, 2004, Xu et al., 2005, Deák et al., 2006, Kesavan et al., 2007, Bretou et al., 2008, Lu et al., 2008, Stein et al., 2009, Fdez et al., 2010, Guzman et al., 2010, Ngatchou et al., 2010, Risselada et al., 2011 and Shi et al., 2012). However, no direct test of this conclusion in a physiological context has been presented. Here, we demonstrate that the SNARE

TMRs are unlikely to be essential for fusion since lipid-anchored syntaxin-1 and synaptobrevin-2 both were fully competent to support synaptic vesicle fusion in a physiological context. The lipid-anchored SNAREs completely rescued the impairment in spontaneous fusion in syntaxin- and synaptobrevin-deficient neurons, http://www.selleckchem.com/screening/pfizer-licensed-library.html and partially rescued KU-57788 chemical structure evoked release. Although lipid-anchored SNAREs were not as efficient as wild-type SNAREs in restoring the amplitude of evoked release in SNARE-deficient neurons, they reversed the impaired synchronization of evoked release, suggesting that impaired expression levels or incomplete targeting may in part account for the partial activity of lipid-anchored SNAREs in rescuing evoked release (Figure 4). Our results

suggest that a prevalent model whereby the SNARE TMRs are an essential component of the fusion machinery may need to be revised, and that SNAREs primarily—and maybe exclusively—operate as force generators for membrane fusion. According to this revised model, dehydrating the membrane surfaces of opposing membranes by forcing them closely together during 3-mercaptopyruvate sulfurtransferase SNARE-complex assembly may be sufficient to destabilize the phospholipid membrane surfaces and to induce fusion. Our data are consistent with the observation that protein-free liposomes form electrophysiologically “normal” fusion pores without protein components lining the pores (reviewed in Jahn et al., 2003) and argue against a necessary, direct role of SNARE TMRs in fusion-pore formation. It is tempting to speculate that the continued

association of the SM protein Munc18-1 with SNARE complexes during all stages of fusion (Khvotchev et al., 2007, Rathore et al., 2010 and Zhou et al., 2013) may reflect a contribution of Munc18-1 to the dehydration of the fusing membranes, thereby allowing spontaneous lipid mixing when SNARE-complex assembly forces membranes into close proximity, although no direct evidence supports this notion at present. The experiments in which we tested the functionality of either lipid-anchored syntaxin-1 (Figures 2 and 3) or lipid-anchored synaptobrevin-2 (Figures 4, 5, and 6) did not exclude the possibility that the SNARE TMRs still play a contributory role in fusion whereby only one of the two SNAREs (i.e., either syntaxin-1 or synaptobrevin) needs to be TMR anchored for fusion.