跳到主要內容

臺灣博碩士論文加值系統

(44.210.99.209) 您好!臺灣時間:2024/04/16 03:00
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:羅芊卉
研究生(外文):Chien-Hui Lo
論文名稱:TTBK2藉由磷酸化CEP83進而促進初級纖毛初始化
論文名稱(外文):TTBK2 phosphorylates CEP83 in promoting primary cilia initiation
指導教授:王琬菁
指導教授(外文):Won-Jing Wang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:69
中文關鍵詞:中心體初級纖毛
外文關鍵詞:centrosomeprimary cilia
相關次數:
  • 被引用被引用:0
  • 點閱點閱:252
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
初級纖毛是當細胞進入G0 / G1期時,從細胞膜突出的一種特化結構。突出的纖毛在細胞中扮演訊息接收者的角色,能夠接收外在環境中的化學和機械的信號,因此在細胞增殖和細胞形態上扮演非常重要的角色。在初級纖毛形成的過程中存在一個重要的激酶蛋白,其蛋白為 Tau微管蛋白激酶2(TTBK2),此蛋白在纖毛起始中扮演關鍵作用,此蛋白需要被帶到母體中心粒的遠端,隨後去除CP110蛋白,然而其細節機制尚不清楚。在這裡,我們發現TTBK2被帶到母體中心粒的distal appendage上與細胞週期的時間點有關。透過實驗,我們篩選出幾個可能為TTBK2受質的蛋白,其中distal appendage上的CEP83被鑑定為TTBK2可能的受質蛋白之一。更進一步利用質譜儀進行分析,當有TTBK2 存在的狀況下,CEP83蛋白上會出現四個磷酸化位點。接下來,我們想探討CEP83被磷酸化後與初級纖毛生長的關聯性,首先我們利用CRIPSR/ Cas9的系統去移除掉內生性的CEP83,重新將正常序列的CEP83及四個磷酸化位點序列變異的CEP83蛋白送進移除掉內生性CEP83的細胞中進行觀察。當細胞表現四個磷酸化位點序列變異的CEP83蛋白時,細胞長出初級纖毛的比例大幅下降。因此,根據上述的實驗結果,我們指出在初級纖毛要突出前,TTBK2會被帶到母體中心粒的distal appendage上,並且與CEP83蛋白結合同時磷酸化此蛋白,進而影響到初級纖毛的生長。
Primary cilia are evolutionarily conserved organelle projecting from the plasma membrane as cells enter G0/G1 phase. Cilia serve as sensory hub for the transduction of chemical and mechanical signals from the environment and are thus important for the physiology of cells during proliferation and morphogenesis. The recruitment of Tau-tubulin kinase-2 (TTBK2) to the distal end of mother centriole and subsequent removal of CP110 play crucial role of cilia initiation, however, the detail mechanism is not known. Here, we show that TTBK2 is recruited to the distal appendage of mother centriole in a cell cycle-dependent manner. Through a biochemical screening, CEP83 is identified as one of TTBK2’s in vivo substrates. A mass spectrometry (MS) analysis reveals four phosphorylation sites on CEP83 upon TTBK2 expression. In order to understand the effect of CEP83 phosphorylation in primary cilia assembly, we generate CEP83-/- cells using CRIPSR/ Cas9 gene targeting to inactivate CEP83 in retinal pigment epithelial cell (RPE1) cells. Unlike re-expression of full-length CEP83 in CEP83-/- cells can support cilia assembly, the ability of cilia assembly is significantly reduced in CEP834A expressing cells. Thus, our data suggest that CEP83 is one of TTBK2’s in vivo substrates required for cilia formation.
Contents 目錄 i
Abstract v
摘要 vi

Chapter 1: Introduction 1
1.1 Centriole, centrosome and cilia 1
1.2 The core structure of the cilium 1
1.3 Ciliopathy 2
1.4 Cilia assembly and disassembly is strictly coupled with the cell cycle 2
1.5 Steps of cilia formation 3
1.5.1 The distal appendage of the mother centriole 4
1.5.2 TTBK2 is required for the removal of CP110 from the mother centriole 4
1.5.3 Ciliary vesicles dock to the DAPs 5
1.5.4 The formation of transition zone 6
1.5.5 The recruitment of intraflagellar transport (IFT) proteins 6
1.6 TTBK2 is a key kinase protein to promote cilia formation 7
1.7 The complex formation of TTBK2 and CEP164 and CEP164 phosphorylation. 8

Chapter 2: Materials and Methods 9
2.1 Cell culture 9
2.2 Transfection 9
2.3 Cell lysis and immunoprecipitation 9
2.4 Western blot analysis 10
2.5 Generation of TTBK2-/- cells and CEP83-/- cells 10
2.6 The purification of recombinant protein 10
2.7 Affinity purification of polyclonal antisera 11
2.8 Immunofluoresence and microscopy 12
2.9 Ciliogenesis experiments 12
2.10 Site-directed mutagenesis 12
2.11 Electroporation 13
2.12 Statistical analysis 13

Chapter 3: Results 15
3.1 The localization of TTBK2 at distal appendage is cell-cycle dependent 15
3.2 Generation of TTBK2 -/- cells 15
3.3 TTBK2 kinase activity is required for cilia assembly and the removal of CP110 16
3.4 The TTBK2 kinase activity is not required for its localization at centriole distal appendage at different cell-cycle stages 16
3.5 Identification of TTBK2 in vivo substrates 17
3.6 The relationship between TTBK2 and CEP83 17
3.7 Identification of phosphorylation sites on CEP83 targeted by TTBK2 18
3.8 Generation of CEP83 -/- cells 19
3.9 CEP83 affects the recruitment of TTBK2 and IFT88 to mother centriole upon cilia assembly 20
3.10 CEP83 phosphorylation promotes cilia formation 20
3.11 Generation of hTTBK2 antibody 20

Chapter 4: Discussion 22
4.1 The localization of TTBK2 is strictly regulated at the appropriate time 22
4.2 TTBK2 may interact with other distal appendage proteins 22
4.3 TTBK2KD interact with CEP83 is more stable than TTBK2WT 22
4.4 The phosphorylation of distal appendage protein is important for cilia formation 23
4.5 The phosphorylation on CEP83 affects centriole to vesicle docking, CP110 removal and hierarchy of distal appendage assembly. 23

Chapter 5: References 25

Chapter 6: Figures and Legends 28
Figure 1 Model for cilia assembly 28
Figure 2 TTBK2 locates at distal appendage of centriole 29
Figure 3 The localization of TTBK2 at distal appendage of centriole is cell-cycle dependent 30
Figure 4 Generation of TTBK2 -/- cells 32
Figure 5 TTBK2 kinase activity is essential for cilia formation 34
Figure 6 TTBK2 kinase activity is required for the removal of CP110 36
Figure 7 The kinase activity of TTBK2 does not affect its localization at distal appendage 38
Figure 8 The kinase activity of TTBK2 does not affect its localization at distal appendage 39
Figure 9 Identification of TTBK2 substrates 41
Figure 10 CEP83 interacts with TTBK2, which is not kinase-dependent 42
Figure 11 CEP83 interacts with the c-terminal region of TTBK2 44
Figure 12 CEP83 is phosphorylated by TTBK2 46
Figure 13 TTBK2 phosphorylates CEP83 at S29, T292, T527 and S698 47
Figure 14 The phosphorylation of CEP83 does not affect its binding to TTBK2 49
Figure 15 Generation of CEP83 -/- cells 50
Figure 16 Loss of CEP83 affects the assembly of distal appendage proteins 52
Figure 17 Loss of CEP83 affects the recruitment of IFT88 and TTBK2 to the centriole 55
Figure 18 CEP83 phosphorylation is required for cilia formation 56
Figure 19 Generation of TTBK2 antibody 57
Figure 20 Immunostaining of purified TTBK2 antibody 59

Chapter 7: Tables 62
7.1 Table 1 Primary antibodies information 62
7.2 Table 2 Primers for cloning 63

Chapter 8: Supplementary 66
8.1 The dSTORM imaging reveals the detail localization of TTBK2 and CEP83 66
8.2 The mass spectrometry analyses reveal four TTBK2 phosphorylation sites on CEP83 68
1. Edde, B., et al., Posttranslational glutamylation of alpha-tubulin. Science, 1990. 247(4938): p. 83-5.
2. Wang, W.J., et al., De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly. Elife, 2015. 4.
3. Izquierdo, D., et al., Stabilization of cartwheel-less centrioles for duplication requires CEP295-mediated centriole-to-centrosome conversion. Cell Rep, 2014. 8(4): p. 957-65.
4. Vincensini, L., T. Blisnick, and P. Bastin, 1001 model organisms to study cilia and flagella. Biol Cell, 2011. 103(3): p. 109-30.
5. Baccetti, B., R. Dallai, and B. Fratello, The spermatozoon of arthropoda. XXII. The 12+0', 14+0' or aflagellate sperm of protura. J Cell Sci, 1973. 13(2): p. 321-35.
6. Prensier, G., et al., Motile flagellum with a "3 + 0" ultrastructure. Science, 1980. 207(4438): p. 1493-4.
7. Christensen, S.T., et al., Sensory cilia and integration of signal transduction in human health and disease. Traffic, 2007. 8(2): p. 97-109.
8. Garcia-Gonzalo, F.R., et al., Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling. Dev Cell, 2015. 34(4): p. 400-9.
9. Bettencourt-Dias, M., et al., Centrosomes and cilia in human disease. Trends Genet, 2011. 27(8): p. 307-15.
10. Wang, W.J., et al., The conversion of centrioles to centrosomes: essential coupling of duplication with segregation. J Cell Biol, 2011. 193(4): p. 727-39.
11. Ozlu, N., et al., An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles. Dev Cell, 2005. 9(2): p. 237-48.
12. Goto, H., A. Inoko, and M. Inagaki, Cell cycle progression by the repression of primary cilia formation in proliferating cells. Cell Mol Life Sci, 2013. 70(20): p. 3893-905.
13. Cole, D.G., et al., Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol, 1998. 141(4): p. 993-1008.
14. Benzing, T. and B. Schermer, Transition zone proteins and cilia dynamics. Nat Genet, 2011. 43(8): p. 723-4.
15. Tanos, B.E., et al., Centriole distal appendages promote membrane docking, leading to cilia initiation. Genes Dev, 2013. 27(2): p. 163-8.
16. Chen, Z., et al., CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells. Dev Cell, 2002. 3(3): p. 339-50.
17. Schmidt, T.I., et al., Control of centriole length by CPAP and CP110. Curr Biol, 2009. 19(12): p. 1005-11.
18. Oda, T., et al., Binding to Cep164, but not EB1, is essential for centriolar localization of TTBK2 and its function in ciliogenesis. Genes Cells, 2014. 19(12): p. 927-40.
19. Ikezu, S. and T. Ikezu, Tau-tubulin kinase. Front Mol Neurosci, 2014. 7: p. 33.
20. Goetz, S.C., K.F. Liem, Jr., and K.V. Anderson, The spinocerebellar ataxia-associated gene Tau tubulin kinase 2 controls the initiation of ciliogenesis. Cell, 2012. 151(4): p. 847-58.
21. Bouskila, M., et al., TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development. Biochem J, 2011. 437(1): p. 157-67.
22. Joo, K., et al., CCDC41 is required for ciliary vesicle docking to the mother centriole. Proc Natl Acad Sci U S A, 2013. 110(15): p. 5987-92.
23. Lu, Q., et al., Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation. Nat Cell Biol, 2015. 17(4): p. 531.
24. Zhang, J., N. Naslavsky, and S. Caplan, Rabs and EHDs: alternate modes for traffic control. Biosci Rep, 2012. 32(1): p. 17-23.
25. Reiter, J.F., O.E. Blacque, and M.R. Leroux, The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep, 2012. 13(7): p. 608-18.
26. Chih, B., et al., A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat Cell Biol, 2011. 14(1): p. 61-72.
27. Sorokin, S., Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol, 1962. 15: p. 363-77.
28. Gilula, N.B. and P. Satir, The ciliary necklace. A ciliary membrane specialization. J Cell Biol, 1972. 53(2): p. 494-509.
29. Yang, T.T., et al., Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone. Sci Rep, 2015. 5: p. 14096.
30. Lee, E., et al., An IFT-A protein is required to delimit functionally distinct zones in mechanosensory cilia. Curr Biol, 2008. 18(24): p. 1899-906.
31. Blacque, O.E., et al., The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport. Mol Biol Cell, 2006. 17(12): p. 5053-62.
32. Cajanek, L. and E.A. Nigg, Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole. Proc Natl Acad Sci U S A, 2014. 111(28): p. E2841-50.
33. Xu, Q., et al., Phosphatidylinositol phosphate kinase PIPKIgamma and phosphatase INPP5E coordinate initiation of ciliogenesis. Nat Commun, 2016. 7: p. 10777.
34. Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top