Craig R, Alamo L, Padrón R
Department of Cell Biology University of Massachusetts Medical School, Worcester 01655.
J Mol Biol. 1992 Nov 20;228(2):474-87. doi: 10.1016/0022-2836(92)90836-9.
Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor. Electron microscopy of longitudinal sections of relaxed specimens showed greatly improved preservation of thick filament ultrastructure compared with conventional fixation. This was revealed by the appearance of a clear helical arrangement of myosin crossbridges along the filament surface and by a series of layer line reflections in computed Fourier transforms of sections, corresponding to the layer lines indexing on a 43 nm repeat in X-ray diffraction patterns of whole, living muscles. Filtered images of single myosin filaments were similar to those of negatively stained, isolated vertebrate filaments and consistent with a three-start helix. M-line and other non-myosin proteins were also very well preserved. Rigor specimens showed, in the region of overlapping myosin and actin filaments, periodicities corresponding to the 36, 24, 14.4 and 5.9 nm repeats detected in X-ray patterns of whole muscle in rigor; in the H-zone they showed a disordered array of crossbridges. Transverse sections, whose Fourier transforms extend to the (3, 0) reflection, supported the view, based on X-ray diffraction and conventional electron microscopy, that in the overlap zone of relaxed muscle most of the crossbridges are detached from the thin filaments while in rigor they are attached. We conclude that the rapid freezing technique preserves the molecular structure of the myofilaments closer to the in vivo state (as monitored by X-ray diffraction) than does normal fixation.
快速冷冻后进行冷冻置换，已被用于研究处于松弛和僵直状态的活的及去膜青蛙缝匠肌肌球蛋白丝的超微结构。与传统固定方法相比，对松弛标本纵切片的电子显微镜观察显示粗丝超微结构的保存有了极大改善。这通过肌球蛋白横桥沿丝表面清晰的螺旋排列以及切片的傅里叶变换中的一系列层线反射得以揭示，这些层线与完整活肌肉X射线衍射图谱中43nm重复的层线索引相对应。单根肌球蛋白丝的滤波图像与负染的分离脊椎动物丝的图像相似，且符合三股螺旋结构。M线和其他非肌球蛋白蛋白也保存得非常好。僵直标本在肌球蛋白和肌动蛋白丝重叠区域显示出与整个肌肉僵直状态下X射线图谱中检测到的36、24、14.4和5.9nm重复相对应的周期性；在H区，它们显示出横桥的无序排列。傅里叶变换延伸至(3, 0)反射的横切片支持了基于X射线衍射和传统电子显微镜的观点，即在松弛肌肉的重叠区域，大多数横桥与细丝分离，而在僵直状态下它们是附着的。我们得出结论，与正常固定相比，快速冷冻技术能使肌丝的分子结构更接近体内状态（通过X射线衍射监测）。
