In the present study, we have explored the late stages of cell–cell fusion in which the nascent fusion pore(s) generated by viral fusogens baculovirus gp64 and influenza HA expand(s) to form syncytia. Fusion pores that develop with a tight contact between cell membranes have a strongly bent rim accumulating the elastic energy of membrane bending. Thus the pore expansion is an energy-consuming process. Earlier studies indicated that the enlargement of micron-scale pores in syncytium formation does not proceed spontaneously but rather involves the cell machinery [12
]. Neither release of membrane lateral tension nor disruption of the force generating intracellular systems such as the actin cytoskeleton and microtubular structures inhibits fusion pore expansion in syncytium formation [12
]. We hypothesized that large fusion pores grow because the rim of the pore accumulates proteins that relax its bending energy. The shape of a fusion pore developing in a cell–cell contact zone with an inter-membrane distance of ~20 nm is similar to a half cylinder with a ~20 nm diameter [13
]. Since the comparable curvature radii are characteristic for highly curved membrane compartments such as endocytic vesicles, we proposed that the late stages of fusion pore growth involve CGPs that drive intracellular membrane shaping. To test this hypothesis, in the present study we explored whether syncytium formation can be promoted by raising the intracellular concentrations of membrane-binding and -bending domains from three CGPs (GRAF1, FCHo2 and epsin) in cells by either overexpressing these proteins or by microinjecting them. Indeed, these diverse CGPs (with BAR, F-BAR and ENTH domains) promoted the late stages of HA-initiated cell-to-cell fusion. Note that, although this finding is consistent with our hypothesis as a proof of principle, the specific different CGPs that may have the most potent effect in fusion between HAb2 cells or other cells remain to be identified. A recent study indicates that GRAF1 expression in C2C12 cells promotes both myogenic differentiation and fusion between differentiated myoblasts [55
]. GRAF1-dependent promotion of cell fusion is suggested to involve BAR domain-mediated membrane sculpting. Our findings in a much simpler experimental system are consistent with this mechanism and suggest that it acts downstream of early fusion stages at a stage of fusion pore expansion.
The conclusion that late stages of syncytium formation involve CGPs was strengthened further by experiments with reagents targeting the function of one of the most abundant CGPs: dynamin [22
]. Both free dynamin tetramers and membrane associated dimers, the building blocks of larger oligomers, are rigid elongated structures with a curved shape. Curvature generated by dynamin can result in different membrane shapes. Long helical oligomers constrain the membrane into tubular shapes. Oligomers that are too short to form helices can generate a wider variety of shapes which include tubular and spherical shapes. Blocking of the GTPase activity of dynamin by dynasore [35
], MiTMAB [36
] and Dynole-34-2 [37
] inhibited syncytium formation initiated by either HA or gp64. Dynamin inhibitors did not inhibit lipid mixing and blocked syncytium formation even when applied after a low pH pulse application, that is, at the time when both HA [56
] and, especially, gp64 [14
] have already formed initial fusion pores. Thus dynamin plays its role downstream of the opening of fusion pores. Our findings suggest that the recently reported inhibition of HIV env-mediated cell–cell fusion by dynasore [57
] also reflects dynamin-dependence of late, rather than early, fusion stages.
Dominant-negative dynamin mutants also affected cell-to-cell fusion. Similar to the experiments using inhibitors, syncytium formation was inhibited by stabilization of the GTP-bound form of dynamin by overexpressing a GTPase-defective S61D mutant [40
]. Syncytium formation was also inhibited by the assembly-defective I690K mutant of dynamin, which is impaired in membrane surface affinity [41
]. In contrast, K44A and S45N, two mutants of dynamin defective in GTP binding [42
], promoted syncytium formation. This promotion may indicate that GTP-free dynamins are more likely than GTP–dynamins to form short arc-like oligomers that facilitate fusion pore expansion. However, this hypothesis remains to be tested. Importantly, since impaired GTP binding for these mutants inhibits GTP hydrolysis, the promotion of syncytium formation argues against the hypothesis that fusion pore expansion is somehow driven by the energy derived from the GTP hydrolysis by dynamin.
The finding that diverse dynamin-targeting reagents have notable effects on the efficiency of syncytium formation suggests the involvement of dynamin in cell–cell fusion. However, although the ability of dynamin to shape membranes is well documented, we cannot at the present time offer a specific mechanism by which curvature generation by dynamin promotes fusion pore expansion. In one scenario, dynamin oligomers, too short to form complete circles or helices and thus shaped as open arcs bind to the curved membrane of a nascent fusion pore rim. Owing to their ability to generate membrane curvature similar to that of the pore edge, binding of these arc-like oligomers drives expansion of the pore edge by bending additional portions of the initially flat membrane of the intracellular contact into the bent shape of the pore rim. Stabilization of the GTP-bound form of dynamin may promote the formation of the long helical structures and therefore the membrane tubules. As mentioned above, short oligomers can be accommodated both in tubules and in the fusion pore rim, and thus competition between the tubules and pore rim for the short-arc dynamins is expected to deplete the pool of short oligomers in the rim and inhibit pore expansion. Further research will either further develop this mechanism to explain the specific effects of different dynamin mutants or bring about alternative mechanisms that may couple membrane shaping by dynamin with fusion pore expansion at late stages of cell-to-cell fusion. Importantly, since dynamin both directly and via interactions with other proteins regulates many different processes in cell physiology, the role of dynamin in syncytium formation might be complex and indirect.
The activity of many intracellular CGPs is regulated by polyphosphoinositides. The present study is the first demonstration that PtdIns(4,5)P2 content controls syncytium formation. Inhibition of the late stages of cell fusion by lowering the concentration of accessible PtdIns(4,5)P2 in the plasma membrane is consistent with the hypothesis that CGPs drive the expansion of fusion pores. However, PtdIns(4,5)P2 can also affect late fusion stages by regulating PtdIns(4,5)P2-binding proteins other than CGPs.
To conclude, the late stages of cell-to-cell fusion initiated by well-characterized viral fusogens depend on the functional activity of intracellular CGPs, including dynamin and representatives of the BAR, F-BAR and ENTH protein families. However, a number of important questions remain open. To start with, the mechanism underlying this dependence is yet to be clarified. CGPs can directly facilitate fusion pore expansion by accumulating at the pore edge and lowering its energy. In principle, CGPs can also promote vesiculation of the membrane junction at the edge of the fusion pores [33
]. However, at least in the case of gp64-initiated fusion of Sf9 cells, fusion pores appear to grow by the displacement of membrane material towards the periphery of the contact zone rather than by vesiculation [13
]. The analysis of the role of CGPs in syncytium formation also has to be extended to biologically important examples of cell fusion such as fusion between myoblasts and between macrophages, and then to the identification of specific CGPs involved in these processes. Better understanding of the mechanism and cell machinery responsible for driving fusion pore expansion in cell-to-cell fusion will bring about new ways of controlling fusion in development and in pathological conditions. The dependence of fusion pore expansion on cell machinery can also be of importance for understanding why transient nanotubular connections between plasma membranes of some cells [58
] do not expand to yield multinucleated cells.
The present study emphasizes an interesting overlap between proteins controlling the late stages of cell-to-cell fusion and proteins that drive the oppositely directed process of membrane remodelling, the fission of one cell membrane into two. Dynamin and curvature-generating domains of GRAF1, FCHo2 and epsin that we found to influence syncytium formation are essential components of different endocytic pathways [23
] that culminate in membrane fission. Proteins of the dynamin family have been also implicated in mitochondrial fusion [60
] and fusion of ER membranes [62
]. Dynamin is involved in an as yet unclear mechanism in HIV fusion with the endosomal membrane, as suggested by a fusion inhibition using the dynamin GTPase inhibitor dynasore [64
]. Furthermore, dynasore promotes the release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles, suggesting that dynamin redirects fusion pore evolution from expansion to premature closure in ‘kiss-and-run’ exocytosis [65
]. A recent study confirms that dynamin regulates the rapidity of fusion pore expansion in exocytosis and suggests that dynamin assembly restricts fusion pore expansion until GTP-hydrolysis-stimulated disassembly [66
]. Another component of the endocytic machinery, the BAR domain protein amphiphysin, has also been reported to restrict dilation of fusion pores [67
]. Note that, in terms of membrane curvature and positioning of proteins, the closing of the exocytotic fusion pore by CGPs (dynamin [65
] and amphiphysin [67
]) located outside of the pore lumen is topologically similar to fusion pore expansion in syncytium formation by the proteins located inside the lumen of the pore. Further elucidation of the overlap between the protein players involved in the processes that unite and divide biological membranes is important for finding shared mechanistic principles underlying fusion and fission [68
]. Although our results from the present study demonstrate that different CGPs can control the expansion of fusion pores, we still do not know which of the diverse intracellular CGPs control these stages in biologically important cell-to-cell fusion processes. Our finding that increases in concentration of either of the several CGP domains promote transition from early fusion intermediates to syncytium formation suggests a redundancy of their membrane-bending function.