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
PubMed ID
26008747
Repository Citation
Smith C, Preibisch S, Joseph A, Abrahamsson S, Rieger B, Myers E, Singer RH, Grünwald D. (2015). Nuclear accessibility of beta-actin mRNA is measured by 3D single-molecule real-time tracking. Open Access Publications by UMMS Authors. https://doi.org/10.1083/jcb.201411032. Retrieved from https://escholarship.umassmed.edu/oapubs/2717
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.