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標題: | 以次五奈米超解析多色光學顯微技術定位鞭毛內運輸作用蛋白的結構及訊號傳導方式 Molecular resolution imaging reveals step-wise reduction of axonemal diameter and ultrastructural architecture of intraflagellar transport proteins |
作者: | 卓曼婷 MAN TENG CHEOK |
指導教授: | 楊東霖 Tony Yang |
關鍵字: | 初級纖毛,纖毛內運輸作用蛋白,超解析影像,擴展顯微鏡,多色成像, Ex-dSTORM,dSTORM,Expansion microscopy,Super-resolution,Primary cilia,IFT, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 初級纖毛(Primary cilia)是脊椎動物細胞中用於感覺信號及細胞外環境偵測的細胞胞器。纖毛內運輸作用蛋白(Intraflagellar Transport Protein,IFT protein)負責初級纖毛的生成組裝、維持以及細胞之間訊號傳遞的溝通工作。過去的研究指出初級纖毛缺陷和鞭毛內運輸作用蛋白缺失會引發多種纖毛相關疾病,包括神經發育遲緩以及生理系統功能障礙等。然而,導致初級纖毛缺陷的生長、組裝和拆除機制,以及訊號傳遞的方式仍未完全解析。
在本研究中,我們使用了次五奈米超解析多色光學顯微技術 (EX-dSTORM),對纖毛內運輸作用蛋白的結構進行了全面的解析。透過結合dSTORM (Direct stochastic optical reconstruction microscopy)和EX(Expansion microscopy) 實現5納米解析求的生物分子結構影像,闡釋纖毛的結構模型和IFT蛋白在方式纖毛上的運輸模型。這項研究的結果將有助於更新我們對纖毛,不同的纖毛內運輸作用蛋白共同合作結構的認識,並更深入瞭解初級纖毛的生成和維護過程。冀望未來可以藉由這個纖毛結構模型更好地了解初級纖毛的結構和IFT 轉運機制,並根據其調控機制開發出針對纖毛類疾病的療法。 Primary cilia are microtubule-based organelles essential for mediating mechano- /photo-/chemo-sensations in mammalian cells. Intraflagellar transport (IFT) proteins are crucial components to support ciliary assembly, maintenance, and cellular signaling. The ciliary microtubule doublets of the ‘9+0’ arrangement serve as tracks for molecular motor-based transport along cilia. Although the tapering structure of primary cilia has been examined by electron microscopy, the spatial correlation between the IFT pattern and axonemal structure remains elusive due to insufficient light resolution. Here, we integrated single-molecule localization microscopy and sample expansion techniques achieving two-color imaging with effective localization precision of sub-5 nm to explore the axonemal ultrastructure and molecular organization of IFT and IFT-associated proteins in retinal pigment epithelial cilia. Our results revealed the step-wise reduction of ciliary diameter, different from the well-known paradigm showing a gradual change in the width of axonemal bundles. Surprisingly, our data showed that the IFT proteins congested near the end of the microtubule doubles within the ciliary compartment. In addition, we resolved the molecular architecture of IFT complexes, cargo, and motor proteins to characterize their relative localizations. We found that IFT proteins presented distinct distributions, including isolated units, paired configurations, or alternating arrangements in IFT trains. Together, our ultrastructural light nanoscopic imaging enables us to perform morphological analysis of primary cilia, which guides the localization of IFT complexes at segmented sites of cilia, suggesting the asynchronous axonemal growth of microtubule doubles. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/90724 |
DOI: | 10.6342/NTU202302562 |
全文授權: | 未授權 |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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