Shen B W, Josephs R, Steck T L
J Cell Biol. 1984 Sep;99(3):810-21. doi: 10.1083/jcb.99.3.810.
We have examined fragments of the filamentous network underlying the human erythrocyte membrane by high-resolution electron microscopy. Networks were released from ghosts by extraction with Triton X-100, freed of extraneous proteins in 1.5 M NaCl, and collected by centrifugation onto a sucrose cushion. These preparations contained primarily protein bands 1 + 2 (spectrin), band 4.1 and band 5 (actin). The networks were partially disassembled by incubation at 37 degrees C in 2 mM NaPi (pH 7), which caused the preferential dissociation of spectrin tetramers to dimers. The fragments so generated were fractionated by gel filtration chromatography and visualized by negative staining with uranyl acetate on fenestrated carbon films. Unit complexes, which sedimented at approximately 40S, contained linear filaments approximately 7-8 nm diam from which several slender and convoluted filaments projected. The linear filaments had a mean length of 52 +/- 17 nm and a serrated profile reminiscent of F-actin. They could be decorated in an arrowhead pattern with S1 fragments of muscle heavy meromyosin which, incidentally, displaced the convoluted filaments. Furthermore, the linear filaments nucleated the polymerization of rabbit muscle G-actin, predominantly but not exclusively from the fast-growing ends. On this basis, we have identified the linear filaments as F-actin; we infer that the convoluted filaments are spectrin. Spectrin molecules were usually attached to actin filaments in clusters that showed a preference for the ends of the F-actin. We also observed free globules up to 15 nm diam, usually associated with three spectrin molecules, which also nucleated actin polymerization; these may be simple junctional complexes of spectrin, actin, and band 4.1. In larger ensembles, spectrin tetramers linked actin filaments and/or globules into irregular arrays. Intact networks were an elaboration of the basic pattern manifested by the fragments. Thus, we have provided ultrastructural evidence that the submembrane skeleton is organized, as widely inferred from less direct information, into short actin filaments linked by multiple tetramers of spectrin clustered at sites of association with band 4.1.
我们通过高分辨率电子显微镜检查了人红细胞膜下丝状网络的片段。通过用Triton X-100提取从血影中释放出网络，在1.5 M NaCl中去除无关蛋白质，并通过离心收集到蔗糖垫层上。这些制剂主要包含蛋白带1 + 2（血影蛋白）、带4.1和带5（肌动蛋白）。通过在2 mM NaPi（pH 7）中于37℃孵育使网络部分解体，这导致血影蛋白四聚体优先解离为二聚体。如此产生的片段通过凝胶过滤色谱进行分级分离，并在有孔碳膜上用醋酸铀负染进行可视化。沉降系数约为40S的单位复合物包含直径约7 - 8 nm的线性细丝，从其上伸出几条细长且盘绕的细丝。线性细丝的平均长度为52 +/- 17 nm，其锯齿状轮廓让人联想到F - 肌动蛋白。它们可以用肌肉重酶解肌球蛋白的S1片段以箭头图案进行标记，顺便说一下，这会使盘绕的细丝移位。此外，线性细丝引发兔肌肉G - 肌动蛋白的聚合，主要但并非仅从快速生长的末端开始。在此基础上，我们将线性细丝鉴定为F - 肌动蛋白；我们推断盘绕的细丝是血影蛋白。血影蛋白分子通常以簇的形式附着在肌动蛋白丝上，这些簇倾向于附着在F - 肌动蛋白的末端。我们还观察到直径达15 nm的游离小球，通常与三个血影蛋白分子相关联，它们也能引发肌动蛋白聚合；这些可能是血影蛋白、肌动蛋白和带4.1的简单连接复合物。在更大的组合中，血影蛋白四聚体将肌动蛋白丝和/或小球连接成不规则阵列。完整的网络是片段所呈现的基本模式的一种细化。因此，我们提供了超微结构证据，即如从较少直接信息广泛推断的那样，膜下骨架被组织成由多个血影蛋白四聚体连接的短肌动蛋白丝，这些四聚体聚集在与带4.1结合的位点。
