Chernomordik and co-workers have envisioned membrane fusion and fission as similar processes that pass through analogous intermediate membrane structures, but in opposite directions as dictated by distinct proteins (Kozlov and Chernomordik, 2002
). A key step in endocytosis involves localized invagination of the plasma membrane, allowing a nascent vesicle to form. This process is mediated by a set of proteins that are able to deform membranes (Farsad and De Camilli, 2003
). Interestingly, invagination of the plasma membrane has also been observed during exocytosis in some secretory cells (Monck and Fernandez, 1994
). This remodeling would result in a curved dimple that points toward secretory vesicles, bringing the two membranes into close proximity at a small point of contact to reduce the energy barrier for fusion. Therefore, ‘dimpling’ of the plasma membrane might constitute an essential step in regulated secretion (Monck and Fernandez, 1994
). However, proteins that mediate this putative invagination step during exocytosis have yet to be identified.
Recent studies have shown that synaptotagmin-I (syt), a Ca2+
sensor that triggers rapid neuronal exocytosis, is able to tubulate membranes in response to Ca2+
(Arac et al., 2006
; Martens et al., 2007
). Hence, syt might operate by buckling the plasma membrane to lower the energy barrier for vesicle fusion.
Attempts have been made to correlate syt's ability to tubulate membranes with its ability to promote fusion of small unilamellar vesicles (SUVs) (Martens et al., 2007
). However, the SUV membrane is already highly curved; as reported here, using more physiologically relevant lipid mixtures, syt-induced membrane tubules have diameters that are comparable to SUVs. These findings indicate that SUV-SUV fusion assays are not dependent on the ability of syt to bend membranes, since SUVs are already fully ‘bent’. In addition, previous work on this problem relied on mutant forms of syt, which, as shown in the present study, also affect the interaction of syt with SNARE proteins. Because syt functions in part by engaging SNAREs (Chapman, 2008
), it could not be determined whether the differences in fusion activity observed for these syt mutants were due to changes in membrane-bending or SNARE-binding activity.
Here, we directly test the hypothesis that syt must bend membranes in order to promote fusion. This was achieved by analyzing SNARE-mediated fusion of giant unilamellar vesicles (GUVs, diameter > 1 μm) in parallel with SUVs (diameter ~ 65 nm). We found that a syt mutant with compromised membrane-bending activity failed to stimulate fusion when the membrane was relatively flat (i.e., when using GUVs), but functioned effectively when the membrane was already highly curved (i.e., when using SUVs). Addition of the N-BAR domain of endophilin, which plays a critical role in endocytosis - presumably by bending the plasma membrane to facilitate vesicle budding (Farsad et al., 2001
) - rescued the function of a membrane-bending deficient syt mutant during regulated GUV-GUV fusion. These findings indicate that exo- and endocytosis proceed via common intermediate membrane structures. Finally, we demonstrate that the cytoplasmic domain of syt - when targeted to either synaptic vesicles or the pre-synaptic plasma membrane - can rescue rapid exocytosis in syt knock-out neurons, validating the use of this protein fragment in reconstituted fusion assays.