Molecular Mechanisms of Myoblast Fusion

描述（由申请人提供）：骨骼肌是基因治疗的主要靶器官，因为可以引入工程化成肌细胞与成熟肌肉融合，在成人体内形成稳定的杂交器官。注射的卫星细胞与肌纤维融合的能力直接影响基于细胞的基因治疗的功效。因此，了解控制成肌细胞融合的机制有望在未来提供新的治疗可能性。我们使用果蝇作为遗传模型来研究成肌细胞融合的分子机制。近年来的研究表明，许多涉及肌肉发育的细胞和分子事件在果蝇和脊椎动物之间是进化保守的。这种保守性使得利用果蝇遗传学来剖析肌肉分化成为可能，并揭示了果蝇和脊椎动物中成肌细胞融合所需的必需基因。在肌肉发育突变体的遗传筛选中，我们已经确定了成肌细胞融合所需的一些新的基因座。到目前为止，我们已经从分子水平上确定了两个基因的特征，反社会基因和孤独基因。对这些和其他融合基因的研究已经开始揭示控制成肌细胞融合的信号级联。抗社会蛋白是一种定位于融合亚细胞位点的新型衔接蛋白，它将融合信号从细胞膜传递到肌动蛋白细胞骨架。Loner是一种ARF6的鸟嘌呤核苷酸交换因子，也可以被募集到融合位点。本项目的目标是解决两个主要的问题，有关成肌细胞融合的机制。首先，融合蛋白如何被跨膜受体募集到融合位点？第二，肌动蛋白细胞骨架重排是如何在成肌细胞融合过程中实现的？我们提出了一系列的实验来研究蚂蚁和孤独者的机制有针对性的网站融合。在具体目标I中，我们将确定蚂蚁中负责融合位点定位的结构域。在特定目标II中，我们将使用生物化学和酵母双杂交方法来分离负责将Loner募集到融合位点的中间蛋白。在具体目标III中，我们提出了成肌细胞融合机制的新组分的鉴定和表征。我们将研究它的表型，定位，并与已知的融合蛋白，以及与肌动蛋白细胞骨架的组件的相互作用。

DESCRIPTION (provided by applicant): Skeletal muscle is a prime target organ for gene therapy, as engineered myoblasts can be introduced to fuse with mature muscle, forming a stable hybrid organ within the adults. The ability of injected satellite cells to fuse with myofibers directly affects the efficacy of cell-based gene therapy. Therefore, understanding the mechanisms controlling myoblast fusion promises to offer novel therapeutic possibilities in the future. We are using the fruit fly Drosophila as a genetic model to study the molecular mechanisms of myoblast fusion. Studies in recent years have revealed that many of the cellular and molecular events involved in muscle development are evolutionarily conserved between fly and vertebrates. This conservation has made it possible to dissect muscle differentiation using Drosophila genetics and to uncover essential genes required for myoblast fusion in flies and vertebrates. In a genetic screen for mutants in muscle development, we have identified a number of new loci required for myoblast fusion. To date, we have molecularly characterized two genes, antisocial and loner. Studies of these and other fusion genes have begun to reveal a signaling cascade controlling myoblast fusion. Antisocial is a novel adaptor protein that is localized to subcellular sites of fusion and relays the fusion signal from cell membrane to actin cytoskeleton. Loner, a guanine nucleotide exchange factor for ARF6, can also be recruited to sites of fusion. The goal of this project is to address two major questions concerning the mechanisms of myoblast fusion. First, how are fusion proteins recruited to sites of fusion by transmembrane receptors? Second, how is actin cytoskeletal rearrangement achieved during myoblast fusion? We propose a series of experiments to investigate the mechanisms by which Ants and Loner are targeted to sites of fusion. In Specific Aim I, we will identify domain(s) in Ants that are responsible for its localization to sites of fusion. In Specific Aim II, we will use biochemical and yeast two- hybrid approaches to isolate the intermediary protein(s) responsible for recruiting Loner to sites of fusion. In Specific Aim III, we propose the identification and characterization of a new component of the myoblast fusion machinery. We will examine its phenotype, localization, and interactions with known fusion proteins as well as with components of the actin cytoskeleton.