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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：
9252472
负责人：
Elizabeth H Chen
金额：
$ 33.21万
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/9252472
关键词：
ATP-Binding Cassette Transporters Actins Actomyosin Affect Alpha Cell 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 public health relevance reconstitution skeletal tissue regeneration tumor

项目摘要

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.