Gonsior S, Hinssen H
Biochemical Cell Biology Group, University of Bielefeld, Germany.
Cell Motil Cytoskeleton. 1995;31(3):196-206. doi: 10.1002/cm.970310303.
We have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross-striated myofibrils were permeabilized with Triton X-100 and incubated with gelsolin. Immunofluorescence microscopy localized both endogenous and exogenous gelsolin in the I-Z-I-regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+ dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross-striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin still bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X-100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric actin filaments.
我们研究了凝溶胶蛋白与原位细肌丝的结合及其抗切断稳定性。用Triton X-100使来自含有横纹肌原纤维的鸡骨骼肌的分化肌管通透化，并与凝溶胶蛋白一起孵育。免疫荧光显微镜检查将内源性和外源性凝溶胶蛋白定位在肌节的I-Z-I区域。染色模式表明外源性凝溶胶蛋白沿细肌丝的整个长度结合。这种结合是Ca2+依赖性的，但在随后加入EGTA后凝溶胶蛋白并未被去除。凝溶胶蛋白孵育后肌动蛋白的荧光染色保持不变，表明横纹肌原纤维中的细肌丝对凝溶胶蛋白的切断作用具有抗性，这与应力纤维中的微丝不同。在用高离子强度提取通透化细胞以去除原肌球蛋白和肌球蛋白后，凝溶胶蛋白仍沿整个细肌丝结合，肌动蛋白模式也保持不变。在Triton X-100通透化和高离子强度提取后，发现巨大蛋白伴肌动蛋白仍然作为肌原纤维成分存在。高盐提取后进行凝溶胶蛋白处理对细肌丝中的肌动蛋白和伴肌动蛋白均无影响。因此，我们得出结论，伴肌动蛋白赋予肌节肌动蛋白丝抗凝溶胶蛋白的能力。
