the Base of Clathrin-Coated Pits. Cell. 174(2):325-337. (2018) 22. Liu S, Xiong X, Thomas SV, et al. Analysis for Carom complex, signaling and function by database mining. Front Bioscii. 21(4):856-872. (2016). 23. Murata A, Okuyama K, Sakano S, et al. A Notch ligand, Delta-like 1 functions as an adhesion molecule for mast cells. J Immunol. 185(7):3905-3912. (2010). 24. Wörthmüller J, Rüegg C. MAGI1, a Scaffold Protein with Tumor Suppressive and Vascular Functions. Cells. 10(6):1494. (2021). 25. Carr HS, Zuo Y, Oh W, Frost JA. Regulation of focal adhesion kinase activation, breast cancer cell motility, and amoeboid invasion by the RhoA guanine nucleotide exchange factor Net1. Mol Cell Biol. 33(14):2773-2786. (2013). 26. Deli MA. Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery. Biochim Biophys Acta. 1788(4):892-910. (2009). 27. Swulius MT, Waxham MN. Ca(2+)/calmodulin-dependent protein kinases. Cell Mol Life Sci. 65(17):2637-2657. (2008). 28. Li Y, Karnak D, Demeler B, Margolis B, Lavie A. Structural basis for L27 domain-mediated assembly of signaling and cell polarity complexes. EMBO J. 23(14):2723-2733. (2004). 29. Mui KL, Chen CS, Assoian RK. The mechanical regulation of integrin-cadherin crosstalk organizes cells, signaling and forces. J Cell Sci. 129(6):1093-1100. (2016). 30. Zuidema A, Wang W, Sonnenberg A. Crosstalk between Cell Adhesion Complexes in Regulation of Mechanotransduction. Bioessays. 42(11):e2000119. (2020). 31. Gillespie JM, Hodge JJ. CASK regulates CaMKII autophosphorylation in neuronal growth, calcium signaling, and learning. Front Mol Neurosci. 6:27. (2013). 32. Lu HT, Feng RQ, Tang JK, Zhou JJ, Gao F, Ren J. CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts. Cell Death Dis. 11(5):388. (2020). 33. Fang L, Zhu J, Ma Y, Hong C, Xiao S, Jin L. Neuroepithelial transforming gene 1 functions as a potential prognostic marker for patients with non-small cell lung cancer. Mol Med Rep.12(5):7439-7446. (2015). | - |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90227 | - |
dc.description.abstract | 作為膜結合關聯鸚鵡翁狀激酶(MAGUK)家族的成員,CASK被視為支架蛋白,對神經元突觸形成至關重要。我們之前的研究表明,CASK可能參與了Ca 2+ -依賴性細胞黏附。因此,我們研究了細胞黏附結構與CASK之間的聯繫。通過在頭頸部癌細胞SAS和角質形成細胞 HaCaT中使用免疫螢光(IF),我們注意到CASK敲除導致細胞-細胞黏附結構(即基於 paxillin 的黏著斑復合物)和細胞-基質黏附結構(即基於 cadherin的緊黏結)的增加,表明CASK可能參與了細胞黏附動態。我們進一步研究了在 CASK敲除期間通過抑制 Rho關聯蛋白激酶(ROCK)或鈣蛋白酶是否調節了細胞動力學的形成或溶解。結果顯示,CASK 敲除的效果與細胞黏附上的 ROCK 抑制是獨立的,而鈣蛋白酶的抑制消除了CASK 敲除對細胞黏附的影響。因此,我們推測 CASK 參與了鈣蛋白酶介導的細胞黏附動態。我們目前正在研究其潛在機制,希望能開發基於 CASK 的治療細胞黏附相關疾病的方法。 | zh_TW |
dc.description.abstract | As a member of the membrane-associated guanylate kinases (MAGUK) family, CASK is considered as a scaffold protein, and it is crucial for neuronal synapse formation. Our previous research suggested that CASK is potentially involved in Ca2+-dependent cell adhesion. We thus investigated the association between cell adhesion structures and CASK. By using immunofluorescence (IF) in head and neck cancer SAS cells and keratinocyte HaCaT cells, we noticed that CASK knockdown led to an increase in both cell-cell adhesion structures, i.e. paxillin-based focal adhesion complexes, and cell-matrix adhesion structures, i.e. cadherin-based adherens junctions, suggesting that CASK could be involved in cell adhesion dynamics. We further investigated whether CASK regulated the formation or dissolution of cell dynamics by suppressing Rho-associated protein kinase (ROCK) or calpain during CASK knockdown. Interestingly, effects of CASK knockdown were independent of ROCK suppression on cell adhesion, while calpain inhibition abolished effects of CASK knockdown on cell adhesion. Therefore, we speculate that CASK was involved in calpain-mediated cell adhesion dynamics. We are currently investigating its underlying mechanism, hoping to develop CASK-based treatments cell adhesion-related diseases. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-24T16:06:07Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-09-24T16:06:07Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | Introduction 12
Calcium/calmodulin-dependent serine protein kinase (CASK) 12 The relationship between focal adhesions and adherens junctions 12 CASK mediates adhesion between neurons and probably also between non-neuronal cells. 13 Ca2+-CASK crosstalk indicates the importance of CASK on cell adhesion 13 CASK is associated with the cell adhesion 14 CASJ regulates FAs and E-cadherin by activating calpain 14 Specific Aims 15 Materials and methods 16 Cell culture 16 Generation of knockdown plasmids and overexpression constructs 16 Generation of truncated constructs 17 Lentivirus preparation and infection 17 Cell Counting Kit-8 (CCK-8) assay 18 Double knockdown experiments 18 Immunofluorescent staining (IF) 18 Focal adhesion analysis 19 Western blotting 19 Antibodies 20 Real-time reverse transcription PCR 20 Results 21 CASK perturbations disrupted cell adhesion structures 21 1. CASK knockdown increased cell-cell adhesion and cell matrix. 21 2. CASK effects on adhesion structures might be more related to protein / structural stability than force-mediated structural formation. 21 2a. The effects of CASK on FA and E-cadherin may be calpain-related instead of being regulated by force. 22 2b. CASK effects on adhesion structures may be independent to the Rho-ROCK pathway 23 2c. Force-regulated structural strengthening did not contribute to CASK-regulated cell adhesion 24 3. CASK effects on adhesion structures might not be related to endocytosis or protein retention on the membrane. 24 4. CASK does not affect the transcriptional process of E-cadherin 25 5. CASK-mediated effects may not be carom-dependent 26 CASK-regulated cell-cell adhesion and cell-matrix may be facilitated by the interaction of its N-terminal domain and other proteins 27 Double perturbation of calpain and MAGI-1 provided an accumulation of E-cadherin signals at the cell junction sites 28 Discussion 30 Formation and heterogeneity of focal adhesions 30 CASK knockdown induced the maturation of focal adhesion 30 CASK and the regulation of transcription and translation process 31 Alteration of E-cadherin signals by shCASK and shFCHSD2 may be a summation of their individual effects 32 Possible mechanisms of actions of MAGI-1 and carom 33 HaCaT may not be the best cell model for experiments involving MAGI-1 and carom 34 MAGI-1 is a focal adhesion regulator 34 CASK-mediated adhesion may be Net1 dependent 34 CASK competes with MAGI-1 for the regulation of calpain and Net1 35 The importance of CASK N-terminal domains 37 Conclusion 39 Figures and Legends 40 Figure 1: CASK knockdown (shCASK) increased epidermal growth factor (EGF)-induced Ca2+ signals in HaCaT cells. 40 Figure 2: CASK knockdown affected FA complexes in HaCaT cells. 41 Figure 3: CASK knockdown (shCASK) affected the distribution of E-cadherin in HaCaT cells. 42 Figure 4: FA complexes were not further induced with double knockdown of CASK (shCASK) and calpain (shCalpain). 43 Figure 5: The combination of CASK knockdown and calpain inhibition did not lead to further increase in FA. 44 Figure 6: Double perturbation of CASK and calpain did not lead to further increase in FA. 45 Figure 7: The effects of calpain-KD on E-cadherin may be dependent on CASK expressions in HaCaT cells. 46 Figure 8: CASK-KD (shCASK) provided Y27632-independent effects on focal adhesion (FA) complexes in HaCaT cells. 47 Figure 9: CASK-KD provided Y27632-independent effects on E-cadherin in HaCaT cells. 48 Figure 10: CASK-KD provided CT04-independent effects on E-cadherin in HaCaT cells. 48 Figure 11: CASK knockdown did not affect the distribution of actin in HaCaT cells. 49 Figure 12: CASK knockdown (shCASK) did not affect actin distribution in HaCaT cells. 50 Figure 13: CASK-KD (shCASK) provided EGTA-independent effects on E-cadherin in HaCaT cells. 51 Figure 14: CASK-KD (shCASK) provided endocytosis-independent effects on focal adhesion (FA) complexes in HaCaT cells. 52 Figure 15: CASK-KD (shCASK) provided endocytosis-independent effects on E-cadherin in HaCaT cells. 53 Figure 16: CASK may not be regulating E-cadherin through its transcription. 54 Figure 17: CASK-KD (shCASK) provided carom-independent effects on FA complexes in HaCaT cells. 55 Figure 18: CASK-KD (shCASK) provided carom-independent effects on E-cadherin in HaCaT cells. 56 Figure 19: Double knockdown of CASK and MAGI-1 provided further increase in focal adhesion (FA) complexes in HaCaT cells. 57 Figure 20: N-terminus of CASK is important for the regulation of E-cadherin in HaCaT cells. 58 Figure 21: N-terminus of CASK is important for the regulation of adhesion junction. 63 Figure 22: Double perturbation of CASK and MAGI-1 provided further increase in the total E-cadherin signals, as well as the accumulation of E-cadherin at cell-cell junctions. 64 Figure 23: Double perturbation of Calpain and MAGI-1 provided an accumulation of E-cadherin at cell-cell junctions. 65 Figure 24: Calpain knockdown resulted in increased stress fibres. 65 Figure 25: Possible mechanisms that result in CASK-regulated adhesion structures 67 References 68 Supplementary 73 Primers 73 shRNA sequences 73 CASK overexpression construct 74 CASK plasmid construct with N-terminus truncation 76 CASK plasmid construct with C-terminus truncation 77 List of probable protein X 77 MAGI-1 binding proteins 77 CASK binding proteins 78 MATLAB scripts 79 Fluro correction 79 Analysis of focal adhesions 81 Analysis of the signals between cells 97 | - |
dc.language.iso | en | - |
dc.title | 探討CASK調控的細胞黏附機制及意義 | zh_TW |
dc.title | Mechanism and Significance of CASK-mediated Cell Adhesion | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 郭津岑;涂熊林;林琬琬 | zh_TW |
dc.contributor.oralexamcommittee | Jean-Cheng Kuo;Hsiung-Lin Tu;Wan-Wan Lin | en |
dc.subject.keyword | 黏著斑,細胞骨架,肌動蛋白,細胞黏附,細胞損傷,活細胞影像, | zh_TW |
dc.subject.keyword | CASK,Focal Adhesion (FA),cytoskeleton,actin,E-cadherin,cell adhesion,Ca2+,cell injury,live-cell imaging, | en |
dc.relation.page | 109 | - |
dc.identifier.doi | 10.6342/NTU202303698 | - |
dc.rights.note | 同意授權(全球公開) | - |
dc.date.accepted | 2023-08-09 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 藥理學研究所 | - |
顯示於系所單位: | 藥理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-111-2.pdf | 16.31 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。