Visualization of mRNA translation in living cells.
The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.
Rodriguez
Alexis J
AJ
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. arodrigu@aecom.yu.edu
Shenoy
Shailesh M
SM
Singer
Robert H
RH
Condeelis
John
J
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D017404:In Situ Hybridization, Fluorescence; D007202:Indicators and Reagents / Q000032:analysis; D007365:Intercellular Junctions / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011132:Polyribosomes / Q000502:physiology; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis
D000199:Actins; D015820:Cadherins; D007202:Indicators and Reagents; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins