Membrane fusion is essential to maintain eukaryotic life. Fusion is tightly regulated and relies on a complex network of protein tethers and lipid interactions. This inherent complexity makes mechanistic investigation of membrane fusion challenging. Taking a reductionist in vitro approach has established a fundamental paradigm for most SNARE dependent fusion events. Classically, bulk in vitro reconstitution assays leveraging synthetic lipid vesicles can determine the protein assemblies that drive membrane fusion. However, this bulk approach may overlook the heterogeneity of fusion events found within our cells. Recent advancements in single molecule light microscopy and cryo electron tomography enable visualization of individual fusion events at high temporal and spatial resolutions, respectively. In this review we highlight key features of bulk and single fusion assays with a focus on the variables to be considered within each approach. Additionally, we propose potential avenues to expand the in vitro toolbox to dissect membrane fusion intermediates.
Madison et al. (2026) studied this question.