Abstract
The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.
Highlights
Extracellular Vesicles (EVs) are a highly heterogeneous group of biological nanoparticles, which can be secreted by almost every living organism and can be isolated from any biological fluid[1]
The proposed strategy is based on the use of two reporter proteins, DSP1 and DSP2, which work as tags encoding non-functional split versions of EGFP and Renilla luciferase proteins
With the aim of targeting DSP1 reporter protein to extracellular vesicles (EVs) we designed chimeric proteins encompassing DSP1 fused to either CD9 or CD63 human tetraspanins (Supplementary Fig. S1), which are widely used as EV markers
Summary
Extracellular Vesicles (EVs) are a highly heterogeneous group of biological nanoparticles, which can be secreted by almost every living organism and can be isolated from any biological fluid[1]. Some of the probes used are membrane-specific fluorescent dyes, such as PKH67, PKH26, DiI, DiR, or rhodamine B24 These dyes may affect normal EV behaviour, they have a short half-life and can give non-specific signal in target cells by direct exchange of the dye between both membranes, since they stain lipids that may be present in the membranes of the samples, and can produce false EV signals by forming micelles[25]. We generated DSP1-tagged tetraspanin constructs to direct them into EVs and report the efficacy of different experimental approaches to develop a highly sensitive method to quantify EV uptake This assay will allow future high-throughput analyses to quickly assess the effects of specific drugs or blocking antibodies on EV uptake, which will facilitate the identification of the functional molecules involved in this process and the screening of potential treatments aimed at impairing EV uptake in pathological situations
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