臺灣博碩士論文加值系統

主纖毛是從母體中心粒，向細胞外部延伸所形成的特化結構，由細胞膜包覆9個成對的微管束所組成。主纖毛作為一個訊息感知的受器，負責接收外環境的物理與化學訊號，因此在調控細胞增生以及細胞型態上扮演著十分重要的角色。目前已知，中心粒遠端的蛋白負責調控主纖毛的生成。說明了，中心粒的遠端與主纖毛的形成之間有很重要的關聯性。利用定量的質譜分析（SILAC-based proteomics），我們在一群中心粒遠端的蛋白中，發現一個嶄新的蛋白CCHCR1 (Coiled-Coil -helical Rod protein 1)。然而，對於CCHCR1是否參與調控主纖毛生成，目前還尚未釐清。為了進一步探討CCHCR1在主纖毛生成上的角色，我們製造了兩支CCHCR1抗體，並透過免疫螢光染色，來驗證其在中心粒上的分布。結果顯示，CCHCR1確實是一個中心粒蛋白，且會出現在不同細胞週期的中心粒上。為了進一步探討CCHCR1的功能，透過CRISPR/Cas9這套基因編輯系統，成功地建立出剔除CCHCR1的細胞株，並發現缺少CCHCR1並不會影響中心粒的複製，但卻會影響主纖毛的生成。為了進一步釐清CCHCR1如何影響主纖毛的生成，透過穿透式電子顯微鏡，觀察CCHCR1缺失細胞的中心粒結構，我們發現CCHCR1的缺失，除了影響主纖毛生長之外，同時也影響了主纖毛囊泡的附著，但不會改變中心粒遠端附屬物的結構。上述的實驗結果顯示，CCHCR1參與在主纖毛生長的早期階段，但詳細的作用機制還有待進一步釐清。總結來說，我們驗證了CCHCR1確實是一個中心粒蛋白，並在主纖毛生長的早期扮演著重要的角色。

The primary cilium is a membrane-bound, microtubule-based sensory organelle, composed of 9 doublet microtubules (MTs), the axoneme, nucleated directly from the distal end of centrioles. Primary cilia can sense a wide range of signals in the extracellular surroundings, and thus critically regulate the physiology of cells during proliferation and morphogenesis. CCHCR1 (Coiled-Coil -helical Rod protein 1) is one of centriole-distal-end proteins identified from our previous SILAC-based proteomics screen, but the function of this protein is largely unknown. We first generate two antibodies to look at the endogenous localization of CCHCR1. Our immunofluorescence results show that CCHCR1 is associated with both of centrioles throughout the cell cycle. In ciliated cells, the CCHCR1 signal is located between basal body and the cilium, which is the centriole distal end. To determine the role of CCHCR1 in vivo, we deplete endogenous CCHCR1 by generating CCHCR1-/- cells through CRISPR/Cas9 system. Neither mitosis nor centriole duplication is affected by CCHCR1 depletion. However, in the absence of CCHCR1, ciliogenesis is specific disrupted. Electron microscope is also used to examine the effect of CCHCR1 in more detail. The observation indicates that loss of CCHCR1 affects primary cilia formation at the step of centriole to vesicle docking. Taken together, our data strongly support CCHCR1 functions as a promoting factor in primary cilia assembly.

Contents.....I
摘要.....IV
Abstract.....V
Chapter 1: Introduction.....1
1-1 Centrosome.....1
1-2 Centriole, centrosome and cilia.....1
1-3 Structure and function of cilia.....2
1-4 Ciliopathies.....3
1-5 Ciliogenesis.....3
1-6 Importance of centriole distal-end proteins in primary cilia formation.....5
1-7 SILAC screening of centriole protein.....5
1-8 CCHCR1 is one of centriolar proteins.....6
Chapter 2: Material and Methods.....9
Chapter 3: Result.....16
3-1 CCHCR1 is one of CEP162-interacting molecules.....16
3-2 CCHCR1 localizes at centrioles throughout the cell cycle.....16
3-3 Overexpression of CCHCR1 promotes cilia formation.....17
3-4 Generation of two CCHCR1 antibodies for the detection of endogenous
CCHCR1.....18
3-5 CCHCR1 is a centriole protein.....19
3-6 The CCHCR1 C-terminal is essential for centriole targeting in vivo.....19
3-7 Establishment of knockout cells through CRISPR/Cas9.....20
3-8 Absence of CCHCR1 does not affect centriole duplication.....21
3-9 Loss of CCHCR1 inhibits primary cilia formation.....21
3-10 Loss of CCHCR1 does not affect expression of proteins at centriole
distal-end.....22
3-11 Loss of CCHCR1 does not affect recruitment of proteins at centriole
distal-end.....22
3-12 TEM analysis reveals the influence of CCHCR1 in primary cilia formation.....23
3-13 CCHCR1 promotes CP110 removal.....23
3-14 To identify CCHCR1 interacting proteins by BioID method.....24
Chapter 4: Discussion.....26
4-1 Role of CCHCR1 during ciliogenesis .....26
4-2 The effect of ciliogenesis in defective CCHCR1 cell.....27
4-3 CCHCR1-C antibody recognizes strong signal in nucleus at S phase in
different cells.....27
4-4 Relationship between centrosome and psoriasis.....28
4-5 Functional implications of CCHCR1.....29
Chapter 5: References.....30
Chapter 6: Figures and Legends.....33
Figure 1. Ectopic expression of CCHCR1 is present at both of centrioles throughout the cell cycle.....33
Figure 2. Overexpression of CCHCR1 promotes primary cilia formation.....34
Figure 3. Generation of two CCHCR1 antibodies.....35
Figure 4. Endogenous CCHCR1 are present at centrioles throughout the cell cycle.....37
Figure 5. The C-terminal coiled-coil domain of CCHCR1 is essential for its centriolar localization.....38
Figure 6. Generation of CCHCR1 knockout cells.....39
Figure 7. CCHCR1 is not involved in centriole duplication.....41
Figure 8. CCHCR1 depletion reduces cilia formation.....42
Figure9. Loss of CCHCR1 does not affect the expression of other centrosome proteins.....44
Figure 10. Loss of CCHCR1 does not affect the subcellular localizations of other centrosome proteins.....45
Figure 11. Loss of CCHCR1 blocks CP110 removal.....47
Figure 12.Model summarizes the roles of CCHCR1 in primary cilia assembly.....48
Figure 13. Generation of BirA*-CCHCR1 stable expression cells.....49
Figure 14.CCHCR1 associated proteins were purified by BioID (The proximity-dependent biotin identification) method in RPE1 cells.....50
Chapter 7: Tables.....52
Table 1. DNA consreucts and primer list.....52
Table 2. Antibodies used in this study include.....54
Supplement.....56
Figure S1. CCHCR1 is the interacting protein of CEP162.....56
Figure S2. TEM analysis reveals the influence of CCHCR1 in primary cilia formation.....57

1. Satir, P., Overview of Structure and Function of Mammalian Cilia. 2007: p. 377–400.
2. Singla V, R.J., The primary cilium as the cell's antenna: signaling at a sensory organelle. Science. , 2006 Aug 4. 313(5787): p. 629-33.
3. Zhong*, M.C.a.Q., Cilia in autophagy and cancer. Cilia, 2016.
4. Mitchison, H.M., Motile and non-motile cilia in human pathology: from function to phenotypes. The Journal of Pathology, January 2017 241( 2): p. 294–309.
5. J, K., Cellular Mechanisms of Ciliary Length Control. Cells., 2016 5(1).
6. Vertii, A., Human basal body basics Vertii et al. Cilia 2016. 5(13).
7. T, A.-R., Shared and Distinct Mechanisms of Compartmentalized and Cytosolic Ciliogenesis. Curr Biol. , 2015 25(23).
8. Scholey, J.M., Intraflagellar transport motors in cilia: moving along the cell's antenna. J Cell Biol., 2008. 180(1): p. 23–29.
9. Johnson KA1, R.J., Polarity of flagellar assembly in Chlamydomonas. J Cell Biol. , 1992 119(6): p. 1605-11.
10. Nachury, M.V., Trafficking to the Ciliary Membrane. Annu Rev Cell Dev Biol., 2011. 10( 26): p. 59-87.
11. Wang J, C.X., Wang F, Zhang J, Li P, Li Z, et al., OFD1, as a Ciliary Protein, Exhibits Neuroprotective Function in Photoreceptor Degeneration Models. PLoS One, May 19, 2016. 11(5).
12. Wang, W.-J., CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base. Nat Cell Biol. , 2013 591-601.
13. K, A., Coding haplotype analysis supports HCR as the putative susceptibility gene for psoriasis at the MHC PSORS1 locus. Hum Mol Genet., 2002 11(5): p. 589-97.
14. K, A., Genetic analysis of PSORS1 distinguishes guttate psoriasis and palmoplantar pustulosis. J Invest Dermatol., 2003 Apr. 120(4): p. 627-32.
15. Elomaa, O., Transgenic mouse models support HCR as an effector gene in the PSORS1 locus. Hum Mol Genet., 2004. 13 (15): p. 1551-1561.
16. MH, T., Centrosomal localization of the psoriasis candidate gene product, CCHCR1, supports a role in cytoskeletal organization. PLoS One, 2012. 7(11).
17. YH, L., CCHCR1 interacts with EDC4, suggesting its localization in P-bodies. Exp Cell Res. , 2014. 32712-23(1): p. 12-23.
18. Ling, Y.H., CCHCR1 interacts with EDC4, suggesting its localization in P-bodies. EXPERIMENTAL CELL RESEARCH, 2014. 327(1): p. 12-23.
19. Kyle J. Roux, 2 Dae In Kim,1,2 Manfred Raida,3 and Brian Burke4, A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. Vol. , March 12, 2012. 196 (6 ): p. 801–810
20. Maren Klinger, W.W., Stefanie Kuhns,* Felix Bärenz,† Stefanie Dräger-Meurer, Gislene Pereira,b, and Oliver J. Gruss, The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone. Mol Biol Cell, 2014: p. 495-507.
21. Hunkapiller J, S.V., Seol A, Reiter JF. , The ciliogenic protein Oral-Facial-Digital 1 regulates the neuronal differentiation of embryonic stem cells. Stem Cells Dev. . Stem Cells Dev. , 2010. 20(5): p. 831–41.
22. A. Spektor, W.Y.T., D. Khoo, B.D. Dynlacht, Cep97 and CP110 suppress a cilia assembly program. Cell, 2007. 130 p. 678–690.
23. Kerstin N. Schmidt, S.K., Annett Neuner, Birgit Hub, Hanswalter Zentgraf, Gislene Pereira, Cep164 mediates vesicular docking to the mother centriole during early steps of ciliogenesis. JCB, 2012. 199 (7): p. 1083.
24. Elif Nur Firat-Karalar1, N.R., John R. Yates III2, and Tim Stearns1,3, Proximity Interactions Among Centrosome Components Identify Regulators of Centriole Duplication. Curr Biol., 2014. 24(6): p. 664–670.
25. Anastassiia Vertii, H.H., and Stephen Doxsey, The Centrosome, a Multitalented Renaissance Organelle. Cold Spring Harb Perspect Biol December 1, 2016 8.