A Content Indicator is Critical to Assess Fast Fusion

Abstract

In vitro reconstitution assays are commonly used to study SNARE-mediated membrane fusion. However, many ensemble and single vesicle experiments have been performed with lipid mixing, but not content mixing, indicators. The fundamental assumption of using lipid mixing to study membrane fusion is that there is a direct correlation between lipid and content mixing. Through simultaneous detection of lipid and small content indicators, we found that lipid mixing can occur seconds before content mixing, or without any content mixing during the observation period (50 secs), for calcium-triggered fusion with SNAREs, full-length synaptotagmin 1, and complexin [1]. Our study, as well as the result from a large content mixing assay [2,3], calls into question liposome assays that only rely on a lipid-mixing reporter to assess fast fusion. Our results suggest that liposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators.[1] Diao, J., Grob, P.,⋯, Brunger, A.T. (2012) Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion. eLife, 1, in press.[2] Diao, J., Su, Z.,⋯, Ha, T. (2010) A single vesicle content-mixing assay for SNARE-mediated membrane fusion. Nat. Commun. 1, 54.[3] Diao, J., Ishitsuka, Y.,⋯, Ha, T. (2012) A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins. Nat. Protoc. 7, 921.