Conformation and elasticity of the isolated red blood cell membrane skeleton.
Conformation and elasticity of the isolated red blood cell membrane skeleton.
复制标题
DOI：
10.1016/s0006-3495(92)81644-2
复制
发表时间：
1992-09
影响因子：
3.4
通讯作者：
Karel Svoboda;Christoph F. Schmidt;Daniel Branton;Steven M. Block
中科院分区：
文献类型：
--
作者：
Karel Svoboda;Christoph F. Schmidt;Daniel Branton;Steven M. Block
研究了非离子去污剂提取人红细胞血影过程中及提取后红细胞膜骨架的结构和弹性。使用光镊将单个细胞悬浮在流动室内，远离所有表面;该程序允许完全交换介质，同时通过高分辨率视频增强差分干涉对比（DIC）显微镜观察骨架的低对比度蛋白质网络。在5 mM盐缓冲液中提取后，骨骼立即呈现膨胀的、接近球形的形状，其与其亲本RBC的形状无关。根据热波动的程度和它们在小流场中的变形能力来判断，骨骼的弯曲刚度明显低于红细胞或鬼。在低温（T小于10 ℃）下，在该缓冲液中保持长达40分钟的骨骼没有进一步的变化，但是当缓冲液的离子强度增加时，骨骼收缩。当盐浓度升高到1.5M时，收缩保持可逆约1分钟，但此后变得不可逆。当在1.5 M盐缓冲液中保持较长时间时，骨骼继续收缩，失去灵活性，并呈现不规则的形状：这种硬化是不可逆的。在这个阶段，骨架与标准原液制备中分离的骨架非常相似。我们建议，刚性的，不可逆的收缩状态的转变是一个后果的血影蛋白二聚体-二聚体重新连接，这些结构重排是热激活的。我们还测量了新鲜和大量提取的骨骼的盐依赖性大小。我们的测量结果表明，在原位，血影蛋白系链是灵活的，在150 mM盐的持久性长度约为10 nm。
We studied the structure and elasticity of membrane skeletons from human red blood cells (RBCs) during and after extraction of RBC ghosts with nonionic detergent. Optical tweezers were used to suspend individual cells inside a flow chamber, away from all surfaces; this procedure allowed complete exchange of medium while the low-contrast protein network of the skeleton was observed by high resolution, video-enhanced differential interference-contrast (DIC) microscopy. Immediately following extraction in a 5 mM salt buffer, skeletons assumed expanded, nearly spherical shapes that were uncorrelated with the shapes of their parent RBCs. Judging by the extent of thermal undulations and by their deformability in small flow fields, the bending rigidity of skeletons was markedly lower than that of either RBCs or ghosts. No further changes were apparent in skeletons maintained in this buffer for up to 40min at low temperatures (T less than 10 degrees C), but skeletons shrank when the ionic strength of the buffer was increased. When the salt concentration was raised to 1.5 M, shrinkage remained reversible for approximately 1min but thereafter became irreversible. When maintained in 1.5 M salt buffer for longer periods, skeletons continued to shrink, lost flexibility, and assumed irregular shapes: this rigidification was irreversible. At this stage, skeletons closely resembled those isolated in standard bulk preparations. We propose that the transformation to the rigid, irreversibly shrunken state is a consequence of spectrin dimer-dimer reconnections and that these structural rearrangements are thermally activated. We also measured the salt-dependent size of fresh and bulk extracted skeletons. Our measurements suggest that, in situ, the spectrin tethers are flexible, with a persistence length of approximately 10 nm at 150 mM salt.