REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCL

骨骼肌亚细胞组织的调节

• 批准号: 6823119
• 负责人: Evelyn Ralston
• 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/6823119
• 关键词: Golgi apparatus; animal tissue; cell component structure /function; cell differentiation; cytoskeleton; endoplasmic reticulum; fiber cell; fibrous protein; intermediate filaments; membrane proteins; microtubules; muscle function; myoblasts; myogenesis; myotubes; protein localization; striated muscles; tubulin

1) Background The Golgi complex is a subcellular organelle that is essential to every cell, from yeast to complex mammalian cells. All membrane and secreted proteins transit through the Golgi complex where they undergo chemical modifications such as glycosylation. The Golgi complex is also the site where these proteins are sorted and targeted to their destination, a particularly important function in large cells such as muscle, which contains distinct membrane domains. The multiplicity of functions of this organelle is linked to its peculiar structural organization which in muscle changes dramatically during differentiation and regeneration. Very little is known of the mechanism of these changes. The organization of the Golgi complex of muscle also depends on the pattern of contractile activity and this regulation is not understood at all. Our goal is to understand how the distribution of the Golgi complex in muscle is regulated and is linked to the functional needs of muscle. In the past we have uncoverered basic aspects of the changes that take place during differentiation. We have established that the Golgi complex of each myoblast fragments into hundreds of smaller independent Golgi complexes placed around the nuclei and throughout the cytoplasm of the multinucleated myotubes and muscle fibers. These small Golgi elements are not distributed randomly but are retained next to endoplasmic reticulum sites specialized in the export of proteins to the Golgi complex. This specific localization suggested that the fragmentation of the Golgi complex during muscle differentiation resembles the fragmentation that the Golgi complex undergoes when microtubules are depolymerized. We have indeed demonstrated the similarity between these processes, therefore identifying changes in the microtubule cytoskeleton as a key factor in the changes that take place during differentiation. Patterned activity is an important regulator of muscle metabolism and contraction but it had never been linked to the organization of the protein secretory pathway. We have discovered that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is plastic in mature muscle fibers and responds to the pattern of contractile activity, causing a fiber type-dependent organization. We hypothesize that this plasticity is important and allows muscle to fulfill different metabolic demands depending on patterned activity. 2) Objective of present studies Our past work provided, for the first time, a description of the organization of the Golgi complex in muscle fibers. In order to understand how it is controlled by external factors such as patterned activity, we need to learn more about the interaction between cytoskeletal elements and Golgi complex organization. We also need to determine how their interaction is regulated at the molecular level and uncover the signaling pathways that are involved. 3) Results during the past year We have continued to investigate the changes in the organization of microtubules during muscle differentiation. We have focused on the protein ninein which, in endothelial cells, accompanies microtubules during their redistribution following cell polarization. We have been able to detect ninein in both undifferentiated and differentiated muscle and we have shown that it becomes redistributed during muscle differentiation. However, ninein does not seem to accompany small microtubules released from the centrosome shortly after their synthesis. This suggests that the mechanism of microtubule reorganization in muscle is distinct from that observed in endothelial cells and that ninein may play a different role in muscle. We have also started to explore signaling pathways that may regulate the Golgi complex organization. We have observed that lithium chloride affects the reorganization of both centrosomal proteins and Golgi complex during muscle differentiation. Lithium chloride is an inhibitor of GSK3-beta, an important kinase. It also affects stable microtubules. We are now using pharmacological tools and cDNA constructs to identify effectors of this pathway, which has been studied in epithelial cells migrating into a wound. Interestingly, we have observed that lithium chloride, previously reported to block muscle differentiation, blocks fusion of myoblasts but not their molecular differention as evidenced by normal induction of the muscle protein myogenin. 4) Conclusions and significance The Golgi complex is an essential organelle which shows great plasticity in skeletal muscle in response to changing physiological conditions and in pathological situations. It is therefore important to understand how this organelle is regulated. But because it has mostly been studied in proliferating, non-differentiated cells, we know little of the changes it undergoes in complex differentiated tissues. We have identified tools, such as lithium chloride and other effectors of the GSK3-beta pathway, which affect the organization of the Golgi complex during muscle differentiation and will allow us to start investigating this problem at the molecular level.

1)背景 高尔基复合体是一种亚细胞细胞器，对从酵母到复杂哺乳动物细胞的每一个细胞都是必不可少的。所有的膜和分泌的蛋白质都通过高尔基复合体，在那里它们经历了糖基化等化学修饰。高尔基复合体也是这些蛋白质被分类和定位到目的地的地方，这在肌肉等包含不同膜域的大细胞中是一个特别重要的功能。这种细胞器功能的多样性与其特殊的结构组织有关，这种结构组织在肌肉分化和再生过程中发生了戏剧性的变化。人们对这些变化的机制知之甚少。肌肉高尔基复合体的组织也依赖于收缩活动的模式，而这一规律根本不被理解。我们的目标是了解高尔基复合体在肌肉中的分布是如何调节的，并与肌肉的功能需求有关。 在过去，我们已经发现了分化过程中发生的变化的基本方面。我们已经证实，每个成肌细胞的高尔基复合体分裂成数百个较小的独立高尔基复合体，分布在细胞核周围以及多核肌管和肌纤维的细胞质中。这些小的高尔基体元素并不是随机分布的，而是被保留在内质网点旁边，专门将蛋白质输出到高尔基复合体。这种特殊的定位表明，在肌肉分化过程中高尔基复合体的碎裂类似于微管解聚时高尔基复合体所经历的碎裂。我们确实证明了这些过程之间的相似性，因此确定微管细胞骨架的变化是分化过程中发生变化的关键因素。 模式活动是肌肉新陈代谢和收缩的重要调节因素，但它从未与蛋白质分泌途径的组织联系在一起。我们发现，高尔基复合体、内质网出口部位和微管在成熟肌肉纤维中的分布是可塑性的，并响应收缩活动的模式，导致纤维类型依赖的组织。我们假设这种可塑性是重要的，并允许肌肉根据模式活动满足不同的新陈代谢需求。 2)本研究的目的 我们过去的工作第一次描述了肌纤维中高尔基复合体的组织结构。为了了解它是如何被外部因素控制的，比如模式活动，我们需要更多地了解细胞骨架元素和高尔基复合体组织之间的相互作用。我们还需要确定它们的相互作用是如何在分子水平上受到调控的，并揭示相关的信号通路。 3)过去一年的业绩 我们继续研究肌肉分化过程中微管组织的变化。我们重点研究了在内皮细胞中，微管在细胞极化后重新分布的过程中伴随着微管的蛋白九。我们已经能够在未分化和分化的肌肉中检测到九肽，并且我们已经证明，在肌肉分化过程中，它会重新分布。然而，在合成后不久，从中心体释放的小微管似乎并不伴随着Ninein。这表明，肌肉中微管重组的机制与内皮细胞中观察到的机制不同，九肽在肌肉中可能发挥不同的作用。 我们还开始探索可能调节高尔基复合体组织的信号通路。我们观察到，在肌肉分化过程中，氯化锂影响中心体蛋白和高尔基复合体的重组。氯化锂是GSK3-β的抑制剂，GSK3-β是一种重要的激酶。它也会影响稳定的微管。我们现在正在使用药理学工具和cDNA结构来识别这一途径的效应者，这一途径已经在上皮细胞迁移到伤口中进行了研究。有趣的是，我们观察到之前报道的阻止肌肉分化的氯化锂阻止了成肌细胞的融合，但并没有阻止它们的分子差异，这一点在肌肉蛋白肌肉生成素的正常诱导中得到了证明。 4)结论和意义 高尔基复合体是骨骼肌中重要的细胞器，在生理条件和病理条件的变化中表现出极大的可塑性。因此，重要的是要了解这个细胞器是如何被调节的。但由于它主要是在增殖的、未分化的细胞中进行研究，我们对它在复杂的分化组织中所经历的变化知之甚少。我们已经确定了一些工具，如氯化锂和GSK3-β途径的其他效应器，它们在肌肉分化过程中影响高尔基复合体的组织，并将使我们能够开始在分子水平上研究这个问题。