REGULATION OF SUBCELLULAR ORGANIZATION IN SKELETAL MUSCL

1)背景 骨骼肌纤维是巨大的多核细胞，通过一波又一波的发育变化来构建，影响整体细胞形态和亚细胞组织。人们对了解肌肉特有的过程给予了很大的关注，例如收缩蛋白的组装。相比之下，我们对具有数百个核和几个不同膜域的细胞中基本细胞生物学功能的组织知之甚少。几种不同亚细胞组织和收缩功能的肌肉纤维的存在使问题变得更加复杂。 我们的工作集中在细胞骨架的组织及其对基本亚细胞细胞器分布的影响，特别是高尔基复合体。通过肌肉培养和啮齿动物肌肉纤维的研究，我们已经确定高尔基体在肌肉分化和成熟过程中会分解成数百个相互独立的小分子。这些元素存在于整个肌肉纤维中，靠近和远离细胞核，这使得权力分散？肌肉中的蛋白质合成。然而，高尔基体并不是随机分布的；相反，它们被保留在蛋白质从内质网输出到高尔基复合体的特定位置旁边。我们提出了一个模型，将分化过程中高尔基复合体的重新分布与微管的重新定位和微管成核位置的重新分布联系起来。我们还表明高尔基复合体、内质网出口部位和微管的分布与纤维类型有关，并受收缩活动模式的控制。我们还发现，在微管核物质的重新分布过程中，周中心素和伽马微管蛋白沿着核膜重新分布到相同的位置，但比例不同，这表明它们是独立调节的。 2)本研究的目的 我们的主要目标是通过发现相关的信号通路来了解肌肉中微管、细胞骨架和细胞器的相互作用是如何在分子水平上被调节的。在这一点上，我们认为在分化过程中发生的局部微管稳定是肌肉分化过程中高尔基复合体重组所必需的。 3)过去一年的业绩 激酶GSK3-β是几个涉及微管稳定的信号通路的中心交叉点。我们用来研究其潜在作用的第一个工具是LiCl。LiCl是一种已知的GSK3-β抑制剂，可导致微管的整体稳定。当小鼠肌肉细胞系C2的培养在氯化锂存在下分化时，我们观察到，正如预期的那样，微管的整体稳定。我们还观察到细胞运动完全丧失，成肌细胞几乎完全无法融合。然而，LiCl并没有停止分化，因为我们发现在大约50%的细胞中表达肌生成素和肌球蛋白重链。重要的结果是，在LiCl中分化的细胞中，大多数细胞阻止了高尔基复合体的重组。有趣的是，中心体蛋白(微管的核化)在更大程度上进行了重组。当我们在另一种微管稳定药物紫杉醇存在的情况下分化细胞时，我们得到了类似的结果；100 nM紫杉醇阻止高尔基复合体的重组，但不阻止中心体蛋白的重组。这是首次证明这两个肌肉分化事件是可以解偶联的，并且它们对微管的动态特性有不同的要求。为了确认GSK3-β是氯化锂的靶标，我们使用了一种更特异的GSK3-β的抑制剂SB-415286，得到了定性上相似的结果。LiCl的另一个潜在靶点PI(3)K的抑制剂对高尔基复合体的重分布没有直接影响，也不能阻止LiCl的影响。 我们还试图通过表达与微管稳定有关的蛋白质的cDNA结构来稳定微管。其中之一是微管+末端蛋白EB1。我们已经在肌肉细胞中定位了EB1，并发现其分布与其他类型的细胞相似。然而，EB1的过度表达和EB1的片段EB1-C似乎都不影响分化肌肉培养中微管的稳定，这表明EB1在肌肉中的作用可能不同于它在增殖细胞中的作用。EB1和EB1-C的缺失表明，在肌肉分化过程中发生的微管和中心体的亚细胞重组不同于向伤口迁移的细胞的微管极化和中心体重新定位。 4)结论和意义 我们首次发现GSK3-β可能参与了肌肉分化过程中细胞骨架和亚细胞细胞器的重组。我们还首次能够将这次重组期间发生的一些事件分开。高尔基复合体是一个重要的细胞器；囊泡进出高尔基复合体是蛋白质运输的一个重要方面。然而，由于这种过程主要是在增殖细胞中研究的，我们对它们在肌肉中的组织知之甚少。这些研究将帮助我们更好地了解在许多病理条件和疾病中受到影响的基本过程。

1) Background Skeletal muscle fibers are giant multinucleated cells, built through waves of developmental changes that affect overall cell morphology as well as subcellular organization. Much attention has been given to understanding muscle-specific processes such as the assembly of contractile proteins. In contrast, we understand very little of the organization of basic cell biological functions in cells that have hundreds of nuclei and several distinct membrane domains. The problem is compounded by the existence of several types of muscle fibers that differ in subcellular organization as well as contractile function. Our work focuses on the organization of the cytoskeleton and its effects on the distribution of essential subcellular organelles, particularly the Golgi complex. Working with muscle cultures and with rodent muscle fibers, we have established that the Golgi complex fragments into hundreds of small unlinked elements during muscle differentiation and maturation. These elements are found throughout the muscle fiber, near and away from the nuclei, which allows a ?decentralization? of protein synthesis in muscle. However, the Golgi elements are not distributed randomly; instead they are retained next to specialized sites through which proteins are exported from the endoplasmic reticulum to the Golgi complex. We have proposed a model that links the redistribution of the Golgi complex during differentiation to a reorientation of microtubules and to a redistribution of microtubule-nucleation sites. We have also shown that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is fiber type-dependent and is controlled by the pattern of contractile activity. We have also shown that during redistribution of the microtubule-nucleating material, the proteins pericentrin and gamma-tubulin are redistributed to the same sites along the nuclear membrane, but in different ratios, suggesting independent regulation. 2) Objective of present studies Our main goal is to understand how microtubule cytoskeleton and organelle interactions in muscle are regulated at the molecular level by discovering signaling pathways involved. We believe at this point that local microtubule stabilization, which takes place during differentiation, is required for the reorganization of the Golgi complex during muscle differentiation. 3) Results during the past year The kinase GSK3-beta is the central crossing point of several signaling pathways that involve stabilization of microtubules. The first tool we have used to investigate its potential role is LiCl. LiCl, a known inhibitor of GSK3-beta, causes a global microtubule stabilization. When cultures of the mouse muscle cell line C2 are differentiated in presence of LiCl we observe, as expected, a global stabilization of microtubules. We also observe a complete loss of cell motility, and a near complete failure of myoblasts to fuse. However, LiCl does not stop differentiation as we find expression of myogenin and of myosin heavy chain in about 50% of the cells. The important result is that in cells that differentiate in LiCl, the reorganization of the Golgi complex is prevented in a majority of cells. Interestingly, the centrosomal proteins (which nucleate microtubules) are reorganized to a much greater extent. We obtain a similar result when cells are differentiated in the presence of another microtubule-stabilizing drug, taxol; 100 nM taxol prevents reorganization of the Golgi complex but not of the centrosomal proteins. This is the first evidence that these two events of muscle differentiation can be uncoupled and that they have different requirements as to the dynamic character of microtubules. To confirm that GSK3-beta is the target of LiCl, we used SB-415286, a more specific inhibitor of GSK3-beta, which gave qualitatively similar results. An inhibitor of PI(3)K, another potential target of LiCl, has no direct effect on the Golgi complex redistribution and does not prevent the effects of LiCl. We have also attempted to stabilize microtubules by expressing cDNA constructs of proteins involved in microtubule stabilization. One of these is the microtubule plus-end protein EB1. We have localized EB1 in muscle cells and have found its distribution to be similar to that in other cell types. However, neither overexpression of EB1 nor that of EB1-C, a fragment of EB1 appear to affect microtubule stabilization in differentiating muscle cultures, suggesting that EB1 may play a different role in muscle than in the proiiferating cells in which it was discovered. The absence of effects of EB1 and EB1-C show that the subcellular reorganization of microtubules and centrosome taking place during muscle differentiation differs from the microtubule polarization and centrosome reorientation in cells migrating towards a wound. 4) Conclusions and significance We have, for the first time, shown that GSK3-beta may be involved in the reorganization of cytoskeleton and subcellular organelles during muscle differentiation. We have also, for the first time, been able to uncouple some of the events taking place during this reorganization. The Golgi complex is an essential organelle; vesicular transport from and to the Golgi complex is an essential aspect of protein trafficking. Yet, because such processes have mostly been studied in proliferating cells we understand very little of their organization in muscle. These studies will help us to better understand basic processes which are affected in numerous pathological conditions and diseases.