Abstract
Lipid mixing (redistribution of lipid probes between fusing membranes) has been widely used to study early stages of relatively fast viral and intracellular fusion processes that take seconds to minutes. Lipid mixing assays are especially important for identification of hemifusion intermediates operationally defined as lipid mixing without content mixing. Due to unsynchronized character and the slow rate of the differentiation processes that prime the cells for cell-cell fusion processes in myogenesis, osteoclastogenesis and placentogenesis, these fusions take days. Application of lipid mixing assays to detect early fusion intermediates in these very slow fusion processes must consider the continuous turnover of plasma membrane components and potential fusion-unrelated exchange of the lipid probes between the membranes. Here we describe the application of lipid mixing assay in our work on myoblast fusion stage in development and regeneration of skeletal muscle cells. Our approach utilizes conventional in vitro model of myogenic differentiation and fusion based on murine C2C12 cells. When we observe the appearance of first multinucleated cells, we lift the cells and label them with either fluorescent lipid DiI as a membrane probe or CellTrackerTM Green as a content probe. Redistribution of the probes between the cells is scored by fluorescence microscopy. Hemifused cells are identified as mononucleated cells labeled with both content- and membrane probes. The interpretation must be supported by a system of negative controls with fusion-incompetent cells to account for and minimize contributions of fusion-unrelated exchange of the lipid probes. This approach with minor modifications has been used for investigating fusion of primary murine myoblasts, osteoclast precursors and fusion mediated by a gamete fusogen HAP2, and likely can be adopted for other slow cell-cell fusion processes.