Methodologies in the study of cell-cell fusion.
The process of membrane fusion has been profitably studied by fusing cells that express fusion proteins on their surfaces to the membranes of target cells. Primary methods for monitoring the occurrence of fusion between cells are measurement of formation of heterokaryons, measurement of activation of reporter genes, measurement of transfer of lipidic and aqueous fluorescent dyes, and electrophysiological recording of fusion pores. Fluorescence and electrical methods have been well developed for fusion of a nucleated cell expressing viral fusion proteins to red blood cell targets. These techniques are now being extended to the study of fusion between two nucleated cells. Microscopic observation of spread of fluorescent dyes from one cell to another is a sensitive and convenient means of detecting fusion on the level of single events. In such studies, both the membrane and the aqueous continuities that occur as a result of fusion can be measured in the same experiment. By following spread of aqueous dyes of different sizes from one cell to another, the growth of a fusion pore can also be followed. By labeling cells with fluorescent probes, a state of hemifusion can be identified if probes in outer membrane leaflets transfer but probes in inner leaflets or aqueous spaces do not. Electrical measurements-both capacitance and double-whole-cell voltage-clamp techniques-are the most sensitive methods yet developed for detecting the formation of pores and for quantifying their growth. These powerful single-event methodologies should be directly applicable to further advances in expressing nonviral fusion proteins on cell surfaces.
Cohen
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Department of Molecular Biophysics and Physiology, Rush Medical College, 1653 West Congress Parkway, Chicago, Illinois, 60612, USA.
Melikyan
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D002459:Cell Fusion / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology*; D003584:Cytological Techniques; D004912:Erythrocytes / Q000502:physiology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006822:Hybrid Cells; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D015335:Molecular Probes; D018408:Patch-Clamp Techniques; D014760:Viral Fusion Proteins / Q000378:metabolism
D015335:Molecular Probes; D014760:Viral Fusion Proteins