# Development of a Live‐Cell Imaging Assay to Elucidate Spatiotemporal Dynamics of Extracellular Vesicle Fusion with Target Cells

## TL;DR

A new live-cell imaging method tracks how extracellular vesicles fuse with target cells, helping understand how they deliver their contents.

## Contribution

A novel live-cell imaging assay called EV-FUSIM is developed to directly visualize and quantify extracellular vesicle fusion with target cells.

## Key findings

- EV-FUSIM enables real-time imaging of EV-binding, uptake, and fusion events.
- The method allows quantification of fusion kinetics and subcellular localization of fusion.
- The assay can identify fusogenic EV subsets and factors modulating fusion.

## Abstract

Cells communicate via extracellular vesicles (EVs) containing functional RNAs, proteins, and lipids. Knowledge on the fate of internalized EVs, especially their capacity to fuse with target cell membranes and deliver luminal cargo, is limited. Currently available EV‐cargo delivery assays are indirect and thus unlikely to uncover molecular players and conditions that specifically control the EV‐fusion step. Here, we present a novel live‐cell imaging assay for detection of EV‐binding, ‐uptake, and ‐fusion in time and space. We employed the SunTag system for exceptional signal amplification. EV‐donor cells were engineered to tag the luminal EV‐membrane with a fluorescent label coupled to SunTag peptides. Recipient cells express fluorescent single‐chain anti‐SunTag antibody (STAb), which binds EV‐enclosed SunTag upon its cytosolic exposure. Using SunTagged EVs carrying fusogen VSV‐G, we visualize the EV‐fusion process, quantify fusion kinetics and efficiency, and determine subcellular localization of fusion events. We term this methodology the Extracellular Vesicle Fusion Spatiotemporal Imaging Method (EV‐FUSIM). In the future, this technology can support the identification of fusogenic EV‐subsets, as well as molecular players and drugs that modulate EV‐fusion, without confounding effects of post‐fusion processes. This will extend knowledge on EV‐biology and can aid in the engineering of EVs that efficiently deliver intraluminal therapeutic payloads.

We present a novel live‐cell imaging assay for detection of extracellular vesicle (EV)‐binding, ‐uptake, and ‐fusion in time and space. Using EVs carrying the fusogen VSV‐G, we demonstrate real‐time imaging of EV‐fusion, quantification of fusion kinetics and efficiency, and subcellular localization of fusion events. In the future, this technology can support the identification of fusogenic EV‐subsets and factors controlling EV‐fusion. This will extend knowledge on EV‐biology and can aid in the engineering of EVs that efficiently deliver intraluminal therapeutic payloads.