Schliwa M, van Blerkom J, Porter K R
Proc Natl Acad Sci U S A. 1981 Jul;78(7):4329-33. doi: 10.1073/pnas.78.7.4329.
Treatment of epithelial BSC-1 cells with low concentrations of the detergent Brij 58 results in partial or complete removal of the plasmalemma and partial extraction of internal membrane-bound organelles without causing massive release of "cytosolic" proteins from the cytoplasmic ground substance. Stereoscopic high-voltage electron microscopy of such extracted and fixed cells demonstrates a system of slender (4-20 nm) strands in a three-dimensional "microtrabecular" arrangement similar to that observed in unextracted whole-mount preparations. Extraction of Brij-extracted cells with Triton X-100 dissolves many of the microtrabecular strands, leaving, as a more stable structure, a characteristic cytoskeletal network composed of various filaments and microtubules. Two-dimensional polyacrylamide gel electrophoresis of 35S-labeled polypeptides performed concurrently with the morphological studies demonstrates that Triton extraction of Brij-extracted cells releases a large number of polypeptides. This release parallels the loss of structural components observed by electron microscopy. Labeling of Brij-extracted cells with heavy meromyosin subfragment 1 decorates actin filaments with characteristic arrowhead complexes which are readily visualized only after subsequent Triton extraction. These observations support the concept that many cytoplasmic proteins are structure-bound and, in addition to the components comprising the cytoskeleton, are structure-forming. We conclude that a metastable association of various proteins of the cytoplasmic ground substance exists whose morphological integrity is maintained, at lest temporarily, after removal of the plasmalemma in solutions containing Brij 58.
用低浓度去污剂Brij 58处理上皮细胞系BSC-1，可使质膜部分或完全去除，并使内膜结合细胞器部分被提取，而不会导致“胞质”蛋白从细胞质基质中大量释放。对这些经提取和固定的细胞进行立体高压电子显微镜观察，发现有一个细长（4 - 20纳米）的丝系统，呈三维“微梁”排列，类似于在未提取的整装标本中观察到的情况。用Triton X-100提取经Brij处理过的细胞，会溶解许多微梁丝，留下一个由各种细丝和微管组成的更稳定的特征性细胞骨架网络作为更稳定的结构。在进行形态学研究的同时，对35S标记多肽进行二维聚丙烯酰胺凝胶电泳表明，用Triton提取经Brij处理过的细胞会释放大量多肽。这种释放与电子显微镜观察到的结构成分的损失平行。用重酶解肌球蛋白亚片段1标记经Brij处理过的细胞，可使肌动蛋白丝带有特征性的箭头状复合物，这些复合物只有在随后用Triton提取后才能容易地观察到。这些观察结果支持了这样一种概念，即许多细胞质蛋白与结构结合，并且除了构成细胞骨架的成分外，还参与结构形成。我们得出结论，细胞质基质中各种蛋白质存在一种亚稳态结合，在含有Brij 58的溶液中去除质膜后，其形态完整性至少能暂时维持。
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