STRESS -BEARING STRUCTURES IN MUSCLE AND MOTILE SYSTEMS

肌肉和运动系统中的应力承受结构

• 批准号: 6289048
• 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/6289048
• 关键词: X ray crystallography; biomechanics; crosslink; cytoskeletal proteins; electron microscopy; fish; muscle contraction; muscle proteins; myocardium; myocardium disorder; myofibrils; protein structure function; striated muscles; yeast two hybrid system

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

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。当例如通过神经脉冲刺激时，肌肉从静止状态被激活。它产生收缩力，缩短，然后当刺激停止时重新变回原来的尺寸。众所周知，肌肉通过分子马达（肌球蛋白）循环到收缩机器（肌节）中的肌动蛋白丝来产生主动力。当肌动蛋白丝被拉动滑动通过肌球蛋白粗丝时，肌肉缩短，而不改变任何一条丝的长度。很少有人知道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。同样不清楚的是，当肌节在发育中的肌肉组织中组装时，肌肉细胞如何设法控制粗细细丝的均匀和精确长度。最近对肌肉细胞骨架晶格的研究开始揭示了这两个问题。横纹肌细胞的细胞质除了含有肌动蛋白和肌球蛋白外，还含有至少两个相互连接的晶格。一个中间纤维网格将所有的肌节连接到膜骨架（中脉）、线粒体、细胞核和肌浆网。在肌节内，由一组弹性肌联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结构连续性。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结构传递。一个重要的应力承载结构是Z线，这是一种密集而狭窄的结构，它锚定并组织四种主要的细丝：肌动蛋白、肌联蛋白、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的机械力传递以及中间丝晶格中起着重要作用。然而，它的密集结构带来了技术挑战，蛋白质组成的可变性使得很难将研究结果从一块肌肉推广到另一块肌肉。我们的项目从几个方面解决了Z线的结构和功能。肌联蛋白、星云蛋白（骨骼肌）、星云蛋白（心脏中的一种星云蛋白样蛋白）在脊椎动物肌肉中Z线的组装和完整性中起什么作用？B.什么是组成和结构的异常广泛的Z线声波肌肉的海军军官学校鱼？它与在衰老的心肌中发现的异常杆状Z体有什么关系？在患病的骨骼肌中发现的异常杆状Z体被称为杆状肌病？肌联蛋白，星云蛋白和neblette到肌原纤维和Z线的组装正在研究与荧光技术与这些蛋白质的单克隆抗体，或通过使用在肌肉细胞内合成的荧光融合蛋白。为了鉴定蛋白质组成，特别是与Z线中的titin，nebulin和nebulette相互作用的蛋白质，我们应用分子生物学方法（酵母双杂交筛选）以及生物化学技术技术来寻找相互作用的蛋白质。目前正在用电子显微镜、X射线衍射和生物化学方法研究海军军官学校鱼类的音肌中异常宽的Z带（1毫米，大约是脊椎动物Z线宽度的100倍）的高分辨率结构。宽的Z带由平行的细丝组成，这些细丝通过十字交叉的连接支柱连接，并且具有接近结晶的规则性，没有致密的无定形材料。活的声波肌肉的X射线衍射表明，连接支柱以螺旋的方式围绕平行的细丝排列。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网格的附着点，该网格提供必要的径向力来组装和维持声波肌纤维的管状形状。这些研究对于理解收缩机制在发育过程中如何组装，在肌肉组织重塑过程中如何分解，在肌肉活动过程中如何传递张力以及肌肉在疾病中如何发生故障非常重要。- 肌肉，宽Z线，线状肌病，音速肌，X射线衍射，电子显微镜，海军军官鱼，交联