# In vitro assays to monitor membrane fusion

## TL;DR

This review discusses methods to study how cell membranes fuse, comparing traditional bulk assays with newer single-molecule techniques.

## Contribution

The paper proposes ways to expand in vitro tools for studying membrane fusion intermediates using advanced imaging techniques.

## Key findings

- Bulk in vitro assays using synthetic lipid vesicles identify proteins driving membrane fusion.
- Single molecule microscopy and cryo tomography reveal fusion heterogeneity and intermediates.
- Combining bulk and single fusion assays can improve understanding of membrane fusion mechanisms.

## Abstract

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.