UMMS Affiliation

RNA Therapeutics Institute

Publication Date

2015-05-25

Document Type

Article

Subjects

Actins; Animals; Cell Line; Cell Nucleus; Imaging, Three-Dimensional; Mice; Microscopy, Video; RNA Transport; RNA, Messenger; Single-Cell Analysis

Disciplines

Cell Biology | Molecular Biology

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 beta-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 microm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

Rights and Permissions

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

DOI of Published Version

10.1083/jcb.201411032

Source

J Cell Biol. 2015 May 25;209(4):609-19. doi: 10.1083/jcb.201411032. Link to article on publisher's site

Journal/Book/Conference Title

The Journal of cell biology

Related Resources

Link to Article in PubMed

PubMed ID

26008747

Creative Commons License

Creative Commons Attribution-Noncommercial-Share Alike 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

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