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，因为工程成肌细胞可以与成熟肌肉融合，在成人体内形成稳定的杂交器官。注射卫星细胞与肌纤维融合的能力直接影响细胞基因治疗的效果。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。我们利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，许多与肌肉发育有关的细胞和分子事件在苍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解剖肌肉分化和揭示果蝇和脊椎动物成肌细胞融合所需的必要基因成为可能。在肌肉发育突变的基因筛选中，我们已经确定了许多成肌细胞融合所需的新位点。到目前为止，我们已经从分子上确定了两种基因，反社会基因和孤僻基因。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。Antisocial蛋白是一种定位于融合亚细胞位点并将融合信号从细胞膜传递到肌动蛋白骨架的新型衔接蛋白。ARF6的鸟嘌呤核苷酸交换因子Loner也可以被招募到融合位点。本项目的目的是解决关于成肌细胞融合机制的两个主要问题。首先，融合蛋白是如何被跨膜受体招募到融合位点的？其次，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？我们提出了一系列实验来研究蚂蚁和Loner靶向融合位点的机制。在Specific Aim I中，我们将识别蚂蚁中负责将其定位到融合位点的结构域。在Specific Aim II中，我们将使用生化和酵母双杂交方法分离负责将Loner招募到融合位点的中间蛋白。在Specific Aim III中，我们提出鉴定和表征成肌细胞融合机制的一个新组成部分。我们将研究其表型、定位以及与已知融合蛋白以及肌动蛋白细胞骨架成分的相互作用。

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.