Ultrastructural study of cytoplasmic sides of erythrocyte membranes by quick-freezing and deep-etching method.

(1)介绍了一种新的制备红细胞膜外露细胞质的方法，提高了直接电子显微镜图像的分辨率。用多聚甲醛短暂固定正常人红细胞。在两张盖子之间夹着一滴含有红血球的药丸。将贴壁的红细胞裂开，用戊二醛进行后处理。所有样品都被快速冷冻在异戊烷-丙烷混合物中，深度蚀刻，并用铂和碳旋转阴影。红细胞质侧面可见丝状结构形成网状结构。(2)对遗传性球形红细胞增多症(HS)和遗传性椭圆形红细胞增多症(HE)的球形和椭圆形红细胞进行机械切开，并用抗血影蛋白抗体进行免疫组织化学染色。采用快速冷冻和深度刻蚀的方法制备了复制膜，并用电子显微镜对其进行了检测。正常红细胞的原位膜骨架主要由网状结构的血影蛋白细丝组成，这些网状结构在细胞膜的胞质侧面形成网络。相反，形状异常的红细胞(HS和HE)的膜骨架比正常红细胞的丝状骨架少得多，颗粒多得多。这种异常的组织结构可能是导致HS和HE患者红细胞形状异常的因素之一。(3)用同样的方法研究了蛙和鸡的红细胞膜骨架的超微结构。除血影蛋白网络外，微管和肌动蛋白细丝也定位于红细胞膜的胞质侧。这表明原始的膜骨架不仅含有血影蛋白，还含有细胞骨架蛋白。

(1) A novel method for prepare exposed cytoplasmic aspects of erythrocyte membranes is described, which improves the resolution in direct electron microscopic images. Normal human erythrocytes were briefly fixed with paraformaldehyde. A drop containing the erythrocyte pellets was sandwiched between 2 coverslips. The attached erythrocytes were split open and postfixed with glutaraldehyde. All specimens were quickly frozen in an isopentane-propane mixture, deeply etched and rotary shadowed with platinum and carbon. Filamentous structures were seen to form networks on the cytoplasmic sides of the erythrocyte. This method will be useful in the analysis of the in situ ultrastructure of cytoplasmic sides of normal erythrocyte membranes.(2) Spherocytic and elliptocytic erythrocytes with hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) were split open mechanically and were immunostained with anti-spectrin antibody. Replica membranes were prepared by a quick-freezing and deep-etching method and were checked by electron microscopy. The in situ membrane skeletons of normal erythrocytes consisted mainly of reticular patterns of spectrin filaments, which formed networks on the cytoplasmic sides of membranes. In contrast, the membrane skeletons of abnormally shaped erythrocytes (HS and HE) were much less filamentous and more granular than those of normal erythrocytes. This abnormal organization may be one of the factors that induce abnormally shaped erythrocytes in HS and HE patients.(3) Ultrastructures of membrane skeletons in frog or chicken erythrocytes were investigated in the same procedure. Microtubules and actin filaments were also localized on the cytoplasmic sides of erythrocyte membranes in addition to spectrin networks. It is suggested that primitive membrane skeletons contain not only spectrin, but also cytoskeletal proteins.