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ARTICLE
Received 13 Dec 2014 | Accepted 24 Mar 2015 | Published 13 May 2015 DOI: 10.1038/ncomms8029 OPEN
Visualization and tracking of tumour extracellular
vesicle delivery and RNA translation using
multiplexed reporters
1 2 1 3 2
Charles P. Lai , Edward Y. Kim , Christian E. Badr , Ralph Weissleder , Thorsten R. Mempel ,
1, 1,
Bakhos A. Tannous * Xandra O. Breakefield *
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.
1 Department of Neurology and Radiology, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, 149 13th Street,
C
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