Dissecting the Mechanisms of Cell-Cell Fusion Using A Cell Culture System

描述(申请人提供)：细胞-细胞融合是多细胞生物体发育和生理学的基础，但人们对其机制基础知之甚少。最近在几个模型系统中的研究已经开始揭示细胞-细胞融合的基本原理。特别是，对果蝇的研究揭示了肌动蛋白细胞骨架在成肌细胞融合中的重要功能，即单核成肌细胞融合形成多核肌肉纤维的过程。具体地说，我们揭示了一种细胞类型特异的、富含F-肌动蛋白的足体样结构，它用多个伸出的手指入侵相对的融合伙伴，最终导致融合孔的形成。此外，对圆线虫的研究已经确定了一对假定的融合蛋白，它们是诱导胚胎融合的必要条件和充分条件。此外，在异种昆虫细胞系Sf9细胞中表达蠕虫融合原可以诱导低频率的细胞-细胞融合。我们现在已经建立了一种高效、可诱导的细胞培养系统，通过在正常情况下不发生融合的果蝇细胞系中共表达蠕虫FusoGen和苍蝇细胞黏附分子。与单独表达FusoGen的细胞相比，这种共表达使细胞融合效率提高了10倍。我们发现在培养的细胞中使用类似的足体结构来促进细胞-细胞融合，并且与果蝇胚胎中的肌肉细胞一样，融合培养的细胞需要Arp2/3成核促进因子。因此，我们已经建立了一种非常接近体内成肌细胞融合的细胞培养系统。该项目的目标是进一步确定该细胞培养系统的分子和细胞机制，并将其用作发现参与细胞-细胞融合的新基因的工具。

DESCRIPTION (provided by applicant): Cell-cell fusion is fundamental to the development and physiology of multicellular organisms, but little is known of its mechanistic underpinnings. Recent studies in several model systems have begun to reveal fundamental principles underlying cell-cell fusion. In particular, studies in the fruit fly Drosophila have revealed an essential function of the actin cytoskeleton in myoblast fusion, the process in which mononucleate myoblasts fuse to form multinucleate muscle fibers. Specifically, we have revealed a cell type-specific, F-actin-enriched podosome-like structure that invades the opposing fusion partner with multiple protrusive fingers, which ultimately leads to fusion pore formation. In addition, studies in the round worm C. elegans have identified a pair of putative fusogenic proteins that are both necessary and sufficient to induce fusion in the embyro. Moreover, it has been shown that expressing the worm fusogens in a heterologous insect cell line, Sf9 cells can induce a low frequency of cell-cell fusion. We have now established a high-efficiency, inducible cell culture system by co-expressing the worm fusogen and a fly cell adhesion molecule in a Drosophila cell line that does not normally undergo fusion. Such co-expression results in a >10 fold increase in cell fusion efficiency compared with cells expressing the fusogen alone. We show that similar podosome-like structures are used in cultured cells to promote cell-cell fusion and that the Arp2/3 nucleation promoting factors are required for fusing cultured cells as for muscle cells in Drosophila embryos. Thus we have established a cell culture system that closely mimics myoblast fusion in vivo. The goal of this project is to further characterize the molecular and cellular mechanisms of this cell culture system and to use it as a tool to discover new genes involved in cell-cell fusion.