Simionescu N, Lupu F, Simionescu M
J Cell Biol. 1983 Nov;97(5 Pt 1):1592-600. doi: 10.1083/jcb.97.5.1592.
We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.
我们用多烯抗生素制霉菌素研究了微血管内皮细胞（小鼠胰腺、横膈膜、脑、心脏、肺、肾脏、甲状腺、肾上腺和肝脏）细胞膜中固醇的分布，据报道制霉菌素对游离3-β-羟基固醇具有结合特异性。在一些实验中，同时用阳离子铁蛋白检测细胞表面阴离子位点。用磷酸盐缓冲盐水（PBS）原位灌注血管，然后进行轻度固定并给予制霉菌素10至60分钟。对组织进行进一步处理以用于薄切片和冷冻断裂电子显微镜检查。短时间暴露（10分钟）于制霉菌素-戊二醛溶液会导致在许多区域，大多数质膜小泡、跨内皮通道和窗孔的气孔周围边缘内出现特征性制霉菌素-固醇复合物环。在肝血窦内皮（肝脏、肾上腺）的大开口边缘、有被小窝和吞噬泡中没有这种环。长时间暴露（30 - 60分钟）后，制霉菌素-固醇复合物随机标记其余的质膜（有被小窝除外，连接处的链间区域部分标记），并且还标记了大多数质膜小泡。这些气孔周围的固醇环大多显示在P面上，在充分发育时，围绕小泡气孔由6 - 8个单位组成，围绕窗孔由10 - 12个单位组成。在它们所在的水平，膜内颗粒和细胞表面阴离子位点实际上被排除。在微血管壁的周细胞和平滑肌细胞的质膜上也检测到了气孔周围的固醇环，与内皮质膜相比，否则它们用制霉菌素-固醇复合物标记很差。据推测，胆固醇的气孔周围环可能是质膜局部瞬时稳定以及在细胞膜与小泡膜融合/裂变过程的某个阶段两者相分离的重要因素。
