Investigating mechanisms of vertebrate myoblast fusion using zebrafish as a model

项目摘要 成肌细胞融合，单核成肌细胞融合形成多核收缩性肌肉的过程 纤维是骨骼肌发育、维持和再生所必需的。深入了解分子 迄今为止，成肌细胞融合的细胞机制主要来自于对遗传系统的研究， 果蝇对果蝇的研究发现了一些进化上保守的调节因子， 成肌细胞融合，范围从细胞粘附分子到肌动蛋白聚合调节剂到机械传感器。 更重要的是，果蝇的研究已经确定了一种新的细胞机制，这种机制是成肌细胞融合的基础。 融合位点--一个攻击细胞利用肌动蛋白推进的膜侵略性地侵入它的融合伴侣 突起，而接收细胞增加机械张力以抵抗入侵，导致细胞 膜并置、融合剂接合和质膜融合。除了进化上保守的 融合促进蛋白，最近在斑马鱼和小鼠中的研究已经确定了一对脊椎动物特异性 融合蛋白，Myomaker和Myomixer（也称为Myomerger和Minion）。然而，如何以及在哪里 这些促进成肌细胞融合的蛋白质在很大程度上是未知的。与果蝇研究相比， 阻碍脊椎动物成肌细胞融合机制的研究是缺乏对成肌细胞融合的认识， 精确的融合点而成肌细胞融合似乎发生在沿着一个宽的细胞-细胞 在培养的哺乳动物成肌细胞的接触区中，完整动物中的成肌细胞融合位点完全保留 未知因此，必须在体内鉴定融合位点并提供细胞框架， 未来的研究可以建立。斑马鱼是研究成肌细胞体内融合的一种很好的脊椎动物模型， 大量的小而透明的斑马鱼胚胎及其快速的子宫外发育。在这一提议中， 我们将使用斑马鱼作为体内模型来确定完整脊椎动物中成肌细胞融合的位点， 分子标记此外，我们将研究融合子之间的定位和潜在的相互作用， Myomaker和Myomixer。此外，我们将探讨融合子和细胞之间的相互作用 粘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 用于开发优化卫星细胞介导的肌肉再生的治疗策略 肌肉退行性疾病的患者。

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.