Stress-bearing Structures In Muscle And Muscle Diseases

肌肉和肌肉疾病中的承压结构

• 批准号: 7137997
• 负责人: KUAN WANG
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7137997
• 关键词: X ray crystallography; alternatives to animals in research; biological signal transduction; biomechanics; cell component structure /function; congenital disorders; cytoskeletal proteins; cytoskeleton; electron microscopy; fish; genetic disorder; intermediate filaments; intermolecular interaction; magnetic resonance imaging; microfilaments; molecular assembly /self assembly; molecular pathology; muscle contraction; muscle disorders; muscle function; muscle proteins; muscle tension; myocardium; myofibrils; protein structure function; sarcomeres; striated muscles

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, 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 are now known to play important roles in the structural organization of sarcomere, the transmission of mechanical forces as well as the stress signaling pathways. 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 are the roles of protein kinases, nonmuscle myosin and other signaling proteins in the function of the Z lines? 4. 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 distribution of titin, nebulin, nebulette nonmuscle myosin IIB and 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 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 and junctional complexes at the Z band that provide the necessary radial force to assemble and maintain the tubular shape of the sonic muscle fiber. We also discovered novel assembly/disassembly intermediates (myosin flares and leptomeres) that suggest a new mechanism of muscle growth by splitting longitudinally along the myotubes. The myofibrillogenesis is being approached by establishing a culture system for the sonic muscle. 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) and analyzed the sound producing characterized by laser vibrometry. These studies are important in the understanding of how contractile machinery of this superfast muscle assembles during development, how it dissembles during remodeling of muscle tissues, how tension are transmitted during muscle activities and how muscles malfunction in nemaline myopathy and other muscle diseases.

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、线粒体、细胞核和肌浆网。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。目前已知Z线在肌节的结构组织、机械力的传递以及应力信号通路中发挥着重要作用。 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、肌球蛋白(骨骼肌)、星云(心脏中的一种类似于星云蛋白的蛋白质)在脊椎动物肌肉中Z线的组装和完整性中起什么作用？2.中船鱼异常宽阔的声肌Z线的组成和结构是什么？3.蛋白激酶、非肌肉肌球蛋白和其他信号蛋白在Z线的功能中起什么作用？4.它与衰老的心肌中发现的异常的线状杆状Z小体有什么关系？ 肌动蛋白、星云蛋白、星云状非肌肉肌球蛋白IIB的分布以及肌原纤维和Z线的分布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研究。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和Z带上的连接复合体的附着点，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。我们还发现了新的组装/拆解中间体(肌球蛋白喇叭和柔肌节)，这表明了一种新的肌肉生长机制，即沿肌管纵向分裂。肌原纤维的形成是通过建立一个声波肌肉的培养系统来探讨的。为了可视化声波肌肉的收缩，我们还成功地利用磁共振成像(MRI)显示了完整声波肌肉中肌肉纤维的详细组织，并用激光测振仪分析了声音产生的特征。这些研究对于了解这种超快肌肉在发育过程中的收缩机制是如何组装的，在肌肉组织的重塑过程中是如何伪装的，在肌肉活动期间张力是如何传递的，以及线虫性肌病和其他肌肉疾病中肌肉功能障碍是如何进行的，这些研究都是重要的。