Visualization of mRNA translation in living cells.

@article{citeulike:895548, abstract = {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.}, address = {Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461.}, author = {Rodriguez, A. J. and Shenoy, S. M. and Singer, R. H. and Condeelis, J. }, citeulike-article-id = {895548}, comment = {Use of FlAsH/ReAsH + cycloheximide Rationale : eGFP fluorescence comes after maturation, whereas ReAsH labeling is seen as soon as the peptide has been translated}, doi = {http://dx.doi.org/10.1083/jcb.200512137}, issn = {0021-9525}, journal = {J Cell Biol}, keywords = {cytosqueleton, local\_synthesis, protein\_labeling, technique, trafficking}, month = {October}, number = {1}, pages = {67--76}, posted-at = {2006-10-13 17:00:06}, priority = {0}, title = {Visualization of mRNA translation in living cells.}, url = {http://dx.doi.org/10.1083/jcb.200512137}, volume = {175}, year = {2006} }

TY - JOUR ID - citeulike:895548 L3 - citeulike-article-id:895548 TI - Visualization of mRNA translation in living cells. JF - J Cell Biol VL - 175 IS - 1 SP - 67 EP - 76 AD - Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461. SN - 0021-9525 N2 - 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. KW - cytosqueleton KW - local_synthesis KW - protein_labeling KW - technique KW - trafficking N1 - Use of FlAsH/ReAsH + cycloheximide Rationale : eGFP fluorescence comes after maturation, whereas ReAsH labeling is seen as soon as the peptide has been translated AU - Rodriguez, AJ AU - Shenoy, SM AU - Singer, RH AU - Condeelis, J PY - 2006/10/09/ UR - http://dx.doi.org/10.1083/jcb.200512137 ER -