METHODOLOGIES IN THE STUDY OF CELL-CELL FUSION

The process of membrane fusion has been profitably studied by fusing c ells that express fusion proteins on their surfaces to the membranes o f target cells. Primary methods for monitoring the occurrence of fusio n between cells are measurement of formation of heterokaryons, measure ment of activation of reporter genes, measurement of transfer of lipid ic and aqueous fluorescent dyes, and electrophysiological recording of fusion pores. Fluorescence and electrical methods have been well deve loped 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 observati on of spread of fluorescent dyes from one cell to another is a sensiti ve and convenient means of detecting fusion on the level of single eve nts. In such studies, both the membrane and the aqueous continuities t hat 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 labe ling cells with fluorescent probes, a state of hemifusion can be ident ified if probes in outer membrane leaflets transfer but probes in inne r leaflets or aqueous spaces do not. Electrical measurements-both capa citance and double-whole-cell voltage-clamp techniques-are the most se nsitive methods yet developed for detecting the formation of pores and for quantifying their growth. These powerful single-event methodologi es should be directly applicable to further advances in expressing non viral fusion proteins on cell surfaces. (C) 1998 Academic Press.