Investigating mechanisms of vertebrate myoblast fusion using zebrafish as a model

项目总结 成肌细胞融合，即单核成肌细胞融合形成多核收缩肌肉的过程 纤维对于骨骼肌的发育、维持和再生是必不可少的。对分子的洞察 到目前为止，成肌细胞融合的细胞机制主要来自于对遗传系统水果的研究 会飞的果蝇。对果蝇的研究揭示了几个在进化上保守的调节因子 成肌细胞融合，从细胞黏附分子到肌动蛋白聚合调节器再到机械传感器。 更重要的是，果蝇的研究已经确定了一种新的细胞机制，这种机制支持成肌细胞在 融合部位-攻击细胞使用肌动蛋白推进的膜侵袭其融合伙伴 突起，而接收细胞增加机械张力以抵抗入侵，导致细胞 膜并列、FusoGen接合和质膜融合。除了进化上的保守性 融合促进蛋白，最近在斑马鱼和老鼠身上的研究已经确定了一对脊椎动物特有的 融合蛋白，Myomaker和Myomixer(也称为Mymer和Minion)。然而，如何以及在哪里 这些蛋白质促进成肌细胞融合在很大程度上是未知的。与果蝇研究相比，一个主要问题是 阻碍脊椎动物成肌细胞融合潜在机制的研究是缺乏对 精确的融合地点。而成肌细胞的融合似乎发生在一个广泛的细胞-细胞的未确定的位置(S) 在培养的哺乳动物成肌细胞中，完整动物的成肌细胞融合部位保持完整 未知。因此，当务之急是确定体内的融合部位，并提供在其上的细胞框架 未来的研究是可以建立的。斑马鱼是研究体内成肌细胞融合的优秀脊椎动物模型，因为 大量小而透明的斑马鱼胚胎及其快速的体外发育。在这份提案中， 我们将使用斑马鱼作为活体模型，以确定在一个完整的脊椎动物中成肌细胞融合的位置 分子标记。此外，我们还将研究融合菌之间的定位和潜在的相互作用， Myomaker和Myomixer。此外，我们还将探索融合子与细胞之间的相互作用 黏附分子和肌动蛋白细胞骨架。拟议研究的见解将对以下方面产生广泛影响 了解肌肉发育和再生的基本原理，最终可能是 用于开发治疗策略，以优化卫星细胞介导的肌肉再生 在肌肉退行性疾病患者中。

PROJECT SUMMARY Myoblast fusion, the process in which mononucleate myoblasts fuse to form multinucleate, contractile muscle fibers, is essential for skeletal muscle development, maintenance and regeneration. Insights into the molecular and cellular mechanisms of myoblast fusion to date have mainly come from studies of a genetic system, the fruit fly Drosophila. Studies in Drosophila have uncovered a handful of evolutionarily conserved regulators of myoblast fusion, ranging from cell adhesion molecules to actin polymerization regulators to mechanical sensors. More importantly, Drosophila studies have identified a novel cellular mechanism underlying myoblast fusion at the site of fusion – an attacking cell aggressively invades its fusion partner using actin-propelled membrane protrusions, whereas the receiving cell increases mechanical tension to resist the invasion, leading to cell membrane juxtaposition, fusogen engagement and plasma membrane fusion. Besides evolutionarily conserved fusion-promoting proteins, recent studies in zebrafish and mouse have identified a pair of vertebrate-specific fusogenic proteins, Myomaker and Myomixer (also known as Myomerger and Minion). However, how and where these proteins facilitate myoblast fusion is largely unknown. Compared to Drosophila studies, a major issue that hinders the study of the mechanisms underlying vertebrate myoblast fusion is the lack of knowledge of the precise sites of fusion. While myoblast fusion appears to occur at undefined location(s) along a broad cell-cell contact zone in cultured mammalian myoblasts, the sites of myoblast fusion in an intact animal remain completely unknown. Thus, it is imperative to identify the sites of fusion in vivo and provide a cellular framework upon which future studies can be built. Zebrafish is an excellent vertebrate model to study myoblast fusion in vivo, due to the large number of small and transparent zebrafish embryos and their rapid ex-utero development. In this proposal, we will use zebrafish as an in vivo model to define the sites of myoblast fusion in an intact vertebrate animal with molecular markers. In addition, we will study the localization and potential interaction between the fusogens, Myomaker and Myomixer. Furthermore, we will explore the interaction between the fusogens and the cell adhesion molecules and the actin cytoskeleton. Insights from the proposed studies will have a broad impact on understanding the fundamental principles of muscle development and regeneration, and ultimately may be exploited for the development of therapeutic strategies to optimize satellite cell-mediated muscle regeneration in patients with muscle degenerative diseases.