Ursitti J A, Pumplin D W, Wade J B, Bloch R J
Department of Physiology, School of Medicine, University of Maryland, Baltimore 21201.
Cell Motil Cytoskeleton. 1991;19(4):227-43. doi: 10.1002/cm.970190402.
We attached paraformaldehyde-fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5-6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/microns 2 of membrane. In contrast, we found 3-4 filaments at each intersection and approximately 400 intersections/microns 2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. Our results suggest that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material. Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by approximately 3 nm.
我们将多聚甲醛固定的人红细胞血影附着在包被的盖玻片上，并对其进行剪切以暴露细胞骨架。快速冷冻、深度蚀刻、旋转复制，或单宁酸/锇固定及塑料包埋显示，细胞骨架是由相互交叉的直丝组成的致密网络。先前对铺展骨架进行的负染研究发现，在每个肌动蛋白寡聚物处有5 - 6个血影蛋白四聚体交叉，估计每平方微米膜上有250个这样的交叉点。相比之下，我们发现每个交叉点有3 - 4根细丝，每平方微米膜上约有400个交叉点。免疫金标记证实这些细丝是血影蛋白，但其长度（29 - 37纳米）约为伸展的血影蛋白二聚体长度的三分之一。细丝的长度和直径足以容纳血影蛋白二聚体，但不能容纳血影蛋白四聚体。我们的结果表明，在原位，血影蛋白二聚体可能以六聚体和八聚体的形式结合，而不是四聚体。我们提出了几种解释，可以使我们对完整细胞骨架的观察结果与先前关于铺展材料的报道相协调。用低离子强度溶液提取剪切后的血影可去除细胞骨架，从而揭示膜细胞质表面的突起。如免疫金标记所示，这些突起含有带3蛋白，当去除细胞骨架时，它们的聚集程度与膜内颗粒（IMP）相似，表明这些结构之间存在直接关系。定量分析表明，每个细胞质突起的化学计量比为2个IMP。细胞质突起可能除了带3蛋白外还含有其他蛋白质，因为用高离子强度溶液进一步处理可提取外周蛋白并使突起直径减小约3纳米。
