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

 描述(申请人提供)：细胞间融合是多细胞生物体的概念、发育和生理学的基础。与囊泡融合和病毒-细胞融合相比，细胞-细胞融合的机制基础知之甚少。了解细胞-细胞融合的机制不仅对基础生物学很重要，而且可能为其在治疗环境中的操作提供基础。为了了解细胞-细胞融合的机制，我们通过在不融合的果蝇细胞系中共表达果蝇细胞黏附分子SNS和线虫FusoGen EFF-1来重建细胞融合。我们使用这种培养系统的研究揭示了肌动蛋白推动的膜突起在促进融合部位融合蛋白参与方面的一般功能，即所谓的融合突触。该培养系统还为细胞-细胞融合的新成分提供了一个很好的功能获得和功能丧失筛查平台，否则由于母体贡献和功能冗余，功能丧失遗传筛查很难发现这些成分。此外，这种培养系统使细胞-细胞融合的生物物理操作成为可能，这在Intat生物体中具有技术挑战性。我们最近利用该培养系统进行的功能筛选导致了细胞-细胞融合中胆固醇外流ATP结合盒(ABC)转运蛋白的鉴定，开辟了该领域令人兴奋的新研究方向。我们将通过测试果蝇ABC转运蛋白是否与传统的ABC转运蛋白一样发挥功能；这些ABC转运蛋白是否特异地转运胆固醇；以及这些ABC转运蛋白是否调节融合突触的胆固醇分布，来阐明ABC转运蛋白在S2R+细胞融合中的分子功能。我们最近的研究也确定了膜骨架蛋白在细胞-细胞融合中的作用。血影蛋白与MyoII共同定位于融合突触，是连接质膜和肌动蛋白网络的理想机械转导分子。此外，血影蛋白还需要限制细胞黏附分子DUF的定位结构域。我们将使用生物化学、遗传学和超分辨率成像的组合方法来探索血影蛋白如何限制DUF的定位。我们还将研究血影蛋白在细胞-细胞融合过程中的机械转导中的潜在功能 遗传学、细胞生物学和生物物理学方法。这些拟议的研究有望为细胞间融合过程中脂质的功能，特别是胆固醇，以及膜-细胞骨架的机械转导带来新的机械性见解。

 DESCRIPTION (provided by applicant): 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 intat 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.