細胞藉由調控微管蛋白的轉譯後修飾去控制不同的生理機制，而這些微管蛋白轉譯後修飾在細胞中的動態分佈及組成變化增加了研究其在特定細胞區域所扮演角色的困難度。為了解決此長久以來的研究困境，我們發展了一套新的技術，STRIP (Spatiotemporally Rewriting Intraciliary PTMs)，其可以快速地改寫在活細胞中初級纖毛上微管蛋白的轉譯後修飾。我們誘導一個改造過的去谷氨酰化酵素CCP5快速地移動到初級纖毛上，其可消除在初級纖毛軸絲上谷氨酰化的轉譯後修飾。藉此技術我們進一步發現去除微管上的谷氨酰化會在初級纖毛新生時期抑制微管的增長，但不影響到初級纖毛的長度或在微管上其他的轉譯後修飾。我們並發現去除微管上的谷氨酰化會抑制由Kinesin-2運動蛋白介導的順向鞭毛内運輸和Hedgehog信號。我們的研究直接證實了谷氨酰化在初級纖毛的結構及功能上所扮演的角色，未來也可以藉由這項技術來研究細胞中其他區域的微管蛋白轉譯後修飾。

Tubulin Post-Translational Modifications (PTMs) occur spatiotemporally in cells and has been suggested to be involved in a wide range of cellular activities. The complexity and dynamic distribution of tubulin PTMs have hampered people from understanding their physiological roles in specific subcellular regions. Here we developed a new method termed STRIP (SpatioTemporally Rewriting Intraciliary PTMs) enabling us to rapidly and locally rewrite tubulin PTMs in living cells. More specifically, we rapidly deplete one tubulin PTM, polyglutamylation, inside cilia by inducibly recruiting an engineered deglutamylase onto ciliary axonemes. The resulting de novo deglutamylation negatively impacted axoneme elongation during ciliogenesis without noticeably affecting cilia length or other tubulin PTMs in the steady state. In addition, axonemal deglutamylation inhibits kinesin-2- mediated anterograde intraflagellar transport and Hedgehog signaling. Our study demonstrates direct evidence of the causal relationship between the polyglutamylation of ciliary axonemes and ciliary functions. By extending the repertoire of de novo PTM modifiers in the future, a thorough cracking of their pleiotropic roles may become possible.

Abstract……………………………………………………………………………………………………ii
中文摘要……………………………………………………………………………………………………iii
CONTENTS……………………………………………………………………………………………………iv
Chapter 1 Introduction…………………………………………………………………1
Chapter 2 Methods……………………………………………………………………………10
Chapter 3 Result………………………………………………………………………………16
Chapter 4 Discussion and Conclusion……………………………28
REFERENCE…………………………………………………………………………………………………33

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