Single molecule tracking of quantum dot-labeled mRNAs in a cell nucleus.
Single particle tracking (SPT) is a powerful technique for studying mRNA dynamics in cells. Although SPT of mRNA has been performed by labeling mRNA with fluorescent dyes or proteins, observation of mRNA for long durations with high temporal resolution has been difficult due to weak fluorescence and rapid photobleaching. Using quantum dots (QDs), we succeeded in observing the movement of individual mRNAs for more than 60 s, with a temporal resolution of 30 ms. Intronless and truncated ftz mRNA, synthesized in vitro and labeled with QDs, was microinjected into the nuclei of Cos7 cells. Almost all mRNAs were in motion, and statistical analyses revealed anomalous diffusion between barriers, with a microscopic diffusion coefficient of 0.12 microm2/s and a macroscopic diffusion coefficient of 0.025 microm2/s. Diffusion of mRNA was observed in interchromatin regions but not in histone2B-GFP-labeled chromatin regions. These results provide direct evidence of channeled mRNA diffusion in interchromatin regions.
Ishihama
Yo
Y
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Funatsu
Takashi
T
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000737:chemistry; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006657:Histones / Q000737:chemistry; D045663:Quantum Dots; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D013194:Staining and Labeling
D002843:Chromatin; D006657:Histones; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins