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发育和生理的基础，但对其机制基础知之甚少。最近对几个模型系统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，在成肌细胞融合形成多核肌纤维的过程中。具体来说，我们发现了一种细胞类型特异性的、富含f -肌动蛋白的足多体样结构，该结构通过多个突出的手指侵入相反的融合伴侣，最终导致融合孔的形成。此外，在蛔虫秀丽隐杆线虫的研究中发现了一对假定的促融合蛋白，它们在胚胎中诱导融合是必要的和充分的。此外，研究表明，在异源昆虫细胞系中表达蠕虫融合原的Sf9细胞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的，可诱导的细胞培养系统，通过在果蝇细胞系中共同表达蠕虫的融合原和苍蝇的细胞粘附分子，而果蝇细胞系通常不会进行融合。与单独表达融合原的细胞相比，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，类似的podosome样结构被用于促进细胞间融合，并且Arp2/3成核促进因子对于培养细胞的融合是必需的，就像果蝇胚胎中的肌肉细胞一样。因此，我们建立了一种模拟成肌细胞融合的细胞培养系统。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，并将其作为发现参与细胞-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.