Stabilization and the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition.
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.
D000818:Animals; D002478:Cells, Cultured; D002592:Cetomacrogol; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000648:ultrastructure*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron
D003902:Detergents; D002592:Cetomacrogol