利用基於原子力顯微鏡之硬度斷層成像探測 HEK293T 細胞之膜力學構造

Abstract

於本研究中，為了量測貼覆型真核細胞的細胞膜力學構造，我建立了基於原子力顯微鏡 (Atomic force microscopy, AFM) 之測量與分析方法。由於量測時細胞膜和其底下細胞骨架可能同時被量測到，我利用與前人不同之分析方法，利用將力圖漸進分段分析之方式得到細胞表面之硬度分布。利用螢光標定位於細胞膜之膜蛋白或醣脂質後，利用將相同位置之螢光圖與硬度分佈相疊合的方式，將分子和硬度之分布相關聯。利用此方法，我測量了位於醣脂質 monosialotetrahexosyl- ganglioside (GM1) 以及膜蛋白GABAA subtype (alpha1)2(beta2)2 gamma2受體周圍之細胞膜硬度及高度，發現醣脂質 GM1 以及膜蛋白GABAA subtype (alpha1)2(beta2)2gamma2受體所在之位置，相較於周圍皆於較硬且較高。

In this study, I established an atomic force microscopy-based method to study the mechanical structure of the membrane of adherent eukaryotic cells. Since both the membrane and the underneath cytoskeletons could be simultaneously measured, I adapt a new analytical method for the force curve analysis. With a progressive truncation-refitting force curve analysis, the distribution of surface stiffness can be calculated. After the fluorescence labeling of sphingolipids or membrane protein, I correlate the stiffness and the molecular distribution by overlapping the fluorescence image with the stiffness distribution. With this method, I studied the membrane on the glycosphingolipid monosialotetrahexosylganglio (GM1) and the membrane around the membrane protein, gamma-aminobutyric acid type A receptor (GABAA receptor), subtype (alpha1)2(beta2)2 gamma2. I observed that both the GM1 and the GABAA receptor, subtype (alpha1)2(beta2)2 gamma2, are partitioned into a stiffer and higher domain than adjacent region.