Fujimoto T, Hayashi M, Iwamoto M, Ohno-Iwashita Y
Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Japan.
J Histochem Cytochem. 1997 Sep;45(9):1197-205. doi: 10.1177/002215549704500903.
[symbol: see text]-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BC theta) to eliminate hemolyzing activity and was used as a cytochemical probe. In fixed cells, binding of BC theta was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BC theta and then with either fluorescein-avidin D or colloidal gold-streptavidin, the labeling in fine dots was distributed on the cell surface without local concentration as long as cells were kept on ice. When the temperature was raised to 37 C after treatment, the probe formed discrete large patches and became sequestered to caveolae. Binding of BC theta alone without the secondary reagents did not cause redistribution even at 37 C. Because the plasma membrane maintains integrity even after binding of BC theta, the probe can be used not only for cytochemical labelling of fixed cells but for pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that crosslinked cholesterol in the plasma membrane is sequestered to caveolae.
[符号：见正文]-毒素（产气荚膜梭菌溶血素O），一种胆固醇结合毒素，经部分蛋白酶解和生物素化（BC θ）以消除溶血活性，并用作细胞化学探针。在固定细胞中，BC θ在质膜中结合强烈，尤其是在顶端微绒毛基部和侧向突起处。用制霉菌素、洋地黄皂苷或番茄碱预处理可消除标记。当活培养细胞先用BC θ处理，然后用荧光素抗生物素蛋白D或胶体金链霉抗生物素蛋白处理时，只要细胞保持在冰上，细点状标记就会分布在细胞表面而无局部聚集。处理后当温度升至37℃时，探针形成离散的大斑块并被隔离到小窝中。即使在37℃下，单独使用BC θ而不使用二级试剂的结合也不会导致重新分布。因为即使在BC θ结合后质膜仍保持完整性，该探针不仅可用于固定细胞的细胞化学标记，还可用于追踪活细胞中交联胆固醇分子的行为。通过使用这种新探针，本研究揭示了质膜中的交联胆固醇被隔离到小窝中。
