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

使用细胞培养系统剖析细胞与细胞融合的机制

基本信息

批准号：
9894439
负责人：
Elizabeth H Chen
金额：
$ 13.75万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894439
关键词：
ATP-Binding Cassette Transporters Actins Actomyosin Affect Binding Biochemistry Biological Biological Process Biology Biophysics Bone remodeling Caenorhabditis elegans Cell Adhesion Molecules Cell Culture System Cell Line Cell fusion Cell membrane Cells Cellular Structures Cholesterol Conceptions Cytoskeleton Defect Development Drosophila genus Fertilization Genetic Genetic Screening Goals Human Hydrolysis Image Immune response Label Link Lipids Mechanics Mediating Membrane Membrane Fusion Methods Microscopy Molecular Mus Muscle Fibers Myoblasts Myosin Type II Neoplasm Metastasis Organelles Organism Pathway interactions Physiological Physiology Placenta Placentation Positioning Attribute Process Proteins Research Resolution Role Site Spectrin Stem cells Synapses System Tertiary Protein Structure Testing Therapeutic Time Vesicle Virus biophysical techniques driving force experimental study fly gain of function human disease insight knock-down loss of function mechanotransduction myogenesis novel protein protein interaction reconstitution skeletal tissue regeneration tumor

ATP-Binding Cassette Transporters Actins Actomyosin Affect Binding Biochemistry Biological Biological Process Biology Biophysics Bone remodeling Caenorhabditis elegans Cell Adhesion Molecules Cell Culture System Cell Line Cell fusion Cell membrane Cells Cellular Structures Cholesterol Conceptions Cytoskeleton Defect Development Drosophila genus Fertilization Genetic Genetic Screening Goals Human Hydrolysis Image Immune response Label Link Lipids Mechanics Mediating Membrane Membrane Fusion Methods Microscopy Molecular Mus Muscle Fibers Myoblasts Myosin Type II Neoplasm Metastasis Organelles Organism Pathway interactions Physiological Physiology Placenta Placentation Positioning Attribute Process Proteins Research Resolution Role Site Spectrin Stem cells Synapses System Tertiary Protein Structure Testing Therapeutic Time Vesicle Virus biophysical techniques driving force experimental study fly gain of function human disease insight knock-down loss of function mechanotransduction myogenesis novel protein protein interaction reconstitution skeletal tissue regeneration tumor

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项目摘要

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.

项目摘要细胞间融合是多细胞生物的概念，发展和生理的基础。与蔬菜融合和病毒细胞融合相比细胞 - 细胞融合。对细胞细胞融合的机械理解不仅对基本生物学很重要，而且很重要还可以为其在治疗环境中的操纵提供基础。以理解的长期目标我们通过共表达果蝇的基础细胞融合的机制，已重新组成细胞融合细胞粘附分子Sns和秀丽隐杆线虫在飞行细胞系中，否则不融合。我们的使用该培养系统的研究揭示了肌动蛋白 - 偏膜突起的一般功能促进融合部位的融合蛋白参与，称为“融合突触”。这种文化系统还为新组件的功能障碍和功能丧失屏幕提供了一个很好的平台细胞 - 细胞融合，否则由于母体造成的功能丧失遗传筛选而难以发现它们贡献和功能冗余。此外，该培养系统使生物物理操纵可能的细胞 - 细胞融合，在完整的生物体上在技术上挑战了。我们最近的功能屏幕使用这种培养系统导致鉴定胆固醇-Efflux ATP结合盒（ABC）转运蛋白在细胞融合中，打开了该领域令人兴奋的新研究方向。我们将阐明分子通过测试果蝇ABC转运蛋白是否功能，ABC转运蛋白在S2R+细胞融合中的功能作为常规的ABC转运蛋白；这些ABC转运蛋白是否专门运输胆固醇；和这些ABC转运蛋白是否在融合突触中调节胆固醇分布。我们最近的研究还确定了膜骨骼蛋白质谱在细胞细胞融合中的作用。 Spectrin与在融合式突触处的myoii，理想地将其定位为连接等离子的机械传感器肌动球蛋白网络的膜。此外，限制限制的本地化域需要光谱蛋白细胞粘附分子DUF。我们将探讨Spectrin如何使用合并的包括生物化学，遗传学和超分辨率成像的方法。我们还将调查潜力使用遗传，细胞生物物理和生物物理学的细胞融合过程中光谱蛋白在机械转导中的功能方法。这些拟议的研究有望将新型的机械见解带入脂质的功能，尤其是胆固醇和膜 - 细胞骨架机械转导过程中的胆固醇。