Stress-bearing Structures In Muscle And Muscle Diseases

在没有任何刺激的情况下，休息的肌肉在伸展和释放时会非常有弹性。当受到神经冲动等刺激时，肌肉从静止状态被激活。当刺激停止时，它会产生收缩力量，缩短，然后恢复到原来的尺寸。众所周知，肌肉通过一个分子马达，肌球蛋白，循环到收缩机械(肌节)中的肌动细丝而产生积极的力量。当肌动蛋白细丝被拉动以滑过肌球蛋白粗丝时，肌肉会缩短，而不改变任何一根肌球蛋白粗丝的长度。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做出反应，人们知之甚少。当肌节在发育中的肌肉组织中组装时，肌肉细胞是如何设法控制粗丝和细丝的统一和精确长度的，目前还不清楚。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细丝外，还含有至少两个相互连接的晶格。中间纤维格子包裹所有肌节，并将其连接到膜骨架(胞壁)、线粒体、核和肌浆网。在肌节内，由一组弹性肌动蛋白细丝和一组不可伸展的星云蛋白细丝组成的细胞骨架基质提供了结构连续性。这两个晶格都会产生恢复力。主动力和弹性力通过肌节的特殊锚定结构传递。一个重要的承压结构是Z线，这是一种密集而狭窄的结构，锚定并组织了四种主要的细丝：肌动蛋白、肌动蛋白、星云蛋白和结蛋白细丝。因此，Z线在肌节的结构组织和肌节以及中间纤维晶格的机械力传递中都起着重要的作用。然而，其致密的结构构成了技术挑战，而且蛋白质组成的多样性使得很难从一块肌肉到另一块肌肉概括研究结果。我们的项目从几个方面阐述了Z线的结构和功能。1.肌动蛋白、星云蛋白(骨骼肌)、星云蛋白(心脏中的一种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起什么作用？2.学员鱼异常宽阔的Z线的组成和结构是什么？3.它与在老化的心肌中发现的异常的线状杆状Z小体有什么关系？在患病的骨骼肌中被称为线状肌病？肌动蛋白、星云和星云组装成肌原纤维和Z线的研究正在用针对这些蛋白质的单抗或使用在肌肉细胞内合成的荧光融合蛋白的荧光技术进行。为了确定蛋白质组成，特别是与Z线上的肌动蛋白、星云蛋白和星云相互作用的蛋白质，我们应用了分子生物学方法(酵母双杂交筛选)以及生化技术技术来寻找相互作用的蛋白质。我们已经成功地解决了异常宽的Z带(1微米，大约是脊椎动物Z线宽度的20倍)的高分辨率结构，目前正在用电子显微镜、X射线衍射和生物化学方法对Midseman Fish的音速肌肉进行研究。有趣的是，Z带也是一个非常精细的中间细丝晶格的附着点，它提供了组装和保持声波肌肉纤维管状形状所需的径向力。我们正在开始克隆与Z带结构有关的蛋白质，并比较线虫肌病肌肉的蛋白质和转录谱。为了可视化声波肌肉的收缩，我们还成功地通过磁共振成像(MRI)可视化了完整声波肌肉中肌肉纤维的详细组织。这些研究对于了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分解，在肌肉活动中如何传递张力，以及线状肌病和其他肌肉疾病中肌肉功能障碍具有重要意义。

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 play 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 prospectives. 1. 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? 2. What are the composition and structure of the unusually broad Z line of sonic muscle of Midshipman fish? 3. 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. We have succeeded in resolving the high-resolution structure of the unusually broad Z band (1 micron, roughly 20 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. 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. We are initiating the cloning of proteins involved in the structure of the Z bands and the comparison of protein and transcript profiles of nemaline myopathy muscles. To visualize the contraction of the sonic muscle, we have also succeeded in visualizing the detailed organization of muscle fibers in intact sonic muscle by magnetic resonance imaging (MRI). 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 nemaline myopathy and other muscle diseases.