- 學位論文
與脂筏交互作用的Galectin8調控初級纖毛生長之研究
Lipid raft interacting galectin 8 regulates primary ciliogenesis
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摘要
在細胞分化後的頂端區域有著一個特殊的胞器稱為初級纖毛。在之前的研究中發現，初級纖毛有別於運動用的纖毛是屬於靜態的、能感知和傳遞環境訊息的胞器，初級纖毛的生成及長度受到各種機制的嚴格調控，例如纖毛的生成及伸長會受到物質在鞭毛內輸送蛋白運輸的頻率或大小來控制，這些輸送蛋白運送材料也包含纖毛生長所必需的微管蛋白和訊息分子等。在本次研究中，我們發現在初級纖毛的細胞膜上被唾液酸聚醣修飾的蛋白質或是脂質與半乳糖凝集素8的交互作用會調控初級纖毛的生長。在細胞進行極化的過程中，我們觀察到分泌到細胞頂端區域外部的半乳糖凝集素8會隨著時間增加，同時這些細胞外的半乳糖凝集素8會結合到初級纖毛及初級纖毛根部的過度區，根據我們的觀察，這樣的結合會使初級纖毛出現快速生長的現象。透過實驗分析，我們發現半乳糖凝集素8會和脂筏的組成分子有一定程度的交互關係，如：神經節苷脂和小窩蛋白1等，這個結果可以支持我們的假說，即半乳糖凝集素8會結合在初級纖毛及初級纖毛根部的過度區。此外我們觀察到半乳糖凝集素8結合在初級纖毛後會擾亂初級纖毛過渡區的屏障功能，促使初級纖毛的快速生長。我們的研究還發現，初級纖毛過渡區的屏障功能是否健全取決於初級纖毛過度區中脂筏結構的完整性，在小窩蛋白1基因敲除和脂筏的藥理學抑制實驗均表現出類似於頂端添加重組半乳糖凝集素8的效果，即初級纖毛快速生長和初級纖毛過度區屏障功能受損，因此我們推論半乳糖凝集素8在初級纖毛短期快速生長上扮演一個重要的角色。後續，我們透過轉錄組分析和蛋白質組分析也發現，半乳糖凝集素8會和胰島素樣生長因子1受體有高度的親和性，同時胰島素樣生長因子1受體下游的蛋白激酶B被活化、Myc的訊號路徑活化和Myc蛋白質表現量增加，這些都暗示著半乳糖凝集素8和初級纖毛的交互作用會誘導細胞重新進入有絲分裂的循環週期。
並列摘要
Primary cilium is a specialized sensory organelle that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials such as tubulin subunits essential for the growing cilia. Here we show the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis. As the epithelia become polarized, there are more galectin 8 being apically secreted and these extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia through interacting with lipid raft components ganglioside and caveolin 1. Furthermore, the binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of transition zone. Our study also demonstrates the functionality of this barrier depends on intact organization of lipid rafts at the cilia as genetically knockout of Cav1 and pharmacologically inhibition of lipid raft both phenocopy the effect of apical addition of recombinant galectin 8; i.e., rapid elongation of primary cilia and redistribution of cilial proteins from transition zone to the growing cilial trunk. We also investigated the cellular response after galectin 8 interacted with primary cilia through transcriptome and proteome analysis. The proteomics analysis implied Gal8 might interact with insulin-like growth factor 1 receptor (IGF1R) while the transcriptomic analysis showed Myc signaling pathway was up-regulated. Moreover, the protein kinase B (AKT) which had been reported as a downstream target of IGF1R was activated and the Myc protein level was increased. Our results imply that the interaction of galectin 8 with primary cilia induces cell entry into the cell cycle.
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