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

速冻深蚀法研究红细胞膜胞质侧面的超微结构。

基本信息

批准号：
03670007
负责人：
OHNO Shinichi
金额：
$ 1.47万
依托单位：
Yamanashi Medical University (1992-1993)Shinshu University (1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

(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.

(1)介绍了一种制备红细胞膜暴露胞质的新方法，它提高了直接电镜图像的分辨率。正常人红细胞用多聚甲醛简单固定。将含有红细胞团块的液滴夹在2个盖玻片之间。将附着的红细胞切开，并用戊二醛后固定。所有标本都在异戊烷-丙烷混合物中快速冷冻，用铂和碳进行深度蚀刻和旋转阴影。在红细胞的胞质侧观察到丝状结构形成网络。该方法可用于正常红细胞膜胞质侧的原位超微结构分析。(2)将患有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）的球形红细胞和椭圆形红细胞机械切开，并用抗血影蛋白抗体进行免疫染色。采用快速冷冻法和深腐蚀法制备了纳米复合膜，并进行了电镜观察。正常红细胞的原位膜骨架主要由网状的血影蛋白丝组成，在膜的胞质侧形成网络。与此相反，异常形状的红细胞（HS和HE）的膜骨架少得多的丝状和更多的颗粒比正常红细胞。这种异常组织可能是导致HS和HE患者红细胞形状异常的因素之一。(3)用同样的方法观察了蛙和鸡红细胞膜骨架的超微结构。微管和肌动蛋白丝也定位于细胞质侧的红细胞膜，除了血影蛋白网络。这表明，原始膜骨架不仅包含血影蛋白，但也细胞骨架蛋白。