Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.

Charles P Lai,Ralph Weissleder,Xandra O Breakefield,Bakhos A Tannous,Christian E Badr,Thorsten R Mempel,Edward Y Kim

doi:10.1038/ncomms8029

Abstract

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.

Highlights

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies
As EVs are derived from the plasma membrane either via inward invagination of endosomes to form multivesicular bodies releasing exosomes[18] or outward budding and shedding for microvesicles and apoptotic blebs released from the cell surface[19,20,21], we hypothesized that tagging the plasma membrane with fluorescent proteins would enable labelling of multiple EV types
Live-cell confocal microscopy of 293T-PalmGFP cells showed that PalmGFP uniformly labels the plasma membrane and reveals budlike structure on their surface and processes (Fig. 1b)

Summary

Introduction

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. Microvesicles and apoptotic blebs as a conduit for intercellular communication without direct cell-to-cell contacts1–4 {Colombo:2013bt, Camussi:2010uk, Vader:2014fq, ELAndaloussi:2013ca}-CITATION_IS_EMPTY These vesicles, collectively termed extracellular vesicles (EVs), are capable of horizontal transfer of lipids, (glyco)proteins and nucleic acids from EV-donor cells to neighbouring and/or distal recipient cells[5,6]. Gaussia luciferase (Gluc), which is over 1,000-fold brighter than commonly used luciferases[23], catalyses flash bioluminescence with rapid signal decay, making it an ideal reporter to study temporal properties of minute events, such as translation of EV-delivered mRNAs

Methods

Results

Conclusion