REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCLE

骨骼肌亚细胞组织的调节

• 批准号: 7969925
• 负责人: Evelyn Ralston
• 金额: $ 116.48万
• 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/7969925
• 关键词: Actins; Affect; American; Animals; Biochemical; Caliber; Cardiac; Cell Line; Cell Nucleus; Cells; Cellular Morphology; Cellular biology; Centrosome; Contractile Proteins; Cytoskeleton; Defect; Digestion; Disease; Dominant-Negative Mutation; Duchenne muscular dystrophy; Dystrophin; Elements; Fiber; Foundations; Glycogen storage disease type II; Goals; Golgi Apparatus; Health; Human; Immunofluorescence Immunologic; Intermediate Filament Proteins; LAMP-1; Laboratories; Length; Life; Link; Lysosomes; Mammalian Cell; Manuscripts; Microtubule Proteins; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Minnesota; Modality; Modeling; Movement; Mus; Muscle; Muscle Cells; Muscle Development; Muscle Fibers; Muscle Proteins; Mutation; Myoblasts; Myopathy; Natural regeneration; Nocodazole; Nuclear Envelope; Organ; Organelles; Osteoclasts; Pathology; Play; Positioning Attribute; Preparation; Process; Proteins; Regulation; Resistance; Role; Skeletal Muscle; Societies; Source; Staining method; Stains; Structure; Subcellular structure; System; Techniques; Tissues; Tubulin; Utrophin; Work; Yeasts; base; cell type; disease characteristic; flexor digitorum brevis; glucose metabolism; in vivo; knock-down; light microscopy; mdx mouse; meetings; mouse model; posters; precursor cell; prevent; research study

Over the past year we have completed a collaborative study of the interaction between the muscle protein dystrophin and the microtubule constituent protein tubulin (Prins et al., 2009). Dystrophin is a large protein whose mutations are responsible for the most prevalent muscle disease, Duchenne Muscular Dystrophy (DMD). Dystrophin was already known to interact with the actin cytoskeleton and with intermediate filament proteins, suggesting that it may act as a cytolinker, a protein that serves to link all constituents of the cell cytoskeleton. Using muscle extracts from control and from mdx mouse, the mouse model for DMD, our collaborator James Ervasti (U. Minnesota) and his laboratory obtained biochemical evidence supporting a direct interaction between dystrophin and tubulin. Amisha Mehta and, later, Victoria Tate in LI performed immunofluorescence staining of single whole muscle fibers from control and mdx mice, as well as from utrophin knockdown mice and mdx-utrophin double knockdown mice. Microtubules lost their normal organization in mdx and mdx-utrophin double knockdown muscles, but not in the utrophin knockdown muscles. In addition, we showed that microtubule defects are already present in young (3 week-old) mice which have not started undergoing the cycles of degeneration-regeneration characteristic of the disease. These experiments support a specific link between dystrophin and microtubules in vivo. We also showed that microtubules in fast muscles of a normal mouse course along transverse and longitudinal bands of dystrophin, confirming that dystrophin contributes to organizing microtubules. This work thus classifies dystrophin as a cytolinker in muscle fibers and may help to understand some of the consequences of the absence of dystrophin, in the mdx mouse and in DMD. We have also progressed towards our goal of understanding the organization of microtubules in mature muscle fibers. Victoria Tate has been using a culture system using muscle fibers detached from the flexor digitorum brevis mouse muscle by enzymatic digestion. She has determined conditions to depolymerize the dynamic microtubules from the live fibers and to then observe their repolymerization. These experiments are important because microtubules are typically nucleated at the centrosome, but several cell types, such as skeletal muscle, lack a typical centrosome. Some cell types also show microtubule nucleation at the Golgi complex, apart from that seen at the centrosome. In skeletal muscle, centrosomal proteins, microtubules, and Golgi complex are all redistributed, first during differentiation, and then further during maturation of muscle fibers. Differentiated muscle cultures show microtubules forming at the nuclear membrane but microtubule dynamics have never been explored in muscle fibers in vivo. Interestingly, nucleation of microtubules in muscle fibers appears to originate at the Golgi complex elements. Victoria has also examined the role of microtubules in the close association between Golgi elements and lysosomes (LAMP-1 positive structures) in muscle fibers. Most Golgi elements (75%) have juxtaposed lysosomes in control and nocodazole-treated fibers but cold and nocodazole, each, increase the fraction of Golgi-unassociated lysosomes. This suggests that both dynamic microtubules and stable, nocodazole-resistant, glutamylated microtubules are involved in Golgi-lysosome association. Our findings provide the foundation of a model to understand microtubule and Golgi complex organization in muscle fibers. They also extend Golgi complex-based microtubule nucleation to a highly differentiated tissue. The mechanisms may not be identical. A poster (Tate et al.) will be presented at the 2009 meeting of the American Society for Cell biology. In parallel to our work on muscle fibers, we have pursued our work on muscle cultures which have provided us with a basic understanding of the changes taking place during differentiation of muscle cells. Tan Zhang together with Kristien Zaal has been investigating the role of the microtubule-associated protein EB1 in muscle differentiation. In other mammalian cells, EB1 is necessary for microtubule stabilization. We hypothesized that EB1 may play a similar role during muscle development. Indeed, we found that dominant-negative constructs of EB1 affect myoblast elongation. Knocking down EB1 permanently prevents microtubule stabilization and, unexpectedly, prevents differentiation of the muscle cells (Zhang et al., 2009). In addition, Tan Zhang has now demonstrated that there is an EB1 pool associated with the Golgi complex and that it may be play a role in the integrity of the Golgi complex (manuscript in preparation). We have also continued to collaborate with Drs. Plotz and Raben on the study of Pompe Disease, a lysosomal storage disorder in which cardiac and skeletal muscles are the source of the pathology (Raben et al., 2008 & 2009).

在过去的一年里，我们完成了一项关于肌肉蛋白肌营养不良蛋白和微管组成蛋白微管蛋白之间相互作用的合作研究（Prins 等，2009）。肌营养不良蛋白是一种大蛋白，其突变导致最常见的肌肉疾病杜氏肌营养不良症 (DMD)。已知抗肌营养不良蛋白与肌动蛋白细胞骨架和中间丝蛋白相互作用，表明它可能充当细胞接头，一种连接细胞骨架所有成分的蛋白质。我们的合作者 James Ervasti（美国明尼苏达州）和他的实验室使用对照小鼠和 DMD 小鼠模型 mdx 小鼠的肌肉提取物，获得了支持肌营养不良蛋白和微管蛋白之间直接相互作用的生化证据。阿米莎·梅塔 (Amisha Mehta) 和后来的维多利亚·泰特 (Victoria Tate) 对对照小鼠和 mdx 小鼠、以及 utropin 敲低小鼠和 mdx-utropin 双敲低小鼠的单个完整肌纤维进行了免疫荧光染色。在 mdx 和 mdx-utropin 双敲低肌肉中，微管失去了正常组织，但在 utropin 敲低肌肉中却没有。此外，我们还发现，年轻（3周大）小鼠中已经存在微管缺陷，这些小鼠尚未开始经历该疾病特征的变性-再生周期。这些实验支持肌营养不良蛋白和体内微管之间的特定联系。我们还表明，正常小鼠快速肌肉中的微管沿着肌营养不良蛋白的横向和纵向带移动，证实肌营养不良蛋白有助于组织微管。因此，这项工作将肌营养不良蛋白归类为肌纤维中的细胞链接剂，可能有助于了解 mdx 小鼠和 DMD 中缺乏肌营养不良蛋白的一些后果。 我们还朝着了解成熟肌纤维中微管组织的目标取得了进展。维多利亚·泰特（Victoria Tate）一直在使用一种培养系统，该系统使用通过酶消化从小鼠指短屈肌上分离出来的肌纤维。她确定了从活纤维中解聚动态微管的条件，然后观察它们的再聚合。这些实验很重要，因为微管通常在中心体成核，但几种细胞类型（例如骨骼肌）缺乏典型的中心体。除了在中心体处观察到的微管成核外，一些细胞类型还在高尔基复合体处显示出微管成核。在骨骼肌中，中心体蛋白、微管和高尔基复合体首先在分化过程中重新分布，然后在肌纤维成熟过程中进一步重新分布。分化的肌肉培养物显示微管在核膜处形成，但从未在体内肌纤维中探索过微管动力学。有趣的是，肌纤维中微管的成核似乎起源于高尔基复合体元件。 Victoria 还研究了微管在肌纤维中高尔基体元件和溶酶体（LAMP-1 阳性结构）之间密切​​联系中的作用。大多数高尔基体元件（75％）在对照和诺考达唑处理的纤维中具有并置的溶酶体，但冷和诺考达唑各自增加了与高尔基体无关的溶酶体的比例。这表明动态微管和稳定的诺考达唑抗性谷氨酰化微管都参与高尔基体-溶酶体关联。我们的研究结果为理解肌纤维中微管和高尔基复合体组织的模型奠定了基础。他们还将基于高尔基复合体的微管成核扩展到高度分化的组织。机制可能不相同。海报（Tate 等人）将在 2009 年美国细胞生物学学会会议上展示。 在我们对肌肉纤维的研究的同时，我们还开展了肌肉培养的工作，这使我们对肌肉细胞分化过程中发生的变化有了基本的了解。张谭和 Kristien Zaal 一直在研究微管相关蛋白 EB1 在肌肉分化中的作用。在其他哺乳动物细胞中，EB1 对于微管稳定是必需的。我们假设 EB1 可能在肌肉发育过程中发挥类似的作用。事实上，我们发现 EB1 的显性失活结构影响成肌细胞伸长。敲除 EB1 会永久性地阻止微管稳定，并且出人意料地阻止肌肉细胞的分化（Zhang 等人，2009）。此外，张谭现已证明存在一个与高尔基复合体相关的EB1库，并且它可能在高尔基复合体的完整性中发挥作用（手稿正在准备中）。 我们还继续与博士合作。 Plotz 和 Raben 对庞贝氏病的研究，庞贝氏病是一种溶酶体贮积症，其中心肌和骨骼肌是其病理来源（Raben 等人，2008 年和 2009 年）。