中心體是一種由微管蛋白質組成的胞器，其作為微管形成中心並調控包含細胞移行、細胞分裂、基因穩定性和纖毛生成等細胞生理機制。中心體缺陷會導致腫瘤形成、神經退化疾病和纖毛缺失症。中心體的微管蛋白質會受到谷氨醯化已被發現數十年，但其所扮演的生理角色仍然未知，主要是因為缺乏可以在活細胞內的特定
時空間去調控微管蛋白後轉譯修飾的技術所致。為了克服這個長期存在的問題，我們發展了一套新的技術能將感興趣的蛋白質快速地轉移到中心體上。藉此系統，我們將一個去谷氨醯化的酵素 CCP5 快速地轉移到中心體上來去除該處的谷氨醯化。研究發現中心體去谷氨醯化後並不影響中心體的結構，但卻會抑制中心體不同的功能如破壞初級纖毛。此外，中心體去氨醯化還會減少微管形成中心的關鍵蛋白質 γ-tubulin 在中心體的含量，並減短從中心體生長出來的微管長度。我們與此研究中發展了一個調控中心體谷氨醯化的嶄新技術，並藉此首次發現中心體谷氨醯化對於中心體的多項功能如初級纖毛生長及其結構維持扮演重要角色，但卻與中心體結構無關。未來將繼續探討中心體谷氨醯化調控初級纖毛結構的分子機制。

Centrosome, a tubulin-based organelle, functions as a microtubule organizing center (MTOC) and tightly regulates the wide range of cellular activities including cell migration, cell division, genome stability, and ciliogenesis. Defects in centrosomes lead to many human diseases such as tumors, neurodegenerative diseases, ciliopathies, and so on. It is known for decades that centrosomal tubulin are subject to glutamylation. However, the physiological roles of centrosomal glutamylation are largely unknown primarily owing to a lack of techniques for spatiotemporally manipulating tubulin post-translational modifications (PTMs) in living cells. To address this long-standing issue, we developed a new technique enabling us to translocate the protein of interest (POI) onto centrosomes within minutes. Taken advantage of this, we rapidly depleted centrosomal glutamylation by inducibly recruiting an engineered deglutamylase onto centrosomes. Rapid deglutamylation in centrosomes does not affect the structural integrity of centrosome. Interestingly, centrosomal deglutamylation disturbs the ciliogenesis and cilia maintenance. Moreover, deglutamylation reduces the level of γ-tubulin, a key component of MTOC, and attenuates the growth of centrosome-derived microtubule. Our study has established a new tool for spatiotemporally perturbing glutamylation in centrosome and has uncovered that glutamylation is important for the centrosomal functions regarding to ciliogenesis, cilia maintenance and microtubule nucleation but plays little role in the centrosome integrity of centrosome. The detail mechanisms of how glutamylation regulates cilia structure will be evaluated in the near future.

口試委員審定書 ……………………………………………………….. 1
致謝 …………………………………………………….........….. 2
Abstract ………………………………………………………....... 3
中文摘要 ………………………………………………………........ 4
目錄 ………………………………………………………............ 5
List of Figures ……………………………………………………….7
Chapter 1 Introduction………………………………………… 8
1.1 Overview of centrosome……………………………… 8
1.2 Centrosomal tubulin undergoes various post-translational
modifications……………………………………………………….... 8
1.3 Glutamylation and modifying enzyme…8
1.4 Convectional approaches for studying the physiological roles of tubulin glutamylation……………………………... 9
1.5 The technical limitation hampers the understanding of centrosomal glutamylation………………………….. 10
1.6 A new tool for spatiotemporal tubulin PTMs in living cells………............................. 11
Chapter 2 Methods…………………………………………………. 13
2.1 Cell culture and transfection………… 13
2.2 Live cell-imaging………………………………………. 13
2.3 Measurement of centrosomal microtubule………….. 14
2.4 Immunofluorescence staining……………………………………….. 14
2.5 Statistical analysis…………………………………………………… 15
Chapter 3 Results………………………………………………………… 15
3.1 Centrosomal tubulin undergoes glutamylation………………………. 15
3.2 Rapidly recruiting soluble proteins onto centrosomes…………......................... 15
3.3 Translocation of the CCP5 catalytic domain onto centrosome for
locally depleting glutamylated tubulin.. 17
3.4 Glutamylation is not required for the structural integrity of
centrosomes…………………………………………………………... 18
3.5 Centrosomal deglutamylation inhibits the ciliogenesis and abolishes
the cilia maintenance…………………………………………19
3.6 Centrosomal deglutamylation attenuates the growth of cytosolic
microtubules………………………………………………………….. 20
Chapter 4 Discussion……………………………………………22
Chapter 5 References……………………………………………43

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