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

项目摘要 细胞间融合是多细胞生物的概念、发育和生理学的基础。 与囊泡融合和病毒-细胞融合相比，对病毒-细胞融合的机制基础知之甚少。 细胞-细胞融合对细胞-细胞融合的机理理解不仅对基础生物学很重要， 也可以为治疗环境中的操作提供基础。长期目标是了解 细胞-细胞融合的机制，我们已经重建细胞融合共表达果蝇 细胞粘附分子Sns和C.线虫融合原Eff-1在蝇细胞系中，否则不会融合。我们 使用该培养系统的研究已经揭示了肌动蛋白推进膜突起的一般功能， 促进融合蛋白在融合位点的接合，称为“融合突触”。这种文化 系统还为新组件的功能获得和功能丧失屏幕提供了一个很好的平台， 细胞-细胞融合，否则很难通过母性功能丧失遗传筛查发现 贡献和功能冗余。此外，这种培养系统使生物物理操纵 细胞-细胞融合是可能的，这在完整的生物体中是技术上具有挑战性的。我们最近的功能屏幕 使用这种培养系统导致了胆固醇流出ATP结合盒（ABC）转运蛋白的鉴定 在细胞间融合中，开辟了该领域令人兴奋的新研究方向。我们将阐明 通过测试果蝇ABC转运蛋白是否起作用来研究ABC转运蛋白在S2 R+细胞融合中的作用 作为常规ABC转运蛋白;这些ABC转运蛋白是否特异性转运胆固醇;以及 这些ABC转运蛋白是否调节胆固醇在融合突触的分布。我们最近的研究 还鉴定了膜骨架蛋白Spectrin在细胞-细胞融合中的作用。Spectrin与 MyoII位于融合突触处，理想地定位为连接血浆 膜到肌动球蛋白网络。此外，需要Spectrin来限制 细胞粘附分子Duf。我们将探索Spectrin如何使用组合的 方法包括生物化学，遗传学和超分辨率成像。我们还将调查 利用遗传学、细胞生物学和生物物理学研究Spectrin在细胞-细胞融合期间的机械转导中的功能 方法.这些拟议中的研究有望为脂质的功能带来新的机制见解， 特别是胆固醇和细胞间融合过程中的膜-细胞骨架机械转导。

PROJECT SUMMARY Intercellular fusion is fundamental to the conception, development and physiology of multicellular organisms. Compared with vesicle fusion and virus-cell fusion, relatively little is known of the mechanistic underpinnings of cell-cell fusion. A mechanistic understanding of cell-cell fusion is not only important for fundamental biology but may also provide basis for its manipulation in therapeutic settings. With the long-term goal of understanding the mechanisms underlying cell-cell fusion, we have reconstituted cell fusion by co-expressing a Drosophila cell adhesion molecule Sns and a C. elegans fusogen Eff-1 in a fly cell line that otherwise does not fuse. Our studies using this culture system have revealed a general function for actin-propelled membrane protrusions in promoting fusogenic protein engagement at the site of fusion, known as the “fusogenic synapse”. This culture system also provides a great platform for gain-of-function and loss-of-function screens for new components of cell-cell fusion, which are otherwise difficult to uncover by loss-of-function genetic screens due to maternal contributions and functional redundancy. Moreover, this culture system makes biophysical manipulations of cell-cell fusion possible, which is technically challenging in an intact organism. Our recent functional screens using this culture system led to the identification of cholesterol-efflux ATP-binding cassette (ABC) transporters in cell-cell fusion, opening up an exciting new research direction in the field. We will elucidate the molecular function of ABC transporters in S2R+ cell fusion by testing whether the Drosophila ABC transporters function as conventional ABC transporters; whether these ABC transporters specifically transport cholesterol; and whether these ABC transporters regulate cholesterol distribution at the fusogenic synapse. Our recent studies have also identified a role for membrane skeletal protein Spectrin in cell-cell fusion. Spectrin co-localizes with MyoII at the fusogenic synapse and is ideally positioned as a mechanotransducer that links the plasma membrane to the actomyosin network. Moreover, Spectrin is required for restricting the localization domain of the cell adhesion molecule Duf. We will explore how Spectrin restricts Duf localization using a combined approach including biochemistry, genetics and super-resolution imaging. We will also investigate the potential function of Spectrin in mechanotransduction during cell-cell fusion using genetic, cell biological and biophysical methods. These proposed studies promise to bring novel mechanistic insights into the function of lipids, especially cholesterol, and membrane-cytoskeleton mechanotransduction during intercellular fusion.