Single-molecule imaging of β-actin mRNAs in the cytoplasm of a living cell

Abstract: β-Actin mRNA labeled with an MS2–EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most β-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D MACRO) at the leading edge was 0.3 μm2/s. On the other hand, D MACRO in the perinuclear region was 0.02 μm2/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize β-actin mRNAs, led to an increase in D MACRO to 0.2 μm2/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of β-actin mRNA. [Copyright &y& Elsevier]