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J Cell Biol. 2015 May 25;209(4):609-19. doi: 10.1083/jcb.201411032.

Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking.

Author information

1
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605.
2
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461 Howard Hughes Medical Institute Janelia Farm, Ashburn, VA 20147 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany.
3
Howard Hughes Medical Institute Janelia Farm, Ashburn, VA 20147 The Rockefeller University, New York, NY 10065.
4
Department of Imaging Sciences, Technical University Delft, Delft 2628CJ, Netherlands.
5
Howard Hughes Medical Institute Janelia Farm, Ashburn, VA 20147 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany.
6
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461 Howard Hughes Medical Institute Janelia Farm, Ashburn, VA 20147.
7
RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605 david.grunwald@umassmed.edu.

Abstract

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

PMID:
26008747
PMCID:
PMC4442804
DOI:
10.1083/jcb.201411032
[Indexed for MEDLINE]
Free PMC Article

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