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

描述（由申请人提供）：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，在果蝇果蝇中的研究揭示了肌动蛋白细胞骨架在成肌细胞融合中的基本功能，在该过程中单核成肌细胞融合形成多核肌纤维。具体来说，我们已经揭示了一种细胞类型特异性的、富含F-肌动蛋白的podosome样结构，该结构以多个扩张指侵入相对的融合伴侣，最终导致融合孔的形成。此外，对蛔虫C.秀丽线虫已经鉴定出一对推定的融合蛋白，它们对于诱导胚胎融合是必需的并且是足够的。此外，已经显示在异源昆虫细胞系Sf 9细胞中表达蠕虫融合子可以诱导低频率的细胞-细胞融合。我们现在已经建立了一个高效率的，诱导型细胞培养系统，共表达的蠕虫fusogen和果蝇细胞粘附分子在果蝇细胞系，通常不进行融合。与单独表达融合子的细胞相比，这种共表达导致细胞融合效率增加>10倍。我们发现，在培养的细胞，以促进细胞-细胞融合和Arp 2/3成核促进因子所需的相似的豆荚状结构的融合培养的细胞在果蝇胚胎中的肌细胞。因此，我们已经建立了一个细胞培养系统，密切模仿成肌细胞融合在体内。该项目的目标是进一步表征这种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。 公共卫生相关性：细胞-细胞融合是多细胞生物的概念，发育和生理学的基础。它参与受精、肌生成、骨重塑、胎盘发育、免疫应答和肿瘤转移等多种过程。因此，了解细胞-细胞融合的机制不仅对基础生物学很重要，而且还可以为人类疾病的治疗环境中的操纵提供基础。

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. PUBLIC HEALTH RELEVANCE: Cell-cell fusion is fundamental to the conception, development and physiology of multicellular organisms. It is involved in processes as diverse as fertilization, myogenesis, bone remodeling, placental development, immune response and tumor metastasis. Thus understanding the mechanisms of cell-cell fusion is not only important for fundamental biology, but may also provide basis for its manipulation in therapeutic settings for human diseases.