臺灣博碩士論文加值系統

纖毛是一個由微管所組成，並且是由母中心粒所延伸長出的構造。纖毛在細胞中像是一個天線，是細胞的訊息接收者，它能夠接收外在環境中物理性以及化學性的訊號並促進細胞做出反應，促進下游相關基因的表現。除此之外，纖毛上具備許多跟胚胎發育以及組織型態發育有關的訊號傳遞相關受器。纖毛的生長與細胞週期是緊密相關的。纖毛會在細胞進入G1/Go 時從細胞膜突，並在進入分裂期前縮回去。而纖毛的形成倚靠一個重要的激酶的活性，Tau 微管蛋白激脢(TTBK2)。Tau 微管蛋白激脢的活性會啟動纖毛的形成。另外，纖毛形成的初期會觀察到囊泡聚集在母中心粒頂端的特化構造，此特化構造稱之為centriole distal appendages。這都是纖毛形成的必備條件。而centriole distal appendages 共由六個蛋白所組成(CEP83, CEP89, SCLT1, FBF1, LRRC45, CEP164)，他們的共通點就是當基因敲弱這些蛋白，都會抑制纖毛的形成，然而詳細的機轉尚不清楚。在此篇論文，我們透過建立了CEP89 基因剔除的RPE1 細胞，發現CEP89 基因的剔除會在不影響細胞週期的前提下，影響纖毛的形成。我們進一步發現CEP89是利用調節囊泡聚集在母中心粒頂端的步驟進而調控纖毛的形成。除此之外，我們發現CEP89在中心粒上的訊號會隨著細胞週期改變。當細胞進入分裂期，中心粒上是觀察不到CEP89的訊號的。CEP89的訊號出現在中心粒的時間點和纖毛形成的時間相關，這意味著CEP89有可能也會調控纖毛的拆解。

Cilia are microtubule-based organelles extending from the mother centrioles, which act as signal sensors in cells and play important roles in development and tissue morphogenesis. Ciliogenesis is tightly coupled with cell cycle that occurs in G0 or G1 phase and follows a series of highly ordered steps. In the initiating stage, it requires the kinase activity of tau tubulin kinase 2 (TTBK2) and the docking of membrane vesicles by the centriole distal appendages (DAs). The centriole distal appendage is a conserved structure composed of CEP83, CEP89, SCLT1, FBF1, LRRC45, and CEP164. It is known that knockdown of distal appendage proteins (DAPs) inhibits ciliogenesis. However, the underline mechanism of how DAPs regulate ciliogenesis and how membrane vesicles are stably attached at centriole distal end remains unclear. Here, we generate CEP89 knockout RPE1 cells to investigate the molecular regulation of CEP89 in ciliogenesis. We find that CEP89 knockout significantly inhibits ciliogenesis without affecting cell-cycle progression. By analyzing the key steps during ciliogenesis, we uncover that CEP89 promotes cilia formation at the step of ciliary vesicle formation. The localization analysis of CEP89 at the centrioles shows that CEP89 disappears from the mother centriole in mitosis. As the localization of CEP89 at the centrioles is coupled to cell cycle, as well as cilia cycle, our results suggest that the removal of CEP89 from the centrioles may also contribute to the control of cilia disassembly.

致謝：……………...……………………….....………………………….................i
Contents 目錄：........................................................................ii
Abstract：..................................................................................v
摘要：........................................................................................vi
Chapter 1. Introduction…............................................................1
1.1 Centriole, centrosome, and cilium…….................................1
1.2 Cilia and Ciliopathy……………………..…………………………………..2
1.3 Cilia biogenesis is strictly regulated and coupled with cell cycle………..................................................................................3
1.4 Ciliogenesis…………………………………………………………………4
1.4.1 The centriole distal appendages........................................4
1.4.2 TTBK2 is recruited to the centriole by CEP164 upon cilia initiation…...........................................................................5
1.4.3 The removal of CP110………..……..................................5
1.4.4 The docking of membrane vesicles to the centriole distal appendages………....……………………………………………………….6
1.4.5 The assembly of transition zone……...........................6
1.4.6 The intraflagellar transport machinery........................7
1.5 Centrosomal protein of 89 kDa..........................................7
Chapter 2. Materials and methods.............................................9
2.1 Cell culture.........................................................................9
2.2 Transfection in 293T and U2OS cells.................................9
2.3 Transfection in RPE1 cells..................................................9
2.4 Generation of CEP89-/- RPE1 cells...................................10
2.5 Cell lysis............................................................................11
2.6 Protein quantification........................................................11
2.7 Western blot analysis........................................................12
2.8 Immunofluorescence, microscopy....................................13
2.10 Ciliogenesis experiments..................................................15
2.11 Cell proliferation rate assay..............................................15
2.12 Cell cycle synchronization and proteasome inhibitor treatment…...........................................................................….16
2.13 Treatment of Eg5 inhibitor and PLK1 inhibitor...................17
2.15 Statistical analysis............................................................18
Chapter 3. Results....................................................................19
3.1 Generation of CEP89 antibody.........................................19
3.2 Generation of CEP89-/- RPE1 cells..................................20
3.3 CEP89 inactivation does not affect cell proliferation.......20
3.4 CEP89 inactivation inhibits ciliogenesis...........................21
3.5 CEP89 inactivation does not affect the assembly of the centriole distal appendages.....................................................22
3.6 CEP89 inactivation does not affect the recruitment of TTBK2 to the centrioles during ciliogenesis…………………….......22
3.7 CEP89 inactivation does not affect the removal of CP110 during ciliogenesis...................................................................23
3.8 CEP89 inactivation inhibits the formation of ciliary vesicle……........................................................................………23
3.9 The localization of CEP89 at the centrioles is cell-cycle dependent….............................................................................24
3.10 The global expression of CEP89 is cell-cycle dependent...............................................................................25
3.11 CEP89 is phosphorylated during G2/M phases................26
3.12 Inhibition of Plk1 blocks CEP89 removal from the centrioles during mitosis….......................................................26
3.13 Overexpression of wild-type Plk1 induces degradation of CEP89…...................................................................................27
Chapter 4. Discussion..............................................................28
4.1 The role of CEP89 in cilia formation.................................28
4.2 The relationships between CEP89 localization dynamics, cilia, and PLK1….……............................………………………………….28
4.3 The degradation of CEP89 during mitosis.......................29
4.4 The relationship between TTBK2 and CEP89...................30
4.5 The relationships between CEP89, centriole, and mitochondria............................................................................31
Chapter 5. Figure legends.......................................................32
Figure. 1. Homemade CEP89 antibody successfully detects endogenous CEP89 in Western blot and immunostaining.......33
Figure. 3. CEP89 inactivation does not affect cell proliferation……................................................................………36
Figure. 4. CEP89 inactivation inhibits ciliogenesis……….......….38
Figure. 5. CEP89 inactivation does not affect the recruitment of centriole distal appendages.....................................................41
Figure. 6. CEP89 inactivation does not affect the recruitment of TTBK2 to the centrioles...........................................................43
Figure. 7. CEP89 inactivation does not affect CP110 removal during ciliogenesis...................................................................45
Figure. 8. CEP89 inactivation inhibits the formation of ciliary vesicle……………........................................................................47
Figure. 9. The diagram shows that CEP89 affects ciliary vesicle formation in promoting ciliogensis..........................................48
Figure. 10. The recruitment of CEP89 to the centrioles is not totally occurred in G2 phases..................................................50
Figure. 11. The localization of CEP89 at the centrioles is cell-cycle dependent......................................................................54
Figure. 12. CEP89 undergoes proteasome-dependent protein degradation in mitosis.............................................................55
Figure. 13. Inhibition of Plk1 blocks CEP89 removal from the centrioles during mitosis………………………………….........……………57
Figure. 14. Overexpression of wild-type Plk1 induces degradation of CEP89. ..........................................................58
Chapter 6. References.............................................................59
Chapter 7. Tables.....................................................................64
Table 1. Primary antibodies information..................................64
Table 2. Secondary antibodies information.............................65
Table 3. Primers for cloning....................................................66

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