STRESS -BEARING STRUCTURES IN MUSCLE AND MOTILE SYSTEMS

在没有任何刺激的情况下，休息的肌肉在拉伸和释放时具有显著的弹性。当受到神经冲动的刺激时，肌肉从静止状态被激活。它产生收缩力，当刺激停止时，它会缩短，然后延长到原来的尺寸。众所周知，肌肉通过一种分子马达（肌凝蛋白）与收缩机械（肌节）中的肌动蛋白丝的循环而产生动力。当肌动蛋白丝被拉过肌凝蛋白粗丝时，肌肉变短，而不改变任何一条丝的长度。对于收缩的肌肉如何恢复其长度以及休息的肌肉如何对拉伸和压缩作出反应，我们所知甚少。当肌节在发育中的肌肉组织中组装时，肌肉细胞如何控制粗细细丝的均匀和精确长度也尚不清楚。最近对肌肉细胞骨架晶格的研究开始揭示这两个问题。横纹肌细胞的细胞质除含有肌动蛋白和肌球蛋白丝外，还含有至少两个相互连接的晶格。中间的纤维晶格包裹并连接所有的肌节与膜骨架（肋柱）、线粒体、细胞核和肌浆网。在肌节内部，由一组弹性肌蛋白丝和一组不可扩展的星云蛋白丝组成的细胞骨架基质提供了结构的连续性。两个晶格都产生恢复力。主动力和弹性力通过肌节的专门锚定结构传递。一个重要的受力结构是Z线，这是一个密集而狭窄的结构，锚定并组织了四种主要的细丝：肌动蛋白、肌动蛋白、星云蛋白和尾纤丝。因此，Z线在肌节的结构组织和肌节机械力的传递以及中间丝晶格中都起着基础作用。然而，它的致密结构带来了技术挑战，蛋白质组成的可变性使得很难将一种肌肉的发现推广到另一种肌肉。我们的项目从几个方面解决了Z线的结构和功能。titin, nebulin（骨骼肌），nebulette（心脏中类似星云蛋白的蛋白质）在脊椎动物肌肉中Z线的组装和完整性中的作用是什么？b.海军军官候补生鱼异常宽的声肌Z线的组成和结构是什么？它与在衰老的心脏肌肉中发现的异常线状棒Z体，在被称为线状肌病的患病骨骼肌中发现的异常线状棒Z体有什么关系？用荧光技术研究肌原纤维和Z线中titin、nebulin和nebulette的组装，或者使用这些蛋白质的单克隆抗体，或者使用在肌肉细胞内合成的荧光融合蛋白。为了确定蛋白质的组成，特别是在Z线上与titin， nebulin和nebulette相互作用的蛋白质，我们正在应用分子生物学方法（酵母双杂交筛选）以及生化技术来寻找相互作用的蛋白质。通过电子显微镜、x射线衍射和生物化学方法，研究了海军军官候补生鱼声音肌肉中异常宽的Z波段（1毫米，大约是脊椎动物Z线宽度的100倍）的高分辨率结构。宽Z带由纵横交错的连接杆连接的平行细丝组成，具有接近结晶的规律性，没有致密的非晶态物质。活体超声肌的x射线衍射表明，连接杆围绕平行细丝呈螺旋状排列。生化和免疫染色显示，肌动蛋白和α -肌动蛋白是Z带的主要成分。有趣的是，Z带也是一个非常复杂的中间纤维晶格的附着点，它提供必要的径向力来组装和维持音肌纤维的管状形状。这些研究对于理解收缩机械在发育过程中如何组装，在肌肉组织重塑过程中如何拆卸，在肌肉活动中张力如何传递以及肌肉在疾病中如何功能障碍具有重要意义。-肌肉，宽z线，线状肌病，声波肌，X射线衍射，电子显微镜，海军军官候补鱼，交联

The resting muscle, in the absence of any stimulation, is remarkably elastic when stretched and released. When stimulated e.g. by nerve impulses, muscle is activated from the resting state. It develops contractile force, shortens and then relengthens to its original dimension when stimulation ceases. It is well known that muscle develops active force by the cycling of a molecular motor, myosin, to actin filaments in the contractile machinery (sarcomere). Muscle shortens when actin filaments are pulled to slide pass myosin thick filaments, without changing the length of either filament. Very little is known of how contracted muscle restores its length and how resting muscle responds to stretch and compression. It is also unclear how muscle cells manage to control the uniform and precise length of thick and thin filaments when sarcomeres are assembled in developing muscle tissues. Recent studies of muscle cytoskeletal lattices begin to shed lights on both questions. The cytoplasm of striated muscle cells contains, besides actin and myosin filaments, contains at least two interconnected lattices. An intermediate filament lattice envelops and links all sarcomeres to the membrane skeleton (costamere), mitochondria, nuclei, and sarcoplasmic reticulum. Inside the sarcomere, a cytoskeletal matrix consisted of a set of elastic titin filaments and a set of inextensible nebulin filaments provides structural continuity. Both lattices generate restoring force. Active force and elastic force are transmitted through specialized anchor structures of the sarcomere. One important stress-bearing structure is the Z line, a dense and narrow structure that anchors and organizes four major filaments: actin, titin,nebulin and desmin filaments. The Z lines therefore plays a fundamental role in both the structural organization of sarcomere and the transmission of mechanical forces of the sarcomere as well the intermediate filament lattice. Its dense structure however poses technical challenges and the variability of protein composition made it difficult to generalize findings from one muscle to the next. Our projects address the Z line structure and function from several prospective.a. What are the roles of titin, nebulin (skeletal muscles), nebulette (a nebulin-ike protein in the heart) in the assembly and integrity of the Z line in vertebrate muscle? b. What are the composition and structure of the unusually broad Z line of sonic muscle of Midshipman fish? What is its relationship to the anomalous nemaline rod Z bodies found in aging heart muscle, in diseased skeletal muscle known as nemaline myopathy ? The assembly of the titin, nebulin and nebulette into the myofibrils and the Z lines are being studied with fluorescence techniques with either monoclonal antibodies to these proteins, or by the use of fluorescent fusion proteins synthesized within the muscle cells. To identify protein composition, especially the proteins that interact with titin, nebulin and nebulette in the Z line, we are applying both molecular biological methods (yeast two hybrid screening), as well as biochemical techniques techniques to search for interacting proteins. The high-resolution structure of the unusually broad Z band (1 mm, roughly 100 times the wide of vertebrate Z lines) in the sonic muscle of Midshipman fish is being studied by electron microscopy, X-ray diffraction and biochemical methods. The broad Z band is consisted of parallel filaments that are linked by criss crossing linking struts and has a nearly crystalline regularity without the dense amorphous material. X-ray diffraction of live sonic muscle indicated that the linking struts are arranged in a helical fashion around the parallel filaments. Biochemical and immunological staining revealed that actin and alpha-actitnin are major components of the Z band. Interestingly, the Z band are also attachment sites of a very elaborate intermediate filaments lattice that provides the necessary radial force to assemble and maintain the tubular shape of the sonic muscle fiber.These studies are important in the understanding of how contractile machinery assemble during development, how it dissemble during remodeling of muscle tissues, how tension are transmitted during muscle activities and how muscles malfunction in diseases. - Muscle, broad Z-line, nemaline myopathy, sonic muscle, X ray diffraction, electron microscopy, Midshipman fish, crosslinking