专业外语教学(吉林大学)Visualization and tracking of tumour extracellular.pdfVIP

专业外语教学(吉林大学)Visualization and tracking of tumour extracellular.pdf

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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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