Background

Fusion of membrane lipid bilayers in cell biological processes as diverse as fusion of intracellular membranes in exocytosis, and fusion of viral and cell membranes in enveloped virus infection, and myoblast fusion in skeletal muscle development apparently involves similar lipid rearrangements (Figure 1) (Brukman et al., 2019). First, a merger of the apposing, contacting monolayers of two fusing bilayers generates a hemifusion connection and allows redistribution of lipid probes between these monolayers. A subsequent merger of the distal monolayers generates a fusion pore and allows redistribution of aqueous probes between fusing membrane compartments.


Figure 1. Schematic representation of the lipid rearrangements during hemifusion and fusion pore formation (modified from Figure 1 in Brukman et al., 2019).

Hemifusion can either transition into a fusion pore or represent a dead end of the fusion reaction aborted before fusion pore formation. In the latter case, hemifusion connections can then dissociate yielding two distinct bilayers. In their turn, nascent fusion pores can either close or expand advancing fusion towards its completion with full unification of the membrane compartments. The rates of formation and dissociation of the key fusion intermediates, hemifusion and fusion pores, and the rates of the transition between these intermediates vary between different fusion processes and are determined by the activity of the proteins involved and lipid compositions of the fusing membranes.

In most of the experimental studies, pores large enough to pass content probes in the range from ~1 kDa to ~100 kDa are detected by fluorescence microscopy. Hemifusion, operationally defined as lipid mixing without content mixing, is detected using fluorescence microscopy or spectrofluorometry. Different modifications of lipid mixing assays have been developed in studies on fusion of protein-free lipid bilayers, and fusion mediated by viral fusogens and intracellular fusogens (Brukman et al., 2019). The successful application of the lipid mixing assay in all these systems has been facilitated by a relative rapidness of these fusion processes with characteristic times of lipid mixing varying from seconds to minutes.

Application of lipid mixing assays to developmental cell-cell fusion, the subject of this work, is critically important for clarification of the pathways of the membrane rearrangements but more challenging than for faster viral and intracellular fusion reactions. For instance, formation of multinucleated myotubes, one of the best characterized examples of cell-cell fusion processes (Sampath et al., 2018), is preceded by myogenic differentiation that prepares the cells for fusion and takes days. As a result, by the time fusion is evaluated, fluorescent lipid probes added before fusion to label plasma membranes are already partially internalized. Moreover, labeling of intracellular membrane compartments containing internalized probes may appear brighter than labeling of the plasma membrane because of a higher membrane content and contrast. An additional concern in developing lipid mixing assays for very slow fusion processes is related to the ability of lipid probes to be transferred from membrane to membrane by lipid-exchanging proteins, lipid micelles or extracellular vesicles (reviewed in Merklinger et al., 2016), i.e., by mechanisms that neither involve hemifusion or fusion nor depend on the fusion machinery.

Here we present a protocol used to assay lipid mixing in fusion between C2C12 cells, immortalized mouse myoblasts that proliferate in high-serum medium and differentiate and fuse in low-serum medium. We also discuss applications of the modified versions of this protocol to other slow cell-cell fusion processes.