Single-molecule imaging of beta-actin mRNAs in the cytoplasm of a living cell.
Beta-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 beta-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 microm(2)/s. On the other hand, D(MACRO) in the perinuclear region was 0.02 microm(2)/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize beta-actin mRNAs, led to an increase in D(MACRO) to 0.2 microm(2)/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of beta-actin mRNA.
Yamagishi
Mai
M
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ishihama
Yo
Y
Shirasaki
Yoshitaka
Y
Kurama
Hideki
H
Funatsu
Takashi
T
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003593:Cytoplasm / Q000378:metabolism*; D004058:Diffusion; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051722:ADAM Proteins; C068490:ADAM8 protein, human