Molecular Mechanisms of Myoblast Fusion

描述(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的成肌细胞与成熟的肌肉融合，在成年人体内形成稳定的杂交器官。注入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效果。因此，了解成肌细胞融合的控制机制有望为未来的治疗提供新的可能性。我们正在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，参与肌肉发育的许多细胞和分子事件在进化上在苍蝇和脊椎动物之间是保守的。这种保守性使得利用果蝇遗传学剖析肌肉分化成为可能，并揭示了果蝇和脊椎动物的成肌细胞融合所需的必要基因。在肌肉发育突变的遗传筛选中，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，我们已经从分子上表征了两个基因：反社会基因和孤独者基因。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。反社会是一种新的接头蛋白，它定位于融合的亚细胞位置，将融合信号从细胞膜传递到肌动蛋白细胞骨架。Loner是ARF6的一种鸟嘌呤核苷酸交换因子，也可以被招募到融合部位。本项目的目标是解决有关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部位的？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？我们提出了一系列实验来研究蚂蚁和Loner被靶向融合位点的机制。在特定目标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(S)。在特定的目标II中，我们将使用生化和酵母双杂交的方法来分离负责招募孤独者到融合位点的中间蛋白(S)。在特定的目标III中，我们提出了一种新的成肌细胞融合机制的成分的鉴定和表征。我们将研究它的表型、定位以及与已知融合蛋白和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.