Möbius Wiebke, Ohno-Iwashita Yoshiko, van Donselaar Elly G, Oorschot Viola M J, Shimada Yukiko, Fujimoto Toyoshi, Heijnen Harry F G, Geuze Hans J, Slot Jan W
Department of Cell Biology, University Medical Center Utrecht and Center for Biomedical Genetics, Utrecht, The Netherlands.
J Histochem Cytochem. 2002 Jan;50(1):43-55. doi: 10.1177/002215540205000105.
We used a proteolytically modified and biotinylated derivative of the cholesterol-binding Theta-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-beta-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the Theta-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.
我们使用了一种经过蛋白水解修饰并生物素化的胆固醇结合θ毒素（产气荚膜梭菌溶素O）衍生物，通过电子显微镜在培养的人淋巴母细胞（RN）的冷冻切片中定位富含胆固醇的膜。我们开发了一种固定和免疫标记程序，以改善膜的保存，并最大限度地减少薄切片上胆固醇的提取和移位。我们还标记了活细胞的表面，并应用了高压冷冻以及解冻过程中冷冻切片的后续固定。在质膜上发现了胆固醇标记，在丝状伪足样突起上标记最强。强烈的标记也与多囊泡体（MVBs）的内部囊泡以及分泌后（外泌体）细胞表面的类似囊泡有关。靠近内体和高尔基体复合体的微管泡状结构通常也呈阳性，但MVBs的周围膜和高尔基体潴泡大多呈阴性。用甲基-β-环糊精处理细胞完全消除了胆固醇标记。我们的结果表明，当与改进的固定和高压冷冻结合使用时，θ毒素衍生物是在超薄冷冻切片中定位膜胆固醇的有用工具。
