THICK AND INTERMEDIATE FILAMENTS IN STRIATED MUSCLE

这些研究的目的是详细了解两个 横纹肌中的肌丝有多种--含肌球蛋白的粗丝 丝和含结蛋白的中间丝。 长丝 将在结构组织的三个层次上进行研究- 组装，长丝本身，以及在厚长丝的情况下， 细丝晶格 一种独特的形态学方法--快速冷冻后冷冻蚀刻 和旋转复制-将用于制备电子样品 显微镜 为了研究粗丝和横桥结构， 肌肉纤维以及具有“体内” 交叉桥的补充将被快速冷冻，通过电子检查 显微镜检查并通过图像处理技术进行分析。 的位置 肌球蛋白分子中的轻链也将被确定 形态学上，并使用针对 轻链表位。 中间纤维从它们的结构单元组装而成 （原丝）将使用体外系统进行研究。 的影响 两种针对结蛋白的单克隆抗体，已知其与原丝末端结合， 也将定量评估结蛋白的聚合 如通过电子显微镜。 集会的可能存在 将通过组装纯化的波形蛋白原丝来探测中间体 在亚生理盐浓度下， 种类并测试它们调节进一步IF聚合的能力。 将确定聚合的速率常数和方向性。 从这些研究中获得的信息将有助于了解分子 肌肉收缩过程中发生的事件，以及肌肉收缩是如何 在细胞骨架的水平上协调。 肌肉的说明 中间细丝结构也可能具有一般 适用于上皮细胞中细丝的其他亚类， 神经系统的细胞，因为所有的中间丝蛋白是 现在已知共享同源氨基酸序列。

The aim of these studies is to understand in detail the structure of two kinds of filaments in striated muscle--the myosin-containing, thick filaments and the desmin-containing intermediate filaments. The filaments will be studied at three levels of structural organization--the subunits of assembly, the filaments themselves and, in the case of thick filaments, the filament lattice. A unique morphological approach--quick-freezing followed by freeze-etching and rotary replication--will be used to prepare samples for electron microscopy. To study thick filament and crossbridge structure, intact muscle fibers as well as fractionated thin filaments with an "in vivo" complement of crossbridges will be quick-frozen, examined by electron microscopy and analyzed by image processing techniques. The location of the light chains in the myosin molecule will also be determined morphologically and verified using monoclonal antibodies directed against light chain epitopes. The assembly of intermediate filaments from their building blocks (protofilaments) will be studied using in vitro systems. The effects of two monoclonal antibodies to desmin, known to bind to protofilament ends, upon the polymerization of desmin will be assessed quantitatively as well as by electron microscopy. The possible existence of assembly intermediates will be probed by assembling purified vimentin protofilaments at sub-physiological salt concentrations, isolating the intermediate species and testing their ability to modulate further IF polymerization. Rate constants and directionality of polymerization will be determined. Information obtained in these studies will help to understand the molecular events during muscular contraction, and how muscle contraction is coordinated at the level of the cytoskeleton. The elucidation of muscle intermediate filament structure will also likely be of general applicability to other subclasses of filaments in epithelial cells and cells of the nervous system, since all intermediate filament proteins are now known to share homologous amino acid sequences.